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Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation

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†Department of Biological Engineering, ‡Department of Chemical Engineering, §Center for Gynepathology Research, ∥Department of Chemistry and ⊥̂Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts United States
Cite this: Biomacromolecules 2015, 16, 8, 2316-2326
Publication Date (Web):June 22, 2015
https://doi.org/10.1021/acs.biomac.5b00549
Copyright © 2015 American Chemical Society
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Abstract

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Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.

1 Introduction

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The need for in vitro physiological models and for clinical therapies has driven the development of synthetic biomaterials that recapitulate key features of the extracellular matrix (ECM).(1) Polymer hydrogels are an attractive foundation for synthetic ECM, as their biophysical and biochemical properties can be tuned in modular fashion to control cellular phenotype. Hydrogels built from macromers containing highly flexible, hydrophilic poly(ethylene glycol) (PEG) have found especially widespread use due to their biocompatibility, relative inertness to nonspecific cell interactions, relative ease of functionalization with biomolecular motifs, and commercial availability of reactive PEG macromers of various molecular weights and branching configurations.(2-5) However, unmodified PEG hydrogels possess limited or no intrinsic biological function and thus may fail to support desired cell behaviors.
Many strategies have been explored to modify PEG hydrogels with ECM-derived biological cues such as adhesion peptides and growth factors,(6-9) but facile, site-specific attachment of large peptides and proteins in a way that retains bioactivity remains a challenge, particularly if the approaches are intended to be used in the presence of cells. Photopolymerization and conjugation of peptides or proteins through amine groups(10-14) are straightforward, but lack specificity and may alter protein function. Conjugation through click chemistry allows increased intermediate stability and improved efficiency of hydrogel conjugation; however, this approach is limited by the difficulties associated with producing proteins that incorporate precursors for click chemistry in an appropriate site-specific manner.(9)
Here, we describe the use of sortase-mediated ligation of epidermal growth factor (EGF) to PEG-based synthetic ECM hydrogels. Sortases are transpeptidases found in Gram-positive bacteria, which anchor surface proteins to the bacterial cell wall in vivo.(15) The enzyme sortase A from Staphylococcus aureus recognizes a specific LPXTG motif (X = any amino acid except proline), cleaves the amide bond between the threonine and the glycine, and covalently attaches an oligoglycine nucleophile.(16) Moreover, the formed product maintains the LPXTG motif, allowing controlled release or cleavage of the tethered protein through a second sortase-mediated transpeptidase reaction in the presence of a soluble N-terminal oligoglycine nucleophile (e.g., triglycine, GGG).
Sortase-mediated ligation has found numerous applications in purification, modification and immobilization of proteins.(17, 18) It has been used to conjugate proteins to different types of surfaces,(19-27) liposomes(28) and peptides.(29, 30) Here we extend sortase-mediated ligation to the modification of synthetic ECM hydrogels. Compared to other enzymatic approaches,(31) sortase-mediated ligation offers the advantage of enhanced catalytic rate due to the engineering of mutant sortases,(32) enhanced diffusion rate due to the relatively small size of the sortase (23.5 kDa), high yield of recombinant expression and relatively modest propensity to modify known mammalian proteins.(33)
In this study, we ligated EGF to relatively soft, permeable PEG hydrogels prefunctionalized with cell adhesion motifs. The biophysical and cell adhesion properties of hydrogels were designed to support physiological culture of primary human epithelial cells. We formed hydrogels by copolymerizing acrylate-terminated multiarm PEG with a thiol-terminated LPRTG peptide and thiol-terminated multiarm PEG through Michael-type addition (Scheme 1) as reported earlier.(3) This cross-linking was carried out in the presence of a cell adhesion peptide bearing a thiol reactive group to render the resulting hydrogels adhesive to primary human epithelial cells. An evolved triple mutant of the sortase A enzyme (P94S/D160N/K196T) developed by Liu and co-workers(32) was used to tether the previously reported N-terminal Gly3-tagged human epidermal growth factor (GGG-EGF)(30) to the hydrogel (Scheme 1). Characterization of the system included sortase-mediated cleavage of the tethered EGF for quantification in solution. To test the bioactivity of the tethered EGF, we investigated DNA synthesis with EGF-responsive human cryopreserved hepatocytes and primary human endometrial epithelial cells. Our results illustrate several features of the sortase-mediated ligation reaction as implemented for modification of synthetic ECM hydrogels, including the ability to efficiently release tethered proteins via a sortase-mediated reaction with a small soluble oligoglycine substrate, GGG.

Scheme 1

Scheme 1. Process Used to Tether GGG-EGF to the Hydrogela
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aTop: Hydrogels are formed by Michael-type additions of 8-arm PEG-acrylate stars cross-linked with 4-arm PEG-thiol stars. Adhesion peptides (SynKRGD) and sortase motif peptides (LPRTG) can also be cross-linked into the hydrogel if they contain a cysteine residue to react with the acrylate of the 8-arm PEG star. Bottom: (A) Sortase-mediated ligation of GGG-EGF to pre-formed PEG hydrogels containing LPRTG peptide. (B) Sortase diffuses into the hydrogel, cleaves the peptide bond between the threonine and the glycine of the LPRTG peptide, releasing G, then the N-terminus of GGG-EGF, which has diffused into the hydrogel, is ligated to the C-terminus of the LPRT-motif (C). The same sequence of steps can be used to cleave EGF once it is ligated to the hydrogel, by adding sortase and the simple peptide GGG; GGG will displace tethered EGF, releasing it into solution.

2 Experimental Section

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2.1 Materials

Commercially available chemicals, including triglycine (GGG), were purchased from Sigma-Aldrich and used without further purification unless otherwise noted. PEG macromers (10 kDa 8-arm PEG-acrylate and 5 kDa 4-arm PEG-thiol) were purchased from JenKem. All custom peptides were synthesized by Boston Open Laboratories, USA, as follows: Ac-CRGLPRTGG-CONH2 (LPRTG), Ac-CRGLPRTGGK(ε-fluorescein)-CONH2 (LPRTG-fam), PHSRNGGGK(εGGG ERCG-Ac)-GGRGDSPY (synKRGD) and PHSRNGGGK(εGGGERCG-Ac)-GGRDGSPY (scrambled synKRDG). Hydrogels were formed in 96-well angiogenesis plates (0.125 cm2; Ibidi). The recombinant sortaggable GGG-EGF (human EGF fused to a triglycine sequence at the N-terminus) and the evolved triple mutant (P94S/D160N/D165A) of the sortase A enzyme(32) were expressed and purified as previously reported.(30) Reagents from the DuoSet EGF ELISA kit (R&D Systems, DY236-05) were used for EGF detection. Cryopreserved hepatocytes (Human Plateable Hepatocytes, Induction Qualified, lot# Hu1663) and hepatocyte media were purchased from Life Technologies. Human wild type EGF (hEGF) was obtained from Invitrogen (PHG0313).

2.2 Hydrogel Fabrication and Sortase-Mediated Ligation of GGG-EGF

Using Michael-type addition as described earlier(3) nominal (preswelling) 5% w/v polymer hydrogels with 1:1 thiol:acrylate ratio were synthesized by preincubating PEG-acrylate macromers with LPRTG and adhesion peptides in phosphate buffered saline (PBS) at pH 6.9 for 20 min. Adhesion peptides (synKRGD or scrambled synKRDG) were added at a nominal concentration of 500 μM and LPRTG nominal concentrations varied from 0 to 250 μM. PEG-thiol cross-linking macromers were added and 10 μL hydrogels were formed within the inner wells of a 96-well angiogenesis plate. After gelation [around 1 h at room temperature (RT)], hydrogels were covered with PBS and allowed to swell for 90 min at 4 °C with PBS changes every 30 min. They were further allowed to swell in intermediate buffer (50 mM HEPES, 150 mM NaCl, pH 7.9) for 90 min at 4 °C with buffer changes every 30 min. Hydrogels were blocked with 70 μL/well calcium buffer (50 mM HEPES, 150 mM NaCl, 10 mM CaCl2, pH 7.9) containing 0.5% purified bovine casein for 1 h at RT. Tethering solution containing 2 or 20 μM GGG-EGF and 15 μM sortase enzyme in calcium buffer was added to the hydrogels (50 μL per well) and the reaction was allowed to proceed for 1 h at RT with constant agitation at 30 rpm. Tethering solution without sortase was used to test for nonspecific binding of GGG-EGF to hydrogels and calcium buffer was used for control and in soluble EGF conditions. The sortase reaction was stopped by addition of 5 μL/well of ethylenediaminetetraacetic acid (EDTA; 300 mM), then supernatants were collected and frozen at −80 °C for EGF quantification, and hydrogels were extensively washed (3× immediately and every 30 min for 3 h) with 70 μL/well intermediate buffer and soaked overnight at 4 °C.

2.3 Hydrogel Fluorescence Measurements for Quantification of Incorporated or Sortase-Cleaved LPRTG

Hydrogels containing a mix of 96% LPRTG and 4% LPRTG-fam peptides with varying total nominal peptide concentrations of 0, 20, 50, 100, or 250 μM were cross-linked in 96-well angiogenesis plates as described above. After 6 × 30 min washes at 4 °C, intermediate buffer (50 μL) was added on top of each hydrogel and fluorescence (λex = 485 nm; λem = 530 nm) was measured using a microplate reader (SpectraMax M2e, Molecular Devices). A linear standard curve was established by measuring the fluorescence of hydrogels containing 0, 20, 50, 100, or 250 μM of total LPRTG peptide (Supporting Information Figure 1). After incubation for 1 h with either 2 μM or 20 μM GGG-EGF in the presence of 15 μM sortase the reaction was stopped with 5 μL/well EDTA (300 mM) and hydrogels were extensively washed (3× immediately, every 30 min for 3 h and soaked overnight at 4 °C) before fluorescence reading. In order to correct for photobleaching, decreased fluorescence of control hydrogels was measured in the absence of sortase (Supporting Information Figure 2).

2.4 GGG-EGF Detection via Direct ELISA on Hydrogels

Hydrogels were blocked with Odyssey Blocking Buffer (Li-Cor Biosciences) diluted 1:1 with PBS (OBB-PBS) for 1 h at RT and washed 3× with 0.1% Tween-20 in PBS. Hydrogels were incubated with biotinylated goat antihuman EGF detection antibody at a concentration of 50 ng/mL in OBB-PBS for 2 h at RT with constant agitation at 30 rpm. The washing steps were repeated and hydrogels were incubated with streptavidin-HRP diluted 1:40 in OBB-PBS for 20 min at RT with constant agitation at 30 rpm and protected from light. After washes, hydrogels were incubated with substrate solution consisting of a 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (tetramethylbenzidine) (R&D Systems, DY994) for 20–30 min at RT protected from light. The reaction was stopped with 1 M H2SO4. Aliquots (20 μL) of supernatant were transferred to a clear bottom Nunc MaxiSorp 384-well plate (Thermo Scientific) and absorbance at 450 nm was measured using a microplate reader (SpectraMax M2e, Molecular Devices). Absorbance at 540 nm was subtracted to account for optical imperfections of the plate.

2.5 Quantification of Sortase-Mediated Cleavage of Tethered EGF from Hydrogels

A modified sandwich ELISA was used to measure the amount of EGF cleaved from hydrogels in the presence of sortase A and GGG peptide. GGG-EGF-tethered hydrogels were soaked in calcium buffer for 1 h at 4 °C and incubated for 48 h at 4 °C with constant agitation in 50 μL/well cleavage solution containing 20 mM GGG and 200 μM sortase in calcium buffer. Supernatants were collected and frozen. A clear bottom Nunc MaxiSorp 384-well plate (Thermo Scientific) was coated with mouse antihuman EGF capture antibody diluted at the recommended concentration of 4.0 μg/mL in sterile PBS. The plate was covered with an adhesive strip, spun at 1500 rpm for 3 min and incubated overnight at 4 °C with constant agitation. Three washing steps with 100 μL/well 0.1% Tween-20 in PBS were performed at RT using an automatic plate washer (405 Touch Microplate Washer, BioTek). The plate was blocked with 100 μL/well OBB-PBS and incubated for 2 to 6 h at RT with constant agitation at 30 rpm. Samples were first diluted with calcium buffer containing 1% BSA in 1.5 mL Protein LoBind tubes (Eppendorf) and then serially diluted in 0.5 mL Protein LoBind tubes. Standard curves were made by serially diluting GGG-EGF in 1% BSA in calcium buffer. After the washing steps, samples were plated and the plate was covered with an adhesive strip, spun at 1500 rpm for 3 min and incubated for 2 h at RT with constant agitation. This process was repeated with the biotinylated goat antihuman EGF detection antibody diluted at the recommended working concentration of 50 ng/mL in OBB-PBS. The plate was washed, incubated with streptavidin-HRP diluted 1:40 in OBB-PBS and spun at 1500 rpm for 3 min protected from light. After 20 min incubation at RT with constant agitation, the plate was washed and incubated with substrate solution for 20–30 min at RT protected from light. The reaction was stopped with 1 M H2SO4 and absorbance at 450 nm was measured.

2.6 Human Cryopreserved Hepatocyte Culture on Hydrogels

After a PBS wash and UV-sterilization, hydrogels were soaked in human hepatocyte seeding medium (hHSM; Williams E medium supplemented with 5% FBS, 1 μM hydrocortisone, 1% penicillin/streptomycin (P/S), 4 μg/mL human recombinant insulin, 2 mM GlutaMAX, 15 mM HEPES (pH 7.4); CM3000) with or without 20 ng/mL hEGF for 1 h. Cryopreserved hepatocytes were quickly thawed in a 37 °C water bath, transferred into a 50 mL falcon tube with 25 mL prewarmed Cryopreserved Hepatocyte Recovery Medium (CHRM; CM7000) and centrifuged at RT at 100g for 8 min. Warm hHSM (1 mL) was added to the cell pellet and cells were gently rocked, counted (Countess Cell Counter, Invitrogen) and placed on ice. Hepatocytes were seeded on hydrogels and on collagen I-coated (BioCoat, BD Biosciences) wells in 96-well angiogenesis plates at a density of 60 000 cells/cm2 (7500 cells/well) in 50 μL/well hHSM. Hepatocytes were incubated at 37 °C, 95% air, 5% CO2. Twenty-four hours after cell seeding, medium was switched to serum-free human hepatocyte maintenance medium (hHMM; Williams E medium supplemented with 0.1 μM hydrocortisone, 0.5% P/S, ITS+ (human recombinant insulin (6.25 μg/mL), human transferrin (6.25 μg/mL), selenous acid (6.25 ng/mL), bovine serum albumin (1.25 mg/mL), linoleic acid (5.35 μg/mL)), 2 mM GlutaMAX, 15 mM HEPES (pH 7.4); CM4000) with or without 20 ng/mL hEGF and cells were incubated for 24 h.

2.7 Endometrial Biopsy Collection and Isolation

Eutopic endometrial biopsies were obtained from two premenopausal women in the proliferative phase of the menstrual cycle, who were undergoing surgery for benign gynecological diseases. Selective criteria included that the patients had regular menstrual cycles (26 to 35 days) and were not using hormonal treatment for at least 3 months prior to surgery. A standardized questionnaire was used to document all clinical data. Tissues were collected with the approval of the Partners Human Research Committee and the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects and with the informed consent of each patient. Endometrial Pipelle biopsies were dissociated and cells purified as described by Osteen and co-workers(34) with some modifications (Supporting Information).

2.8 Endometrial Epithelial Cell Culture on Hydrogels

Before cell seeding, hydrogels were washed with PBS, UV-sterilized for 15 min and soaked in DMEM/F12/FBS (mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 (Gibco) supplemented with 1% penicillin/streptomycin and 10% v/v dextran/charcoal treated fetal bovine serum (Atlanta Biologicals) with or without 20 ng/mL hEGF for 1 h. Cultured endometrial epithelial cells (EECs) were trypsinized and seeded on hydrogels and on standard tissue-culture polystyrene (TCPS) in 96-well angiogenesis plates at a density of 20 000 cells/cm2 (2500 cells/well) in 50 μL/well DMEM/F12/FBS. EECs were incubated at 37 °C, 95% air, 5% CO2. Twenty-four hours after cell seeding, medium was switched to serum-free medium DMEM/F12 with or without 20 ng/mL hEGF and cells were incubated for 16 h.

2.9 DNA Synthesis Assay

Click-iT EdU Alexa Fluor 488 kit (Life Technologies) was used to quantify cells actively synthesizing DNA. Forty-eight hours after cell seeding, hepatocytes were incubated in hHMM with 10 μM of 5-ethynyl-2′-deoxyuridine (EdU) in with or without 20 ng/mL (3.3 nM) hEGF for 24 h at 37 °C, 95% air, 5% CO2. Similarly, EECs were incubated 40 h post seeding with 10 μM EdU in DMEM/F12 with or without 20 ng/mL (3.3 nM) hEGF for 24 h. Both cell-types were fixed with 3.7% formaldehyde in PBS for 15 min at RT. Cells were washed twice with 3% BSA in PBS and permeabilized with 0.5% Triton X-100 in PBS for 20 min at RT. Click-iT reaction cocktail was prepared as described by the manufacturer and 20 μL per well was added. Cells were incubated for 30 min at RT protected from light. After cells were washed once with 3% BSA in PBS and once with PBS, they were incubated with Hoechst 33342 diluted 1:2000 in PBS for 30 min at RT protected from light. Cells were finally washed twice with PBS and imaged using a Leica DMI 6000 microscope and Oasis Surveyor software. Images were processed, and cell nuclei were counted using ImageJ64 software. The percentage of cells synthesizing DNA was computed as the ratio of cells positively stained with Alexa Fluor 488 divided by the total number of cells given by Hoechst counter-staining.

3 Results and Discussion

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We first evaluated the ability of the evolved triple mutant of the sortase A to tether the solution-phase GGG-EGF nucleophile to LPRTG substrate motifs that were covalently incorporated in PEG hydrogels (LPRTG-gel). Compared to the wild-type sortase, this mutant displays a 3-fold improvement in turnover (kcat = 4.8 ± 0.6 s–1) and a 14-fold improvement in the affinity for the LPXTG substrate (Km LPXTG = 0.56 ± 0.007 mM), resulting in a 43-fold increase of catalytic efficiency (kcat/Km LPXTG = 8600 ± 1500 M–1 s–1), although the affinity for the N-terminal GGG substrate is less favorable.(32) These enhanced properties were predicted to allow efficient catalysis at the relatively low LPXTG substrate concentrations in the hydrogels.
Sortase-mediated ligation was characterized by three complementary methods: measurement of the consumption of the GGG-EGF nucleophile from the reaction solution, measurement of the consumption of the LPRTG-gel substrate, and measurement of GGG-EGF released from the hydrogel after sortase-mediated cleavage of tethered EGF from the hydrogel. We also used a direct on-gel ELISA approach to assess EGF accessibility. The results of these experiments were used to define suitable tethering conditions to create synthetic ECM for modulation of epithelial cell DNA synthesis.

3.1 The Amount of GGG-EGF Consumed from Solution in the Presence of Sortase Depends on LPRTG-Gel Concentration

We first measured the disappearance of the GGG-EGF nucleophile from the supernatants of PEG hydrogels fabricated with systematically varied LPRTG-gel concentrations (0, 20, 50, 100, or 250 μM, corresponding to 0, 200, 500, 1000, and 2500 total pmol LPRTG per hydrogel) and then exposed to coupling solutions containing 15 μM (750 pmol) sortase and GGG-EGF at concentrations of either 2 μM (100 pmol total peptide) or 20 μM (1000 pmol total peptide) to tether EGF via Scheme 1. The amount of GGG-EGF present in the reaction supernatant was measured by sandwich ELISA at the start and end of the reaction.
For control hydrogels (i.e., LPRTG-gel = 0 μM), GGG-EGF diffuses into the hydrogel but does not react even in the presence of sortase. The observed reduction in GGG-EGF concentration in the supernatant of control hydrogels due to this partitioning was 6% for both the 2 and 20 μM GGG-EGF conditions (Figure 1A,B), a value about half that expected based on simple dilution due to the volume of liquid in the hydrogel and consistent with the protein-repulsion properties of PEG. For both the 2 μM and 20 μM GGG-EGF coupling concentrations, GGG-EGF depletion from the coupling solution increased with increasing concentrations of LPRTG-gel (Figure 1A,B) in a manner consistent with enzyme catalysis of the reaction. For the 2 μM GGG-EGF coupling condition (Figure 1A), total LPRTG-gel is always in stoichiometric excess to total GGG-EGF (200–2500 pmol LPRTG: 100 pmol GGG-EGF); hence, the incomplete depletion of GGG-EGF substrate (∼20% at each condition) indicates a kinetic or thermodynamic limit to coupling under these conditions. Interestingly, for the 20 μM GGG-EGF coupling condition (Figure 1B), the proportion of GGG-EGF depleted for each LRPTG-gel concentration is comparable to that in the 2 μM GGG-EGF case; i.e., within the error of the ELISA, 10 times as much GGG-EGF is consumed in the 20 μM versus 2 μM GGG-EGF coupling condition (Figure 1A,B).

Figure 1

Figure 1. Amount of GGG-EGF peptide nucleophile remaining in solution as a function of total LPRTG concentration in hydrogel. GGG-EGF is quantified by sandwich ELISA before and after sortase-mediated ligation of GGG-EGF to preformed hydrogels containing LPRTG, using nucleophile concentrations of 2 μM (A) or 20 μM (B). The amount of GGG-EGF remaining in solution after sortase-mediated ligation decreases with increasing LPRTG concentration in hydrogel. Error bars represent standard error of the mean (n = 2).

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This apparent first-order dependence of the reaction on GGG-EGF concentration is consistent with the reported ping-pong bibi mechanism for sortase A,(35) where a thioacyl intermediate is resolved by the N-terminus of an oligoglycine nucleophile—provided that there is sufficient oligoglycine to render the competing reaction, hydrolysis,(16) relatively unimportant. However, the interpretation of the results from this pilot experiment is inherently limited by the convolution of two rate processes—enzyme kinetics and enzyme/substrate diffusion—occurring simultaneously with a complex reaction mechanism, which can include not only ligation but also hydrolysis. Based on size, the diffusion coefficients of GGG-EGF and sortase in water are about 2.5 × 10–6 cm2/s and 1.5 × 10–6 cm2/s, respectively,(36) and diffusion of molecules of this size in the hydrogels would be expected to be hindered by 50–90%.(36-38) We can thus estimate the characteristic diffusion times, τD, for GGG-EGF and sortase in the hydrogel in the absence of reaction as 0.4–1.8 h and 0.6–3 h, respectively, using the well-established relationship τD ∼ L2/4DA-hydrogel where L is the thickness of the hydrogel (0.8 mm) and DA-hydrogel is the effective diffusion coefficient for either GGG-EGF or sortase in the hydrogel. These characteristic diffusion times represent a lower boundary, as interactions with the LPRTG-gel substrate would be expected to retard diffusion and create gradients in reaction rate during the 1 h incubation. In summary, this pilot experiment provides compelling evidence that sortase is capable of tethering relatively large peptides (∼6 kDa) to PEG hydrogels, motivating further characterization of the reaction process.

3.2 LPRTG Cleavage Product Is Released from the Hydrogel in the Presence of GGG-EGF and Sortase

In order to elucidate how many LPRTG molecules were effectively processed by sortase, 4% of the LPRTG peptides incorporated into the hydrogels were labeled with fluorescein (LPRTG-fam). When the sortase enzyme cleaves the peptide bond between the threonine and the glycine of the LPRTG-fam motif, fluorescein is released, thus resulting in a decrease of fluorescence associated with the hydrogel. Fluorescence intensities after washing for hydrogels before and after incubation for 1 h with either 2 μM or 20 μM GGG-EGF in the presence or absence of 15 μM sortase are shown in Supporting Information Figure 2. After correction for photobleaching, fluorescence intensities were converted to pmol LPRTG in hydrogel (see Experimental Section), and the amount of reacted LPRTG was calculated as the difference between the values before and after the sortase reaction. As shown in Figure 2, LPRTG consumption increased with increasing initial amount of LPRTG in the hydrogels and was greater for the 20 μM GGG-EGF than the 2 μM GGG-EGF condition. This pattern is qualitatively consistent with the trends seen for consumption of GGG-EGF (Figure 1). This confirms that the triple mutant sortase is capable of recognizing the LPRTG motif when it is incorporated in PEG hydrogels; i.e., sortase can cleave the peptide bond between the threonine and the glycine in order to release the fluorescein from the hydrogel.

Figure 2

Figure 2. Amount of reacted LPRTG as a function of total initial LPRTG amount in hydrogel. After correction for photobleaching, fluorescence intensities were converted to pmol LPRTG in the hydrogel and the amount of reacted LPRTG was calculated as the difference between the values before and after sortase-mediated ligation. Error bars represent standard error of the mean (n = 2).

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Interestingly, whereas the amount of GGG-EGF consumed from the supernatant in the 20 μM condition was 10-fold higher than the amount consumed in the 2 μM condition, the amount of reacted LPRTG at the 20 μM GGG-EGF condition was only about 2-fold higher than the reacted LPRTG at the 2 μM condition (Figure 2). We speculate that this discrepancy arises from a difference in the relative rates of hydrolysis and transpeptidation for the different GGG-EGF concentrations. Considering the greatest LPRTG concentration as a representative case, the number of moles of GGG-EGF consumed (20 pmol and 230 pmol for the 2 μM and 20 μM concentrations, respectively) is substantially lower than the amount of reacted LPRTG (140 pmol and 310 pmol for the 2 μM and 20 μM GGG-EGF concentrations, respectively), and the ratio of reacted LPRTG:GGG-EGF consumed is much higher for the lower GGG-EGF concentration. Because the sortase and LPRTG concentrations compared here are identical and only the GGG-EGF nucleophile concentration changes, this observation is consistent with nucleophile starvation at the lower GGG-EGF concentration leading to proportionately greater hydrolysis compared to ligation. This interpretation is plausible as the Km,GGG value is in the millimolar range, and thus the reaction would be expected to be in a regime that depends on the GGG-EGF concentration.

3.3 Specific Tethering of GGG-EGF Increases with LPRTG Concentration in Hydrogels and GGG-EGF Concentration in Solution

A noteworthy aspect of sortase-mediated ligation is that the product formed contains an LPRTGGG sequence that becomes itself a potential substrate if a GGG-containing nucleophile is available. During ligation of GGG-EGF to the hydrogel through the LPRTG motif covalently incorporated into the hydrogel, this secondary reaction is minimized by the presence of a substantial stoichiometric excess of LPRTG substrate compared to GGG-EGF. However, this feature can also be exploited to cleave EGF from the hydrogel following ligation, by adding a high concentration (20 mM) of the simple nucleophile GGG, thus driving the reaction toward replacement of EGF by GGG and freeing EGF to the solution phase. Subsequent measurement of EGF in solution via ELISA allows the amount of ligated (vs nonspecifically associated) EGF to be ascertained.
In order to establish reaction conditions that would lead to complete cleavage of the tethered molecules, we used cleavage of LPRTG-fam as a surrogate measure for reaction progress. As noted above, sortase-mediated reactions occurring in the hydrogel require diffusion of the enzyme and substrate into the hydrogel, a process that likely requires several hours longer than the 1 h reaction time used for ligation. After the initial ligation reaction with 2 or 20 μM GGG-EGF, hydrogels containing 0, 20, 50, 100, or 250 μM of total LPRTG-containing peptide at a ratio of 4% LPRTG-fam and 96% LPRTG were incubated with 20 mM GGG and 200 μM sortase for 48 h at 4 °C. Fluorescence was measured as previously described. After this cleavage step (i.e., cleavage of previously tethered GGG-EGF plus LPRTG and LPRTG-fam), the percentage of residual fluorescence dropped to a similar level of 9–12% of initial fluorescence for all LPRTG concentrations and for both GGG-EGF concentrations (Supporting Information Figure 3). Residual fluorescence is likely caused by adsorption of fluorescein to the plastic of the plate or to the hydrogel, or less likely, by inaccessibility of certain LPRTG-fam molecules to cleavage. The relatively similar low level of residual fluorescence observed for all the conditions suggests that previously tethered GGG-EGF was released completely.

Figure 3

Figure 3. Amount of GGG-EGF released in solution as a function of total LPRTG concentration in hydrogel. GGG-EGF is quantified by sandwich ELISA after hydrogel washes and sortase-mediated hydrogel cleavage following tethering of GGG-EGF at either 2 μM (A) or 20 μM (B). The amount of GGG-EGF released in solution after sortase-mediated cleavage increases with increasing LPRTG concentration in hydrogel. Error bars represent standard error of the mean (n = 2).

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Hydrogel supernatants were collected after all reaction steps, including washes following sortase-mediated ligation and after hydrogel cleavage, in order to quantify GGG-EGF with sandwich ELISA. Figure 3 shows the amount of released GGG-EGF as a function of LPRTG concentration for sortase-mediated ligation at 2 and 20 μM GGG-EGF respectively. For both concentrations, the amount of GGG-EGF released during washes after tethering is low, constant, and proportional to the initial concentration of GGG-EGF used (Figure 3). Cleaved EGF increased with LPRTG concentration in the hydrogels and is also proportional to the GGG-EGF in the initial tethering solution, a trend consistent with previous experiments. The sum of the amounts of GGG-EGF left in solution after ligation, extracted in washes, and released by cleavage should be equal to the amount of GGG-EGF present in the original coupling solution. Indeed, this is the case for most of the conditions, as shown in Figure 4. For sortase-mediated ligation at 2 μM GGG-EGF, a maximal amount of 19 pmol of GGG-EGF was found to be cleaved from hydrogels (Figure 3A), equivalent to the 20 pmol obtained from quantification of depletion from the original reaction solution with a sandwich ELISA after the reaction (Figure 1A). Further, this suggests that at this nucleophile concentration, all the consumed GGG-EGF is tethered to the hydrogel with imperceptible nonspecific binding. At 20 μM GGG-EGF initial concentration, a maximal amount of ∼160 pmol released GGG-EGF was detected, for hydrogels with 250 μM LPRTG-gel (Figure 3B). This means that from the estimated 230 pmol GGG-EGF that disappeared from the initial ligation reaction supernatant (Figure 1B), 160 pmol GGG-EGF was tethered to the hydrogel. Further, ∼11 pmol was recovered from washes (i.e, presumably partitioned into the aqueous phase of the hydrogel without binding), leaving a balance of 59 pmol that is presumably adsorbed to the hydrogel through noncovalent binding mechanisms (Figure 4B). Noncovalent binding, as estimated by the gap between the sum of the recovered material and that in original ligation solution, seems to be higher and more uniform relative to the LPRTG-gel concentration for the 20 μM GGG-EGF condition (Figure 4B).

Figure 4

Figure 4. Nucleophile mass balance for sortase-mediated ligation of GGG-EGF to LPRTG-containing hydrogels as a function of LPRTG concentration in the hydrogel and for GGG-EGF concentrations of either 2 μM (A) or 20 μM (B). The amount of GGG-EGF present in initial tethering solution and in hydrogel supernatant after washes and after cleavage was quantified by sandwich ELISA. Error bars represent standard error of the mean (n = 2).

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3.4 Direct ELISA Reveals Nonlinear Dependence of Surface-Tethered EGF on Substrate Concentration

The results of ELISA analysis on the reaction solutions (Figure 1) and the cleavage products (Figures 3 and 4) provide strong evidence for covalent sortase-mediated ligation of GGG-EGF to PEG hydrogels bearing the LPRTG motif, and suggest that the total amount of EGF tethered to the hydrogel is approximately proportional to the GGG-EGF concentration in the ligation solution. As the objective of tethering EGF to the hydrogels is to stimulate the EGF receptor of cells adhering to the hydrogel on a sustained basis, thus influencing downstream phenotypic responses, we further characterized the EGF tethered to the hydrogel using a direct ELISA approach to quantify the relative amounts of accessible EGF tethered under the different substrate conditions. Direct ELISA was conducted by incubating hydrogels with an anti-EGF antibody for 2 h, and thus it is expected that antibody binding would be restricted to near the surface of the hydrogels due to the slow diffusion of large proteins in the hydrogels. In this analysis, hydrogels incubated with GGG-EGF in the absence of sortase were included as a control for nonspecific binding.
The relative amounts of EGF detected by direct ELISA, as a function of initial LPRTG-gel concentration, GGG-EGF concentration in the ligation solution, and the presence or absence of sortase, are shown in Figure 5A as the net signal after subtraction of background. In agreement with the analysis presented in Figure 4, nonspecific interaction of GGG-EGF with the hydrogel is undetectable at the 2 μM GGG-EGF concentration, but is significant at the 20 μM GGG-EGF concentration, as evidenced by the detectable signal for hydrogels incubated with GGG-EGF in the absence of sortase, the lack of clear dependence of this signal on LPRTG-gel concentration, and the significant increase in the signal for hydrogels incubated with 20 μM compared to 2 μM GGG-EGF (Figure 5A). Interestingly, ELISA-detectable EGF increases with addition of sortase even in the absence of LPRTG-gel (i.e., comparison of the points along the vertical axis for LPRTG-gel = 0 μM in Figure 5A). The magnitude of this increase is comparable for the 2 μM and 20 μM GGG-EGF conditions. A plausible explanation is that sortase adsorbs nonspecifically to the hydrogels and acts as an affinity capture molecule for GGG-EGF.

Figure 5

Figure 5. Detection of GGG-EGF on hydrogels as a function of total LPRTG concentration in hydrogel after sortase-mediated ligation of 2 or 20 μM GGG-EGF. (A) GGG-EGF nonspecific binding was tested after incubation of the hydrogels with 2 or 20 μM GGG-EGF in the absence of sortase. (B) Measurements performed without sortase were subtracted from the ones performed in the presence of the enzyme. Error bars represent standard error of the mean (n = 2).

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In order to compare the relative amounts of EGF tethered to hydrogels via sortase-mediated ligation at different LPRTG-gel concentrations for the 2 and 20 μM GGG-EGF tethering concentrations, the signal associated with incubation in the absence of sortase was subtracted from the total signal for each LPRTG-gel concentration for the 2 μM and 20 μM GGG-EGF cases to normalize the data for nonspecific association of GGG-EGF with the hydrogels. The resulting values are plotted in Figure 5B as a function of LPRTG-gel concentration initially present in the hydrogel. This plot shows that the net ELISA signal above background (including nonspecific adsorption) is roughly comparable at low (<50 μM) LPRTG-gel concentrations for both the 2 μM and 20 μM GGG-EGF substrate conditions and at high (>50 μM) LPRTG-gel concentrations, the net ELISA signal is about 2-fold greater for the 20 μM GGG-EGF than that for the 2 μM GGG-EGF condition. The difference in this result and the data in Figure 4, where measurement of cleaved EGF indicates a 10-fold greater amount of tethered EGF for the higher substrate concentration, can be interpreted in the context of the complex parallel rate processes occurring during the ligation reaction and during the direct ELISA. During reaction, both the substrate GGG-EGF and the sortase enzyme diffuse into the hydrogel, and the ligation reaction competes with a hydrolysis reaction in a manner such that hydrolysis is more prominent at very low GGG-EGF concentrations. Thus, it is likely that the ratio of tethered EGF for the two different substrate concentrations changes with distance from the surface of the hydrogel, such that the ratio of [tethered EGF, 20 μM] to [tethered EGF, 2 μM] increases away from the surface, due to the increase in prominence of hydrolysis in the 2 μM case.

3.5 DNA Synthesis of Primary Human Hepatocytes and Endometrial Epithelial Cells Is Enhanced by Tethered EGF

The central objective of tethering EGF to PEG hydrogels is to present EGF in a mode that engages and activates the EGF receptor on the basal surface of adherent cells in a sustained fashion, and does so in the context of an environment that mimics key adhesion and mechanical features of extracellular matrix. Epithelial cells polarize EGFR and EGFR ligands to the basolateral surface, and disruption of this polarization is associated with disease states.(39) Some ligands for EGFR, such as amphiregulin, are strongly matrix binding and may act as pseudotethered ligands, hence presentation of EGF tethered to PEG hydrogels may mimic a mode of natural stimulation that is deficient in synthetic matrix. We thus focused on assessing the DNA synthesis response of primary epithelial cells to tethered EGF as proof-of-principle for the activity of tethered EGF.
Motivated in part by several studies showing the phenotypic effects of soluble versus tethered EGF on primary hepatocytes,(40-42) we examined the DNA synthesis response of primary human hepatocytes as a metric of tethered EGF activity. We also investigated the DNA synthesis response of primary human endometrial epithelial cells, which express EGFR and respond to EGF stimulation.(43) Expansion and differentiation of primary human endometrial epithelial cells in culture is of great interest for study of endometrial biology and disease. Primary human hepatocytes and primary human endometrial epithelial cells are also representative of epithelial cells that are responsive to EGF but relatively refractory toward in vitro proliferation.
For these proof of principle experiments, we aimed to choose an EGF concentration in a regime that would be expected to maximally stimulate DNA synthesis, as human hepatocytes and endometrial epithelial cells exhibit relatively low rates of DNA synthesis and growth in vitro compared to cell lines and other more highly proliferative primary cell types such as mesenchymal stem cells and keratinocytes. The anticipated low rates of DNA synthesis under optimal conditions is further compounded by the potential for the PEG adhesion environment to further diminish the DNA synthesis response capacity of EGF-stimulated cells: even cells that proliferate robustly in response to EGF culture can exhibit diminished proliferation behaviors when the adhesion context is altered by the presence of relatively poorly adsorptive PEG chains. For example, in the challenging application of PDMS surface modification with PEG-tethered EGF for creation of a confluent corneal epithelial cell monolayer for a biohybrid cornea replacement, tethered EGF improved cell coverage but the PEG chains impeded complete, homogeneous monolayer formation.(44) We have also observed that DNA synthesis by primary rat hepatocytes cultured on glass substrates with PEG-tethered EGF is diminished when the PEG brush inhibits attachment of adhesion proteins(40) and that tethered EGF improves, but does not completely rescue, colony formation by primary human bone marrow stromal cells cultured on PEG-tethered minimal adhesion peptides compared to untreated glass.(45)
Given that hydrogels tethered in the presence of 2 μM GGG-EGF exhibited undetectable (<0.5 pmol) noncovalently bound EGF and presented abundant surface-accessible EGF (Figure 4A), we used a tethering concentration of 2 μM GGG-EGF with hydrogels containing 250 μM LPRTG-gel. This concentration ensures that observed effects were due to tethered EGF and not to noncovalently bound EGF leaching off the hydrogel. Further, based on our previous estimates of tethered ligand density required to stimulate maximal rates of DNA synthesis in hepatocytes,(40) the ligand density in such hydrogels was anticipated to be sufficient to stimulate maximal EGFR signaling on these epithelial cells. Specifically, at bulk concentrations of 250 μM, the LPRTG motif is spaced 19 nm apart on average, yielding an approximate surface density of about 2500 LPRTG/ μm2. EGF is tethered to yield a final average bulk concentration of 2 μM (20 pmol for 10 μL hydrogel, data from Figure 1), resulting in an average spacing between EGF of about 95 nm and average of ∼100 EGF/ μm2. However, the surface appears to be somewhat enriched for tethered EGF than the bulk, thus we can estimate that cells spread to a typical size of 1000 μm2 are exposed to a lower limit of 100 000 and an upper limit of 2 500 000 molecules of EGF (the latter presumed all surface LPRTG are modified by EGF); i.e., comparable to or in excess of the number of cell surface receptors.
During the hydrogel fabrication process, hydrogels were functionalized with a branched peptide containing the PHSRN and RGD sequences derived from the 9th and 10th type III repeats in fibronectin. The canonical RGD motif from the 10th domain interacts primarily with αv integrins and induces mesenchymal-like behavior in epithelial cells, while inclusion of the 9th domain PHSRN synergy site fosters interactions through integrin α5β1 and a more physiological phenotypic response.(46) The synKRGD sequence(47) was incorporated into hydrogels via Michael-type addition through the thiol on the GGGERCG segment at the time of initial hydrogel synthesis to give a concentration of 0.5 mM, as we observed in pilot studies that this concentration was sufficient to induce robust attachment of the epithelial cell types used in this study.
Human primary hepatocytes and endometrial epithelial cells were cultured on hydrogels containing 0 or 500 μM synKRGD adhesion peptide. Because the presence of the adhesion peptide during cross-linking may influence the structure of the hydrogels, we substituted peptides with scrambled adhesion sequences (see Experimental Section) for the active synKRGD to create the control “0 μM synKRGD” peptide condition. Cells were either unexposed to EGF, presented with EGF tethered at 2 μM on 250 μM LPRTG-gel hydrogels, or presented with soluble hEGF at a concentration of 20 ng/mL (3.3 nM). The KD for EGF binding to EGFR is 0.2–1 nM,(40) and the DNA synthesis response is saturated at soluble EGF values >3 nM.(48) DNA synthesis was assessed over a period of 24h, 48 h after seeding for hepatocytes and 40 h after seeding for endometrial epithelial cells.
In the absence of EGF, while only a small number of cells attached to hydrogels containing 500 μM scrambled adhesion peptide (“0 μM synKRGD”), cell attachment on 500 μM active synKRGD hydrogels was comparable to positive controls on collagen I or TCPS (Figures 6A and C). Tethered EGF had no effect on hepatocyte attachment, contrary to soluble hEGF, which slightly enhanced attachment on 500 μM synKRGD hydrogels compared to unexposed hydrogels, and which yielded a 1.8 fold increase on collagen I-coated TCP (Figure 6A). In contrast, tethered EGF was as effective as the soluble form in fostering endometrial epithelial cell attachment to 0 and 500 μM synKRGD hydrogels (Figure 6C), and soluble EGF substantially enhanced endometrial epithelial cell attachment on standard TCPS (Figure 6C).

Figure 6

Figure 6. Cell attachment and DNA synthesis of primary human hepatocytes (A, B) and endometrial epithelial cells (C, D). Cells were seeded on hydrogels containing 0 or 500 μM synKRGD and 250 μM LPRTG and on standard culture substrates. After 48 h (A, B) or 40 h in culture (C, D), cells were incubated with 10 μM EdU for 24 h. Tethered EGF stimulated DNA synthesis compared to unmodified hydrogels or soluble hEGF. Error bars represent standard error of the mean (n = 3).

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Additionally, soluble EGF considerably increased DNA synthesis of primary hepatocytes compared to control cultures without EGF, from 9 to 21% on 500 μM synKRGD hydrogels and from 10 to 22% on collagen I-coated TCPS (Figure 6B). Stimulation of hepatocyte DNA synthesis by tethered EGF tended to be slightly greater: from 9 to 27% on 500 μM synKRGD hydrogels (Figure 6B). In previous studies of primary rat hepatocytes on EGF tethered to glass(40) or to self-assembling peptides hydrogels,(41) DNA synthesis rates stimulated by tethered EGF were comparable to or slightly lower than rates stimulated by a saturating amount of soluble EGF on hydrogels or on collagen-coated tissue culture plastic. Human hepatocytes have previously been reported to exhibit a lower rate of DNA synthesis than rat hepatocytes when stimulated by soluble EGF,(49) hence, the enhanced DNA synthesis response here for EGF tethered to synKRGD-modified hydrogels, compared to stimulation by soluble EGF of hepatocytes on either synKRGD-modified hydrogels or collagen I-coated substrates, may reflect a species difference. It may also arise from a more favorable combination of adhesion and EGFR signaling than was achievable in the glass-tethered or peptide-gel-tethered format. Primary endometrial epithelial cells also responded to soluble EGF stimulation by increasing DNA synthesis, from 6 to 12% on 500 μM synKRGD hydrogels and from 6 to 15% on TCPS (Figure 6D). The effect of tethered EGF was comparable to that of soluble EGF in these cells, increasing DNA synthesis from 6 to 14% compared to control hydrogels (Figure 6D).
These results provide encouraging evidence that EGF tethered to functionalized PEG hydrogels via sortase-mediated ligation stimulates biologically relevant responses in primary human epithelial cells. This study thus provides a foundation for future analysis of more detailed phenotypic responses.

4 Conclusions

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In conclusion, we report sortase-mediated ligation of human EGF to preformed PEG hydrogels. This enzymatic approach is not only simple and relatively inexpensive, but it also displays high specificity and modularity, and may be applied to a variety of PEG hydrogels including those cross-linked in situ in the presence of cells. A compendium of analytical approaches were used to show that the amount of grafted growth factor was readily controlled by the amount of LPRTG substrate incorporated in hydrogels and the amount of GGG-EGF present in solution. While the sortase enzyme was capable of recognizing and processing LPRTG peptides that were incorporated in the hydrogels through Michael-type addition, this method represents a useful tool for specific modification of any type of hydrogel containing the appropriate substrate. The results of this study underscore the challenges in interpreting reaction data for preformed hydrogels, as the combination of kinetic and diffusion phenomena working in concert during the ligation process may create gradients of enzyme and substrate in the hydrogels; indeed, here, the growth factor appeared to be somewhat enriched at the surface of the hydrogels. This study also underscores the challenges in minimizing noncovalent binding during such tethering processes, as even with the well-established antifouling properties of the PEG polymer as well as enzymatic specificity, significant nonspecific binding appeared to occur for high GGG-EGF coupling concentration. One of the more novel aspects of the study is the exploitation of sortase as a cleavage enzyme. Efficient and near complete release of tethered EGF from the hydrogels through sortase-catalyzed reaction of the LPRTG-containing tether with soluble GGG allows near absolute quantification of the previous tethered protein through well-established solution phase techniques. Finally, biological activity of the tethered EGF was confirmed by the phenotypic response of human primary epithelial cells. The well-studied hepatocyte cell system showed a more robust enhancement to tethered EGF (compared to soluble EGF) than had been observed in previously published studies, while preliminary experiments with endometrial epithelial cells constituted a first example of endometrial epithelial cell culture in the presence of a tethered growth factor. This demonstration of sortase-mediated ligation of bioactive molecules to hydrogels will likely contribute to the development of improved culture systems for in vitro models, by expanding the repertoire of bioactive molecules that can be covalently attached to PEG to include large peptides and proteins.

Supporting Information

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Protocol for primary human epithelial cells isolation and purification; (S1) standard curve for conversion of hydrogel fluorescence arbitrary units to amount of LPRTG in pmol; (S2) photobleaching controls; (S3) percentage of retained hydrogel fluorescence as a function of LPRTG in hydrogel after sortase-mediated hydrogel cleavage; (S4) micrographs of hepatocyte DNA synthesis on tethered EGF hydrogels; (S5) micrographs of endometrial epithelial cell DNA synthesis on tethered EGF hydrogels. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00549.

∇Author Present Address

F. Hoffmann-La Roche AG, Basel, CH.

○Author Present Address

Novartis Institute of Biomedical Research, Cambridge, MA, USA.

#Author Contributions

These authors contributed equally. E.C. designed and conducted experiments, analyzed data and wrote the manuscript. K.R. conceived and designed experiments, produced recombinant proteins and wrote the manuscript. C.C.A. conceived and designed experiments and contributed to protocol development. C.D.C. conceived experiments with endometrial cells, designed protocols, and interpreted data. C.K. conceived experiments and developed recombinant protein expression protocols. A.T.K. conceived sortase kinetic experiments, developed protocols, and produced recombinant proteins. B.I. conceived and designed experiments and cosupervised the project. L.G.G. conceived and cosupervised the project and wrote the manuscript. All authors edited the manuscript.

The authors declare no competing financial interest.

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Acknowledgment

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We acknowledge Prof. David Liu, Harvard University, for the sortase mutant plasmids. We are grateful to the study participants and the surgical and study staff, including Drs. Keith Isaacson, Stephanie Morris, and Johanna Frey Renggli, at Newton Wellesley Hospital for endometrial biopsy collection. This work was supported by NIH 5R01EB010246, NIH 5UH2TR000496, the Institute for Collaborative Biotechnologies (W911NF-09-0001), NIH 1T32GM008334, the DARPA Microphysiological Systems Program (W911NF-12-2-0039), the Begg New Horizon Fund for Undergraduate Research at MIT, the NIH Biotechnology Training Program NIH/NIGMS 5T32GM008334, the Biophysical Instrumentation Facility, the Ludwig Postdoctoral Fellowship for Cancer Research for C.K. and a Swiss National Science Foundation Postdoctoral Fellowship for K.R.

References

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This article references 49 other publications.

  1. 1
    Griffith, L. G.; Swartz, M. A. Nat. Rev. Mol. Cell Biol. 2006, 7, 211– 224
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    1
    Capturing complex 3D tissue physiology in vitro
    Griffith, Linda G.; Swartz, Melody A.
    Nature Reviews Molecular Cell Biology (2006), 7 (3), 211-224CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. The emergence of tissue engineering raises new possibilities for the study of complex physiol. and pathophysiol. processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. The authors discuss here some of the 'design principles' for recreating the interwoven set of biochem. and mech. cues in the cellular microenvironment, and the methods for implementing them. The authors emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhs1Kisro%253D&md5=5a957aaf2581e5bcc5fc06690e8883ad
  2. 2
    Poly(ethylene glycol) Chemistry; Harris, J. M., Ed.; Springer US: Boston, MA, 1992.
    [ Crossref], Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A. J. Controlled Release 2001, 76, 11– 25
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    3
    Protein delivery from materials formed by self-selective conjugate addition reactions
    Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A.
    Journal of Controlled Release (2001), 76 (1-2), 11-25CODEN: JCREEC; ISSN:0168-3659. (Elsevier Science Ireland Ltd.)
    A new chem. crosslinking scheme was utilized for the formation of degradable poly(ethylene glycol) hydrogels suitable for the delivery of protein drugs. An aq. soln. contg. a PEG-multiacrylate and solid particles of albumin was mixed with an aq. soln. contg. a PEG-dithiol, rapidly producing a cross-linked hydrogel through a Michael-type addn. reaction. For some formulations, it was obsd. that about 65% of the incorporated protein was released with zero-order kinetics over a period of about 4 days. By changing the functionality of the cross-linker, the release of protein could even be delayed for about 4 days, followed by zero-order release. The mechanism for release appeared to be a combination of slow dissoln. of protein in the presence of PEG and hindered diffusion of protein through the gel. The crosslinking of the gels was studied rheometrically, and the hydrolytic degrdn. of the gels was characterized by measuring the swelling of the gels. Biochem. anal. of the released proteins demonstrated that the polymers reacted with each other, but not with proteins. Utilizing the Flory-Rehner and Peppas-Merrill equations, a framework for modeling the protein release from the gels is described.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXmt1Glsr4%253D&md5=d40f8c15efca8049cf93228dc3a31a71
  4. 4
    Lutolf, M. P.; Hubbell, J. A. Biomacromolecules 2003, 4, 713– 722
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    4
    Synthesis and Physicochemical Characterization of End-Linked Poly(ethylene glycol)-co-peptide Hydrogels Formed by Michael-Type Addition
    Lutolf, M. P.; Hubbell, J. A.
    Biomacromolecules (2003), 4 (3), 713-722CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The synthesis of novel hybrid hydrogels by stepwise copolymn. of multi-arm vinyl sulfone-terminated poly(ethylene glycol) macromers and α-ω cysteine oligopeptides via Michael-type addns. is described. Crosslinking kinetics, studied by in situ rheometry, can be controlled by pH and the presence of charged amino acid residues in close proximity to the Cys, which modulates the pKa of the thiol group. These end-linked networks were characterized by their equil. swelling in water, by their viscoelastic properties in the swollen state, and by their sol. fraction. Structure and properties are sensitive to the prepn. state including stoichiometry and precursor concn. and less sensitive to the pH during crosslinking. For each network the concn. of elastically active chains (ν) was calcd. from exptl. detd. sol fractions using Miller-Macosko theory and compared to values obtained from swelling and rheometry studies and by calcn. from Flory's classical network models. Hydrogels were also prepd. with varying macromer structures, and their properties were shown to respond to both macromer functionality and mol. wt.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsVaqurw%253D&md5=1c7b5b8bb1a980b6489b06da10cdf5ad
  5. 5
    Lutolf, M. P.; Raeber, G. P.; Zisch, A. H.; Tirelli, N.; Hubbell, J. A. Adv. Mater. 2003, 15, 888– 892
    [ Crossref], [ CAS], Google Scholar
    5
    Cell-responsive synthetic hydrogels
    Lutolf, Matthias P.; Raeber, George P.; Zisch, Andreas H.; Tirelli, Nicola; Hubbell, Jeffrey A.
    Advanced Materials (Weinheim, Germany) (2003), 15 (11), 888-892CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Synthetic biomaterial matrixes have been formed in the presence of cells by a Michael-type addn. reaction between thiolate groups of cysteine-contg. peptides and a vinylsulfone functionalized multi-arm poly(ethylene glycol). These materials locally degrade in response to cell-surface proteases, allowing cells to create paths for 3D cell migration. Results demonstrate that synthetic materials can be responsive to cellular influences in vitro and in vivo, including here, as an example, inducing and responding to angiogenic invasion.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkvFCmurg%253D&md5=de92fd194b9c82d5bc065f6f9850f94c
  6. 6
    Zhu, J. Biomaterials 2010, 31, 4639– 4656
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    6
    Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering
    Zhu, Junmin
    Biomaterials (2010), 31 (17), 4639-4656CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    In this review, the authors explore different approaches for introducing bioactivity into poly(ethylene glycol) (PEG) hydrogels. Hydrogels are excellent scaffolding materials for repairing and regenerating a variety of tissues because they can provide a highly swollen 3-dimensional (3D) environment similar to soft tissues. Synthetic hydrogels like PEG-based hydrogels have advantages over natural hydrogels, such as the ability for photopolymn., adjustable mech. properties, and easy control of scaffold architecture and chem. compns. However, PEG hydrogels alone cannot provide an ideal environment to support cell adhesion and tissue formation due to their bio-inert nature. The natural extracellular matrix (ECM) was an attractive model for the design and fabrication of bioactive scaffolds for tissue engineering. ECM-mimetic modification of PEG hydrogels has emerged as an important strategy to modulate specific cellular responses. To tether ECM-derived bioactive mols. (BMs) to PEG hydrogels, various strategies were developed for the incorporation of key ECM biofunctions, such as specific cell adhesion, proteolytic degrdn., and signal mol.-binding. A no. of cell types were immobilized on bioactive PEG hydrogels to provide fundamental knowledge of cell/scaffold interactions. This review addresses the recent progress in material designs and fabrication approaches leading to the development of bioactive hydrogels as tissue engineering scaffolds.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksV2ru7o%253D&md5=ea20a8041483618e93fe71d3642e5f21
  7. 7
    Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47– 55
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    7
    Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering
    Lutolf, M. P.; Hubbell, J. A.
    Nature Biotechnology (2005), 23 (1), 47-55CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    A review. New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrixes (ECMs) and ECM-bound growth factors, both for therapeutic applications and basic biol. studies. Recent advances include nanofibrillar networks formed by self-assembly of small building blocks, artificial ECM networks from protein polymers or peptide-conjugated synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable proteolytic remodeling. These materials have already found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis. Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biol. may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsFGmtg%253D%253D&md5=081fb3c462e35263fedf6690fc67ec07
  8. 8
    Burdick, J. A.; Murphy, W. L. Nat. Commun. 2012, 3, 1269
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    8
    Moving from static to dynamic complexity in hydrogel design
    Burdick Jason A; Murphy William L
    Nature communications (2012), 3 (), 1269 ISSN:.
    Hydrogels are water-swollen polymer networks that have found a range of applications from biological scaffolds to contact lenses. Historically, their design has consisted primarily of static systems and those that exhibit simple degradation. However, advances in polymer synthesis and processing have led to a new generation of dynamic systems that are capable of responding to artificial triggers and biological signals with spatial precision. These systems will open up new possibilities for the use of hydrogels as model biological structures and in tissue regeneration.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s3htlaguw%253D%253D&md5=e50e82f5a80b7732b248d882511c2a62
  9. 9
    Nimmo, C. M.; Shoichet, M. S. Bioconjugate Chem. 2011, 22, 2199– 2209
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    9
    Regenerative Biomaterials that "Click": Simple, Aqueous-Based Protocols for Hydrogel Synthesis, Surface Immobilization, and 3D Patterning
    Nimmo, Chelsea M.; Shoichet, Molly S.
    Bioconjugate Chemistry (2011), 22 (11), 2199-2209CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A review. The click chem. era has generated a library of versatile "spring-loaded" reactions that offer high yields, regio- and stereospecificity, and outstanding functional group tolerance. These powerful transformations are particularly advantageous for the design of sophisticated biomaterials that require high levels of precision and control, namely, materials that promote tissue regeneration such as hydrogels, 2D functionalized substrates, and 3D biomimetic scaffolds. In this review, the synthesis and application of regenerative biomaterials via click chem. are summarized. Particular emphasis is placed on the copper(I)-catalyzed alkyne-azide cycloaddn., Diels-Alder cycloaddns., and thiol-click coupling.
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  10. 10
    Drumheller, P. D.; Hubbell, J. A. Anal. Biochem. 1994, 222, 380– 388
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    10
    Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates
    Drumheller, Paul D.; Hubbell, Jeffrey A.
    Analytical Biochemistry (1994), 222 (2), 380-8CODEN: ANBCA2; ISSN:0003-2697. (Academic)
    Polymer networks of poly(ethylene glycol) (PEG) in densely cross-linked matrixes of acrylic acid (AA) and trimethylolpropane triacrylate were synthesized as biospecific cell adhesive substrates. Networks grafted with synthetic adhesion peptides produced substrates to investigate long-term, receptor-mediated cell/surface interactions, without nonspecific protein adsorption producing spurious adhesion signals. PEG rendered the networks very resistant to cell adhesion in vitro, and AA provided reactive carboxyl moieties for N-terminal grafting of peptides. Networks with higher mass fractions of AA had greater background cell adhesion, which diminished with higher mass fractions of PEG such that complete resistance to cell adhesion could be obtained. Networks grafted with inactive control peptides (GRGES or no peptide) remained completely cell nonadhesive in the presence of serum or even when preincubated with adhesion proteins, while networks grafted with bioadhesive peptides (GRGDS, GYIGSRY, or GREDVY) supported morphol. complete fibroblast adhesion. The amt. of AA in the network readily controlled the amt. of incorporated peptide. These networks may be suitable as anal. tools specifically to investigate long-term cell/substrate interactions in the presence of serum, yet without nonspecific protein adsorption producing adhesion signals other than those immobilized for study.
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  11. 11
    Hahn, M. S.; Miller, J. S.; West, J. L. Adv. Mater. 2005, 17, 2939– 2942
    [ Crossref], [ CAS], Google Scholar
    11
    Laser scanning lithography for surface micropatterning on hydrogels
    Hahn, Mariah S.; Miller, Jordan S.; West, Jennifer L.
    Advanced Materials (Weinheim, Germany) (2005), 17 (24), 2939-2942CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A confocal-based laser scanning lithog. method for controlled, high-fidelity two-dimensional and three-dimensional surface patterning of poly(ethylene glycol) hydrogels. The method can be applied to any optically clear, photoactive substrate. Laser scanning lithog. employs user-defined masks to spatially control laser irradn. during pattern generation, eliminating the need for photomasks and a clean-room. Also, pattern mols. are covalently immobilized onto the substrate surface providing mech. and phys. stable patterns. In this work, the surface properties of deformable, biocompatible poly(ethylene glycol)-diacrylate hydrogels were modified in a controlled manner using laser scanning lithog., with pattern feature sizes down to at least 5 μm. Human dermal fibroblast (HDF) were selectively bind to regions patterned with appropriate cell-adhesive peptides. Patterned substrates functionalized with gradients of different cell-adhesive ligands can be fabricated, providing a tool for investigating cell-biomaterial interactions and the structure-function relationships of tissues.
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  12. 12
    Hahn, M. S.; Taite, L. J.; Moon, J. J.; Rowland, M. C.; Ruffino, K. A.; West, J. L. Biomaterials 2006, 27, 2519– 2524
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    12
    Photolithographic patterning of polyethylene glycol hydrogels
    Hahn, Mariah S.; Taite, Lakeshia J.; Moon, James J.; Rowland, Maude C.; Ruffino, Katie A.; West, Jennifer L.
    Biomaterials (2006), 27 (12), 2519-2524CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    A simple, inexpensive photolithog. method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithog. masks were prepd. by printing the desired patterns onto transparencies using a laser jet printer. Precursor solns. contg. monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor soln. were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradn. time and precursor soln. concn. on the levels of immobilized peptide were characterized, demonstrating that bound peptide concn. can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addn., 3D structures can be generated by incorporating PEG-diacrylate into the precursor soln. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl-PEG-RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnol.
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  13. 13
    Lee, W.; Lee, T. G.; Koh, W.-G. J. Ind. Eng. Chem. 2007, 13, 1195– 1200
    [ CAS], Google Scholar
    13
    Grafting of poly(acrylic acid) on the poly(ethylene glycol) hydrogel using surface-initiated photopolymerization for covalent immobilization of collagen
    Lee, Woojin; Lee, Tae Gyu; Koh, Won-Gun
    Journal of Industrial and Engineering Chemistry (Seoul, Republic of Korea) (2007), 13 (7), 1195-1200CODEN: JIECFI; ISSN:1226-086X. (Korean Society of Industrial and Engineering Chemistry)
    The modification of the protein-repellent poly(ethylene glycol) (PEG) hydrogel surface was achieved by a two-step process using immobilization of benzophenone on the PEG hydrogel as surface initiator and subsequent surface-initiated graft polymn. of acrylic acid by UV irradn. Formation of poly(acrylic acid) (PAA) layer on the PEG hydrogel was demonstrated by confirming the presence of carboxyl groups in the poly(acrylic acid) (PAA) with FTIR/ATR spectroscopy and measuring the height of PAA layers with alpha-step surface profiler. In the grafted region, PAA and PEG hydrogel formed an interpenetrating polymer network extending 200 μm into PEG hydrogel and homo PAA protruded 14∼17 μm above the PEG hydrogel surface. Activation of the carboxyl groups in PAA allowed covalent immobilization of collagen, a cell adhesion protein, on the PAA-grafted PEG hydrogel, which was demonstrated with FTIR/ATR spectroscopy by confirming the formation of a new amide bond. Surface-initiated graft polymn. combined with photolithog. produced well-defined PAA micropatterns on the PEG hydrogels and collagen was immobilized only on the PAA region due to the lack of adhesion for proteins to PEG, producing protein micropattern on the PEG hydrogel.
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  14. 14
    Hynd, M. R.; Frampton, J. P.; Dowell-Mesfin, N.; Turner, J. N.; Shain, W. J. Neurosci. Methods 2007, 162, 255– 263
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    14
    Directed cell growth on protein-functionalized hydrogel surfaces
    Hynd, Matthew R.; Frampton, John P.; Dowell-Mesfin, Natalie; Turner, James N.; Shain, William
    Journal of Neuroscience Methods (2007), 162 (1-2), 255-263CODEN: JNMEDT; ISSN:0165-0270. (Elsevier Ltd.)
    Biochem. surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymd. in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labeled proteins. Soft protein lithog. (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biol. assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphol. on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72 h in vitro, and remained viable on protein-stamped areas for more than 4 wk. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.
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  15. 15
    Mazmanian, S. K.; Liu, G.; Hung, T. T.; Schneewind, O. Science 1999, 285, 760– 763
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    15
    Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall
    Mazmanian, Sarkis K.; Liu, Gwen; Ton-That, Hung; Schneewind, Olaf
    Science (Washington, D. C.) (1999), 285 (5428), 760-763CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Surface proteins of Gram-pos. bacteria are linked to the bacterial cell wall by a mechanism that involves cleavage of a conserved Leu-Pro-X-Thr-Gly (LPXTG) motif and that occurs during assembly of the peptidoglycan cell wall. A Staphylococcus aureus mutant defective in the anchoring of surface proteins was isolated and shown to carry a mutation in the srtA gene. Overexpression of srtA increased the rate of surface protein anchoring, and homologs of srtA were found in other pathogenic Gram-pos. bacteria. The protein specified by srtA, sortase, may be a useful target for the development of new antimicrobial drugs.
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  16. 16
    Ton-That, H.; Mazmanian, S. K.; Faull, K. F.; Schneewind, O. J. Biol. Chem. 2000, 275, 9876– 9881
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    16
    Anchoring of surface proteins to the cell wall of Staphylococcus aureus.: Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates
    Ton-That, Hung; Mazmanian, Sarkis K.; Faull, Kym F.; Schneewind, Olaf
    Journal of Biological Chemistry (2000), 275 (13), 9876-9881CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. Purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH2-Gly3, sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH2-Gly3. In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate.
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  17. 17
    Tsukiji, S.; Nagamune, T. ChemBioChem 2009, 10, 787– 798
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    17
    Sortase-mediated ligation: a gift from gram-positive bacteria to protein engineering
    Tsukiji, Shinya; Nagamune, Teruyuki
    ChemBioChem (2009), 10 (5), 787-798CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review on sortase-mediated ligation and current state of its applications. Topics discussed include: recombinant sol. sortase; transpeptidation mechanism; substrate specificity; introduction of new functionalities into proteins; covalent immobilization of proteins onto solid supports; protein-protein bioconjugation; protein circularization; and self-cleavable tag for one-step purifn. of recombinant proteins. It also tackles the prepn. of oligopeptide-nucleic acid hybrids; chemoenzymic synthesis of neoglycoconjugates; and cell-surface protein labeling/engineering.
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  18. 18
    Ritzefeld, M. Chem.—Eur. J. 2014, 20, 8516– 8529
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    18
    Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy
    Ritzefeld, Markus
    Chemistry - A European Journal (2014), 20 (28), 8516-8529CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Bioorthogonal, chemoselective ligation methods are an essential part of the tools used to study biochem. pathways. Specifically enzymic approaches are valuable methods in this context due to the inherent specificity of the deployed enzymes and the mild conditions of the modification reactions. One of the most common strategies is based on the transpeptidation catalyzed by sortase A derived from Staphylococcus aureus. The procedure is well established and a wide variety of applications have been published to date. Here, implementations of sortase A, which range from protein labeling using fluorescence dyes and the prepn. of cyclic proteins to the modification of entire cells, are summarized. Furthermore, there is a focus on the optimization approaches established to solve the drawbacks of sortase-mediated transpeptidation.
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  19. 19
    Parthasarathy, R.; Subramanian, S.; Boder, E. T. Bioconjugate Chem. 2007, 18, 469– 476
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    19
    Sortase A as a Novel Molecular "Stapler" for Sequence-Specific Protein Conjugation
    Parthasarathy, Ranganath; Subramanian, Shyamsundar; Boder, Eric T.
    Bioconjugate Chemistry (2007), 18 (2), 469-476CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-pos. bacteria. Here, the authors describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chem. modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, the authors show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. The authors find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addn. to the protein immobilization and modification tool kit.
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  20. 20
    Chan, L.; Cross, H. F.; She, J. K.; Cavalli, G.; Martins, H. F. P.; Neylon, C. PLoS One 2007, 2, e1164
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    20
    Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation
    Chan Lilyan; Cross Hannah F; She Joseph K; Cavalli Gabriel; Martins Hugo F P; Neylon Cameron
    PloS one (2007), 2 (11), e1164 ISSN:.
    BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.
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  21. 21
    Ito, T.; Sadamoto, R.; Naruchi, K.; Togame, H.; Takemoto, H.; Kondo, H.; Nishimura, S.-I. Biochemistry 2010, 49, 2604– 2614
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    21
    Highly Oriented Recombinant Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A
    Ito, Takaomi; Sadamoto, Reiko; Naruchi, Kentaro; Togame, Hiroko; Takemoto, Hiroshi; Kondo, Hirosato; Nishimura, Shin-Ichiro
    Biochemistry (2010), 49 (11), 2604-2614CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Recombinant glycosyltransferases are potential biocatalysts for the construction of a compd. library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chem. and enzymic synthetic procedures. The structurally defined glycan-related compd. library is a key resource both in the basic studies of their functional roles in various biol. processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile sepn. of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here, a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto com. available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A is described. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human β1,4-galactosyltranseferase or recombinant Helicobacter pylori α1,3-fucosyltransferase with simple aliph. amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the post-translational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of this strategy based on site-specific transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.
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  22. 22
    Matsumoto, T.; Tanaka, T.; Kondo, A. Langmuir 2012, 28, 3553– 3557
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    22
    Sortase A-Catalyzed Site-Specific Coimmobilization on Microparticles via Streptavidin
    Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko
    Langmuir (2012), 28 (7), 3553-3557CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    A microparticle surface was designed by the unique method incorporating streptavidin-biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG) was immobilized on the surface using streptavidin-biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin-FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-assocd. fluorescence was obsd. using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepd. using chem. modification. The authors' method provides a simple strategy for site-specific coimmobilization on mol. surfaces and expands the use of protein hybrid devices.
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  23. 23
    Sijbrandij, T.; Cukkemane, N.; Nazmi, K.; Veerman, E. C. I.; Bikker, F. J. Bioconjugate Chem. 2013, 24, 828– 831
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    23
    Sortase A as a Tool to Functionalize Surfaces
    Sijbrandij, Tjitske; Cukkemane, Nivedita; Nazmi, Kamran; Veerman, Enno C. I.; Bikker, Floris J.
    Bioconjugate Chemistry (2013), 24 (5), 828-831CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A widely accepted approach to combat surface fouling is based on the prevention of biofoulants to attach to a surface by the functionalization with poly(ethylene glycol) (PEG). The goal of this study was to generate a proof of concept for the enzymic coupling of PEG to a peptide precoated surface by using the enzyme Sortase A (SrtA). A hydrophobic polystyrene surface was primed with anchoring peptide P3 equipped with a pentaglycine acceptor motif for SrtA, to enable subsequent transpeptidation with either biotin or a PEG-tail contg. the sortase recognition motif LPETG. High levels of surface-bound biotin were detected only in cases with biotin-LPETG and SrtA. Little if any reactivity was detected in wells treated with the SrtA scrambled motif EGLTP, or in the absence of SrtA. Conjugation of PEG resulted in a significant decrease of bacterial adherence to the surface.
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  24. 24
    Sinisi, A.; Popp, M. W.-L.; Antos, J. M.; Pansegrau, W.; Savino, S.; Nissum, M.; Rappuoli, R.; Ploegh, H. L.; Buti, L. Bioconjugate Chem. 2012, 23, 1119– 1126
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    24
    Development of an influenza virus protein array using Sortagging technology
    Sinisi, Antonia; Popp, Maximilian Wei-Lin; Antos, John M.; Pansegrau, Werner; Savino, Silvana; Nissum, Mikkel; Rappuoli, Rino; Ploegh, Hidde L.; Buti, Ludovico
    Bioconjugate Chemistry (2012), 23 (6), 1119-1126CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Protein array technol. is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during a bacterial or viral infection. The authors developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as Sortagging. LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular exts. were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purifn. steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technol. has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes to develop protein arrays for high-throughput screening.
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  25. 25
    Jiang, R.; Weingart, J.; Zhang, H.; Ma, Y.; Sun, X.-L. Bioconjugate Chem. 2012, 23, 643– 649
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    25
    End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation
    Jiang, Rui; Weingart, Jacob; Zhang, Hailong; Ma, Yong; Sun, Xue-Long
    Bioconjugate Chemistry (2012), 23 (3), 643-649CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    We report an enzymic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 deriv. was site-specifically modified with N-terminal diglycine contg. mols. such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was obsd. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chem. modification of the target protein. This site-specific covalent modification leads to mols. being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biol. activity.
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  26. 26
    Clow, F.; Fraser, J. D.; Proft, T. Biotechnol. Lett. 2008, 30, 1603– 1607
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    26
    Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A
    Clow, Fiona; Fraser, John D.; Proft, Thomas
    Biotechnology Letters (2008), 30 (9), 1603-1607CODEN: BILED3; ISSN:0141-5492. (Springer)
    The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purifn. protocols.
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  27. 27
    Leung, M. K. M.; Hagemeyer, C. E.; Johnston, A. P. R.; Gonzales, C.; Kamphuis, M. M. J.; Ardipradja, K.; Such, G. K.; Peter, K.; Caruso, F. Angew. Chem., Int. Ed. 2012, 51, 7132– 7136
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    27
    Bio-Click Chemistry: Enzymatic Functionalization of PEGylated Capsules for Targeting Applications
    Leung, Melissa K. M.; Hagemeyer, Christoph E.; Johnston, Angus P. R.; Gonzales, Catalina; Kamphuis, Marloes M. J.; Ardipradja, Katie; Such, Georgina K.; Peter, Karlheinz; Caruso, Frank
    Angewandte Chemie, International Edition (2012), 51 (29), 7132-7136, S7132/1-S7132/5CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The authors demonstrated an efficient method for the covalent and site-specific protein functionalization of polymer capsules by using Staphylococcus aureus enzyme Sortase A (Srt A). Owing to the precision of the ligation of genetically encoded substrates, this bioclick approach allows targeting ligands to be oriented with the antigen-binding sites available for binding, thus retaining their bioactivity. The GPIIb/IIIa-specific scFv-functionalized capsules showed a high level of specific targeting to thrombi, thus making them promising for anti-thrombotic and thrombolytic therapy. This SrtA mediated biofunctionalization technique is expected to find a range of applications in functionalizing materials for use in drug delivery as well as diagnostics and imaging.
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  28. 28
    Guo, X.; Wu, Z.; Guo, Z. Bioconjugate Chem. 2012, 23, 650– 655
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    28
    New Method for Site-Specific Modification of Liposomes with Proteins Using Sortase A-Mediated Transpeptidation
    Guo, Xueqing; Wu, Zhimeng; Guo, Zhongwu
    Bioconjugate Chemistry (2012), 23 (3), 650-655CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A new method was developed for site-specific modifications of liposomes by proteins via sortase A (SrtA)-mediated transpeptidation reactions. In this regard, the enhanced green fluorescent protein (eGFP) was biol. engineered to carry at its polypeptide C-terminus the LPATG motif recognized by SrtA and used as the protein donor for linking to liposomes that were decorated with phospholipids carrying a diglycine motif as the other SrtA substrate and the eGFP acceptor. Under the influence of SrtA, eGFP was efficiently attached to liposomes, as proved by analyzing the enzymic reaction products and the resultant fluorescent liposomes. Increasing the concn. and the distance of the diglycine motif on and from the liposome surface could significantly improve the efficiency of liposome modification by proteins. It is anticipated that this strategy can be widely useful for the modification of liposomes by other proteins.
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  29. 29
    Piluso, S.; Cassell, H. C.; Gibbons, J. L.; Waller, T. E.; Plant, N. J.; Miller, A. F.; Cavalli, G. Soft Matter 2013, 9, 6752– 6756
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    29
    Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool
    Piluso, Susanna; Cassell, Heather C.; Gibbons, Jonathan L.; Waller, Thomas E.; Plant, Nick J.; Miller, Aline F.; Cavalli, Gabriel
    Soft Matter (2013), 9 (29), 6752-6756CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheol. studies and TEM of the corresponding hydrogels (including PNIPAAm-contg. systems) showed no significant variation in properties and hydrogel morphol. compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.
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  30. 30
    Krueger, A. T.; Kroll, C.; Sanchez, E.; Griffith, L. G.; Imperiali, B. Angew. Chem., Int. Ed. 2014, 53, 2662– 2666
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    30
    Tailoring Chimeric Ligands for Studying and Biasing ErbB Receptor Family Interactions
    Krueger, Andrew T.; Kroll, Carsten; Sanchez, Edgar; Griffith, Linda G.; Imperiali, Barbara
    Angewandte Chemie, International Edition (2014), 53 (10), 2662-2666CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chem., which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was obsd. to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways.
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  31. 31
    Ehrbar, M.; Rizzi, S. C.; Hlushchuk, R.; Djonov, V.; Zisch, A. H.; Hubbell, J. A.; Weber, F. E.; Lutolf, M. P. Biomaterials 2007, 28, 3856– 3866
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    31
    Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering
    Ehrbar, Martin; Rizzi, Simone C.; Hlushchuk, Ruslan; Djonov, Valentin; Zisch, Andreas H.; Hubbell, Jeffrey A.; Weber, Franz E.; Lutolf, Matthias P.
    Biomaterials (2007), 28 (26), 3856-3866CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    The mol. engineering of cell-instructive artificial extracellular matrixes is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chem. and the biomol. characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quant. incorporated and released upon cell-derived proteolytic degrdn. of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochem. and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.
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  32. 32
    Chen, I.; Dorr, B. M.; Liu, D. R. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11399– 11404
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    32
    A general strategy for the evolution of bond-forming enzymes using yeast display
    Chen, Irwin; Dorr, Brent M.; Liu, David R.
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (28), 11399-11404, S11399/1-S11399/29CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the mol. life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.
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  33. 33
    Swee, L. K.; Lourido, S.; Bell, G. W.; Ingram, J. R.; Ploegh, H. L. ACS Chem. Biol. 2015, 10, 460– 465
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    33
    One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism
    Swee, Lee Kim; Lourido, Sebastian; Bell, George W.; Ingram, Jessica R.; Ploegh, Hidde L.
    ACS Chemical Biology (2015), 10 (2), 460-465CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. The authors demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. The authors successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymic approach enables engineering of the plasma membrane for research or therapy under physiol. reaction conditions that ensure the viability of the modified cells.
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  34. 34
    Osteen, K. G.; Hill, G. A.; Hargrove, J. T.; Gorstein, F. Fertil. Steril. 1989, 52, 965– 972
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    34
    Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens
    Osteen K G; Hill G A; Hargrove J T; Gorstein F
    Fertility and sterility (1989), 52 (6), 965-72 ISSN:0015-0282.
    Appropriate endometrial maturation is of paramount importance to achieve reproductive success. Practical and ethical considerations require that in vitro methods be available to evaluate regulation of human endometrial function. Additionally, tissue complexity requires separation of individual cell populations. This report describes an improved method for isolation of endometrial epithelial and stromal cells, using biopsy specimens as a tissue source. Separated cells were obtained using selective enzymatic digestion in conjunction with physical separation procedures. Isolated populations exhibited over 95% homogeneity, ascertained immunocytochemically. Using this system, isolated cells from normal endometrium can readily be obtained for in vitro studies. Within the defined conditions of a culture system, important areas of current concern in the endometrium such as ectopic endometrial growth and implantation can be addressed.
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  35. 35
    Huang, X.; Aulabaugh, A.; Ding, W.; Kapoor, B.; Alksne, L.; Tabei, K.; Ellestad, G. Biochemistry 2003, 42, 11307– 11315
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    35
    Kinetic mechanism of Staphylococcus aureus sortase SrtA
    Huang, Xinyi; Aulabaugh, Ann; Ding, Weidong; Kapoor, Bhupesh; Alksne, Lefa; Tabei, Keiko; Ellestad, George
    Biochemistry (2003), 42 (38), 11307-11315CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal contg. a LPXTG motif is cleaved between the Thr and Gly residues. The resulting Thr C-terminus of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-contg. peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. Here, the authors analyzed the steady-state kinetics of these 2 types of reactions catalyzed by sortase. The kinetic results fully supported a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction had a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. The authors also demonstrated that the nucleophile binding site of S. aureus sortase SrtA was specific for diglycine. Whereas the S1' and S2' sites of the enzyme both preferred a Gly residue, the S1' site was exclusively selective for Gly. The lengthening of the polyglycine acceptor nucleophile beyond diglycine did not further enhance the binding and catalysis.
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  36. 36
    Cruise, G. M.; Scharp, D. S.; Hubbell, J. A. Biomaterials 1998, 19, 1287– 1294
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    36
    Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels
    Cruise, Gregory M.; Scharp, David S.; Hubbell, Jeffrey A.
    Biomaterials (1998), 19 (14), 1287-1294CODEN: BIMADU; ISSN:0142-9612. (Elsevier Science Ltd.)
    Poly(ethylene glycol) (PEG) diacrylate hydrogels are being explored as permselective membranes for the transplantation of islets of Langerhans. Hydrogel membranes formed by interfacially photopolymg. PEG diacrylate precursor soln. were prepd. from PEG diacrylate of mol. wts. (MW) ranging from 2000 (2K) to 20,000 (20K) with concns. 10-30% wt./wt. The effects of PEG diacrylate MW and concn. in the membrane precursor soln. upon the diffusivities of vitamin B12, myoglobin, ovalbumin, albumin, and IgG were detd. Regardless of the concn. of the PEG diacrylate in the precursor soln., hydrogels prepd. with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepd. with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepd. with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K and 8K diacrylates were also seen in calcns. of the mol. wt. between crosslinks and the mesh size, with values in the range of 150-170 g/mol and 15-35°A, resp., depending on PEG diacrylate concn. In contrast, hydrogels formed from PEG 20K diacrylate had mol. wt. between crosslinks 1150-2000 g/mol and mesh sizes 45-70°A, with larger values being obsd. in membranes polymd. from more dil. PEG diacrylate precursor. The results indicate that PEG diacrylate hydrogels can prevent the diffusion of albumin and the components specific to an immune response (such as IgG, IgM, and C1q) while maintaining the rapid permeation of insulin and glucose.
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  37. 37
    S, J. J.; Griffith, L. G. Macromolecules 1997, 30, 5255– 5264
    [ ACS Full Text ACS Full Text], Google Scholar
    There is no corresponding record for this reference.
  38. 38
    Zustiak, S. P.; Boukari, H.; Leach, J. B. Soft Matter 2010, 6, 3609– 3618
    [ Crossref], [ CAS], Google Scholar
    38
    Solute diffusion and interactions in cross-linked poly(ethylene glycol) hydrogels studied by Fluorescence Correlation Spectroscopy
    Zustiak, Silviya P.; Boukari, Hacene; Leach, Jennie B.
    Soft Matter (2010), 6 (15), 3609-3618CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    Controlled diffusion and release of sol. mols. is one of the key challenges in developing 3-dimensional (3D) scaffolds for tissue engineering and drug delivery applications in part because current methods to measure dynamic transport properties are difficult to perform directly, are strongly affected by the exptl. setup, and therefore can be a subject to various artifacts. In this work the authors present a method for direct measurement of translational diffusion of solutes, namely Fluorescence Correlation Spectroscopy (FCS), by characterizing the diffusion of model proteins through a 3D cross-linked poly(ethylene glycol) (PEG) hydrogel scaffold. The authors examd. both the dynamics of hydrogel structure (e.g., crosslinking and swelling) as well as protein size and their effect on protein diffusivity. For example, the authors demonstrated that protein diffusivity was closely related to protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., γ-globulin or Ig). The authors validated the FCS protein diffusivity results by comparison to std. bulk diffusion assays. Addnl., due to the nature of FCS measurements, the authors were able to probe for hydrogel-protein interactions during crosslinking that may contribute to the obstructed protein diffusion in the 3D scaffold. The authors detd. that such interactions in this system were not covalent (i.e., were independent of the crosslinking chem.) but may be due to weaker hydrogen bonding or ionic interactions. Also, these interactions were protein specific and contributed up to 25% of the total decrease in protein diffusivity in the hydrogel as compared to diffusivity in water. Though interactions between various proteins and PEG were reported, this is the first study that has explored these effects in detail in cross-linked PEG hydrogels using FCS; the authors' findings question the assumption that PEG hydrogels are completely inert to protein interactions when applied as drug delivery matrixes and tissue engineering scaffolds.
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  39. 39
    Singh, B.; Coffey, R. J. Annu. Rev. Physiol. 2014, 76, 275– 300
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    39
    Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells
    Singh, Bhuminder; Coffey, Robert J.
    Annual Review of Physiology (2014), 76 (), 275-300CODEN: ARPHAD; ISSN:0066-4278. (Annual Reviews)
    A review. A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochem. and functionally sep. apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a crit. regulator of epithelial homeostasis and is perturbed in a no. of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the sol. form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biol. consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of sol. EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiol., and demonstrate the pathophysiol. consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes.
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  40. 40
    Kuhl, P. R.; Griffith-Cima, L. G. Nat. Med. 1996, 2, 1022– 1027
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    40
    Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase
    Kuhl, Philip R.; Griffith-Cima, Linda G.
    Nature Medicine (New York) (1996), 2 (9), 1022-1027CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Co.)
    We have tethered epidermal growth factor (EGF) to a solid substrate in a manner permitting the factor to retain its biol. activity as assessed by both mitogenic and morphol. assays. Mouse EGF was covalently coupled to aminosilane-modified glass via star poly(ethylene oxide) (PEO), which allows the ligand to retain significant mobility and active conformation. Tethered EGF was as effective as sol. EGF in eliciting DNA synthesis and cell rounding responses of primary rat hepatocytes under different surface conditions. In contrast, phys. adsorbed EGF at comparable surface concns. showed no activity. Presentation of growth factors in this manner may help to expedite their clin. use by permitting greater control of temporal and spatial availability in the extracellular environment.
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  41. 41
    Mehta, G.; Williams, C. M.; Alvarez, L.; Lesniewski, M.; Kamm, R. D.; Griffith, L. G. Biomaterials 2010, 31, 4657– 4671
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    41
    Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels
    Mehta, Geeta; Williams, Courtney M.; Alvarez, Luis; Lesniewski, Martha; Kamm, Roger D.; Griffith, Linda G.
    Biomaterials (2010), 31 (17), 4657-4671CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
    The compn., presentation, and spatial orientation of extracellular matrix mols. and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of sol. EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between sol. and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrixes reveal new facets of hepatocellular biol. in culture and may be more broadly useful in culture of other soft tissues.
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  42. 42
    Williams, C. M.; Mehta, G.; Peyton, S. R.; Zeiger, A. S.; Van Vliet, K. J.; Griffith, L. G. Tissue Eng., Part A 2011, 17, 1055– 1068
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    42
    Autocrine-Controlled Formation and Function of Tissue-Like Aggregates by Primary Hepatocytes in Micropatterned Hydrogel Arrays
    Williams, Courtney M.; Mehta, Geeta; Peyton, Shelly R.; Zeiger, Adam S.; Van Vliet, Krystyn J.; Griffith, Linda G.
    Tissue Engineering, Part A (2011), 17 (7 and 8), 1055-1068CODEN: TEPAB9; ISSN:1937-3341. (Mary Ann Liebert, Inc.)
    The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concns. of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mech. compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5β1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsFOgtrc%253D&md5=661a3dd2a88f900756785452195ef534
  43. 43
    Chegini, N.; Rossi, M. J.; Masterson, B. J. Endocrinology 1992, 130, 2373– 2385
    [ PubMed], [ CAS], Google Scholar
    43
    Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF β-receptors in human endometrial tissue: localization and in vitro action
    Chegini, Nasser; Rossi, Michael J.; Masterson, Byron J.
    Endocrinology (1992), 130 (4), 2373-85CODEN: ENDOAO; ISSN:0013-7227.
    Human endometrial tissue and primary stromal cell culture contain immunoreactive EGF, platelet-derived growth factor (PDGF)-AB as well as EGF receptor, and PDGF β-receptors. The immunostaining for EGF, EGF receptor, and PDGF β-receptor was assocd. with endometrial luminal and glandular epithelial and stromal cells, whereas only the stromal cells contained immunoreactive PDGF-AB. The immunostaining intensity of EGF, EGF receptor, and PDGF-AB was similar in both phases of the menstrual cycle, whereas PDGF β-receptor immunostaining was highest in the proliferative phase and was considerably reduced, particularly in luminal and glandular epithelial cells in the secretory phase. In addn., primary stromal cell cultures express EGF, PDGF-AB, and contain EGF and PDGF-β receptors, and very low levels of PDGF-α receptor. [3H]thymidine incorporation indicated that after 48 h of incubation in serum-free medium approx. 75-80% of stromal cells are quiescent. Incubation of quiescent stromal cells with 10% fetal bovine serum stimulated [3H]thymidine incorporation in a time-dependent manner reaching maximal level after 30-48 h, with a doubling time of 38.2 h. EGF (1.5-15 ng/mL) stimulates [3H]thymidine incorporation by quiescent stromal cells. This effect was reduced at concns. >15 ng/mL. PDGF-AB (3-10 ng/mL) and PDGF-BB (0.5-10 ng/mL) also stimulate [3H]thymidine incorporation in quiescent stromal cells compared to controls. The action of EGF (15 ng/mL) and PDGF-AB (10 ng/mL) was time dependent, reaching maximal after 36 and 48 h of incubation. Addn. of PDGF-AB (10 ng/mL) to EGF (15 ng/mL) enhanced the action of EGF or PDGF-AB used individually. 17β-Estradiol or progesterone at 1μM did not stimulate [3H]thymidine incorporation; although they were stimulatory in combination; they did not alter the action of EGF or PDGF when added in combination. These observations provide further evidence that human endometrial tissue contains specific immunoreactive EGF receptors. They also demonstrate the presence of immunoreactive EGF, PDGF-AB, and PDGF-β receptors in endometrial tissue as well as stromal cells in primary culture. Both EGF and PDGF are mitogenic for endometrial stromal cells, suggesting an autocrine/paracrine role in modulation of endometrial cell growth and differentiation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XitVymurk%253D&md5=caa43714a0edf631153bb4adc01bd355
  44. 44
    Klenkler, B. J.; Dwivedi, D.; West-Mays, J. A.; Sheardown, H. J. Biomed. Mater. Res. 2010, 93, 1043– 1049
    [ CAS], Google Scholar
    44
    Corneal epithelial cell adhesion and growth on EGF-modified aminated PDMS
    Klenkler, Bettina J.; Dwivedi, Dhruva; West-Mays, Judith A.; Sheardown, Heather
    Journal of Biomedical Materials Research, Part A (2010), 93A (3), 1043-1049CODEN: JBMRCH; ISSN:1549-3296. (John Wiley & Sons, Inc.)
    Growth factor tethering has significant potential to mediate cellular responses in biomaterials and tissue engineering. We have previously demonstrated that epidermal growth factor (EGF) can be tethered to polydimethylsiloxane (PDMS) substrates and that these surfaces promoted interactions with human corneal epithelial cells in vitro. The goal of the current work was to better understand the specific effects of the tethered growth factor on the cells. The EGF was reacted with a homobifunctional N-hydroxysuccinimide (NHS) polyethylene glycol (PEG) deriv., and then bound to allyamine plasma-modified PDMS. Human corneal epithelial cells were seeded on the surfaces and cultured in serum-free medium for periods of up to 5 days. Cell growth was monitored and quantified by trypsinization and counting with a Coulter counter. Expression of matrix proteins and α6-integrins was assessed by immunostaining and confocal microscopy. A centrifugation assay was used to det. cell adhesion under an applied detachment force. Binding of EGF was found to significantly increase cell nos. and coverage across the surfaces at 5 days of culture in vitro. Immunofluorescence expts. indicate increased expression of fibronectin, laminin, and α6-integrins on the EGF-modified surfaces, and expression is localized at the cell-material interface as obsd. by confocal microscopy. In accordance with these results, the highest quantity of adherent cells is found on the EGF-modified substrates at 5 days of culture. The results provide initial evidence that binding of EGF may be used to improve the epithelialization of and the adhesion of the cells on a polymeric artificial cornea device. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFSmtrc%253D&md5=571242baf9c9084acbcd36df6350f664
  45. 45
    Marcantonio, N. A.; Boehm, C. A.; Rozic, R. J.; Au, A.; Wells, A.; Muschler, G. F.; Griffith, L. G. Biomaterials 2009, 1– 10
    Google Scholar
    There is no corresponding record for this reference.
  46. 46
    Brown, A. C.; Rowe, J. A.; Barker, T. H. Tissue Eng., Part A 2011, 17, 139– 150
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    46
    Guiding Epithelial Cell Phenotypes with Engineered Integrin-Specific Recombinant Fibronectin Fragments
    Brown, Ashley C.; Rowe, Jessica A.; Barker, Thomas H.
    Tissue Engineering, Part A (2011), 17 (1-2), 139-150CODEN: TEPAB9; ISSN:1937-3341. (Mary Ann Liebert, Inc.)
    The extracellular matrix (ECM) provides important cues for directing cell phenotype. Cells interact with underlying ECM through cell-surface receptors known as integrins, which bind to specific sequences on their ligands. During tissue development, repair, and regeneration of epithelial tissues, cells must interact with an interstitial fibronectin (Fn)-rich matrix, which has been shown to direct a more migratory/repair phenotype, presumably through interaction with Fn's cell binding domain comprised of both synergy Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) sequences. We hypothesized that the Fn synergy site is crit. to the regulation of epithelial cell phenotype by directing integrin specificity. Epithelial cells were cultured on Fn fragments displaying stabilized synergy and RGD (FnIII9'10), or RGD alone (FnIII10) and cell phenotype analyzed by cytoskeleton changes, epithelial cell-cell contacts, changes in gene expression of epithelial and mesenchymal markers, and wound healing assay. Data indicate that epithelial cells engage RGD only with αv integrins and display a significant shift toward a mesenchymal phenotype due, in part, to enhanced transforming growth factor-β activation and/or signaling compared with cells on the synergy contg. FnIII9'10. These studies demonstrate the importance of synergy in regulating epithelial cell phenotype relevant to tissue engineering as well as the utility of engineered integrin-specific ECM fragments in guiding cell phenotype.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVCqtg%253D%253D&md5=2d0410c3ad679c4907185d6af96101b8
  47. 47
    Kuhlman, W.; Taniguchi, I.; Griffith, L. G.; Mayes, A. M. Biomacromolecules 2007, 8, 3206– 3213
    [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
    47
    Interplay Between PEO Tether Length and Ligand Spacing Governs Cell Spreading on RGD-Modified PMMA-g-PEO Comb Copolymers
    Kuhlman, William; Taniguchi, Ikuo; Griffith, Linda G.; Mayes, Anne M.
    Biomacromolecules (2007), 8 (10), 3206-3213CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    The effects of tether length on cell adhesion to poly(Me methacrylate)-graft-poly(ethylene oxide), PMMA-g-PEO, comb copolymer films functionalized with the adhesion peptide RGD were investigated. Copolymers having PEO tether lengths of 10 and 22 EO segments were synthesized and coupled with a synthetic peptide that contained both RGD and the synergy sequence PHSRN. Cell spreading assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. Fluorescence resonance energy transfer (FRET) measurements indicated a mean sepn. between integrin-bound peptides of 15.6±1.4 nm for combs with long (22-mer) tethers, compared with 17.5±1.3 nm for short (10-mer) tethers, on films of comparable peptide d. (∼2500 peptides/μm2). The results suggest that the added mobility afforded by the more extensible tethers encouraged the formation of focal adhesions by allowing cells to reorganize tethered peptides on the nanometer length scale. In addn., adhesion peptides were selectively coupled to 10-mer or 22-mer PEO tethers within a bimodal brush to investigate stratification effects on cell adhesion. Peptides bound by short tethers in a bed of long unsubstituted chains resulted in surfaces that resisted, rather than promoted, cell adhesion. By contrast, when long peptide tethers were employed with short unsubstituted chains, cell attachment and spreading were comparable to that found on a monomodal brush of long chains at equiv. peptide d.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVChs7zE&md5=d97c9d091b6ea566e259211970a083a2
  48. 48
    Sand, T. E.; Christoffersen, T. J. Cell. Physiol. 1987, 131, 141– 148
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    48
    Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis
    Sand, Tor Erik; Christoffersen, Thoralf
    Journal of Cellular Physiology (1987), 131 (2), 141-8CODEN: JCLLAX; ISSN:0021-9541.
    Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of EGF and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed 1st-order kinetics both in untreated and hormone-stimulated cells. Addn. of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25-26 h), but the rate const. for the S phase entry increased 5-6-fold. Expts. where the time of hormone addn. was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40-50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin-pretreated hepatocytes was postponed for 44 h after plating in serum-free medium, the cellular sensitivity was increased as compared with EGF treatment at 0 h (a 1-log shift of the dose-effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate const.) was shortened (6-7 vs. 26 h).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXktFyms7c%253D&md5=b3d5087284982a13ecf75f1eff5417a8
  49. 49
    Ismail, T.; Howl, J.; Wheatley, M.; McMaster, P.; Neuberger, J. M.; Strain, A. J. Hepatology 1991, 14, 1076– 1082
    [ Crossref], [ PubMed], [ CAS], Google Scholar
    49
    Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis
    Ismail, Tariq; Howl, John; Wheatley, Mark; McMaster, Paul; Neuberger, James M.; Strain, Alastair J.
    Hepatology (Philadelphia, PA, United States) (1991), 14 (6), 1076-82CODEN: HPTLD9; ISSN:0270-9139.
    Human hepatocytes isolated from normal liver tissue were maintained in culture for up to 5 days, and DNA synthesis was detd. Hydroxyurea reduced [3H]thymidine incorporation into DNA by 95%, indicating replicative DNA synthesis. As previously found with rat hepatocytes, EGF and transforming growth factor-α stimulated DNA synthesis at low nanomolar concns.; transforming growth factor-α was slightly more potent. Although the overall rate of thymidine incorporation was lower than that for rodent cells, human hepatocytes were sensitive to lower concns. of these growth factors, and the degree of stimulation was similar. Transforming growth factor-β inhibited DNA synthesis at low picomolar levels. By contrast (unlike rat hepatocytes), AVP failed to initiate or potentiate DNA synthesis in human cells. Thus, normal human hepatocytes can respond to low concns. of growth promoters or inhibitors previously shown to have activity on rat hepatocytes. These factors may then play a role in control of human liver growth. However, important species differences are apparent, highlighting the limitations of extrapolating from animal studies to humans.
    >> More from SciFinder ®
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  2. Zhi Zou, Elisabeth Gau, Islam El-Awaad, Felix Jakob, Andrij Pich, Ulrich Schwaneberg. Selective Functionalization of Microgels with Enzymes by Sortagging. Bioconjugate Chemistry 2019, 30 (11) , 2859-2869. DOI: 10.1021/acs.bioconjchem.9b00568.
  3. Joshua A. Hammer, Anna Ruta, Aidan M. Therien, Jennifer L. West. Cell-Compatible, Site-Specific Covalent Modification of Hydrogel Scaffolds Enables User-Defined Control over Cell–Material Interactions. Biomacromolecules 2019, 20 (7) , 2486-2493. DOI: 10.1021/acs.biomac.9b00183.
  4. Paige J. LeValley, April M. Kloxin. Chemical Approaches to Dynamically Modulate the Properties of Synthetic Matrices. ACS Macro Letters 2019, 8 (1) , 7-16. DOI: 10.1021/acsmacrolett.8b00808.
  5. Christopher D. Spicer, E. Thomas Pashuck, Molly M. Stevens. Achieving Controlled Biomolecule–Biomaterial Conjugation. Chemical Reviews 2018, 118 (16) , 7702-7743. DOI: 10.1021/acs.chemrev.8b00253.
  6. Joshua A. Hammer, Jennifer L. West. Dynamic Ligand Presentation in Biomaterials. Bioconjugate Chemistry 2018, 29 (7) , 2140-2149. DOI: 10.1021/acs.bioconjchem.8b00288.
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  9. Stephanie A. Fisher, Alexander E.G. Baker, and Molly S. Shoichet . Designing Peptide and Protein Modified Hydrogels: Selecting the Optimal Conjugation Strategy. Journal of the American Chemical Society 2017, 139 (22) , 7416-7427. DOI: 10.1021/jacs.7b00513.
  10. Ulri N. Lee, John H. Day, Amanda J. Haack, Ross C. Bretherton, Wenbo Lu, Cole A. DeForest, Ashleigh B. Theberge, Erwin Berthier. Layer-by-layer fabrication of 3D hydrogel structures using open microfluidics. Lab on a Chip 2020, 20 (3) , 525-536. DOI: 10.1039/C9LC00621D.
  11. Jared A. Shadish, Cole A. DeForest. Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials. Matter 2020, 2 (1) , 50-77. DOI: 10.1016/j.matt.2019.11.011.
  12. Faraz Jivan, Daniel L. Alge. Bio‐Orthogonal, Site‐Selective Conjugation of Recombinant Proteins to Microporous Annealed Particle Hydrogels for Tissue Engineering. Advanced Therapeutics 2020, 3 (1) , 1900148. DOI: 10.1002/adtp.201900148.
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  14. Xiaolin Dai, Alexander Böker, Ulrich Glebe. Broadening the scope of sortagging. RSC Advances 2019, 9 (9) , 4700-4721. DOI: 10.1039/C8RA06705H.
  15. Samuel Lim, Gi Ahn Jung, Riya J. Muckom, Dominic J. Glover, Douglas S. Clark. Engineering bioorthogonal protein–polymer hybrid hydrogel as a functional protein immobilization platform. Chemical Communications 2019, 55 (6) , 806-809. DOI: 10.1039/C8CC08720B.
  16. Matthew R. Arkenberg, Dustin M. Moore, Chien-Chi Lin. Dynamic control of hydrogel crosslinking via sortase-mediated reversible transpeptidation. Acta Biomaterialia 2019, 83, 83-95. DOI: 10.1016/j.actbio.2018.11.011.
  17. Yi Zhang, Keun-Young Park, Kiall F. Suazo, Mark D. Distefano. Recent progress in enzymatic protein labelling techniques and their applications. Chemical Society Reviews 2018, 47 (24) , 9106-9136. DOI: 10.1039/C8CS00537K.
  18. Mariana Oksdath, Sally L. Perrin, Cedric Bardy, Emily F. Hilder, Cole A. DeForest, R. Dario Arrua, Guillermo A. Gomez. Review: Synthetic scaffolds to control the biochemical, mechanical, and geometrical environment of stem cell-derived brain organoids. APL Bioengineering 2018, 2 (4) , 041501. DOI: 10.1063/1.5045124.
  19. Lucca Trachsel, Nicolas Broguiere, Jan-Georg Rosenboom, Marcy Zenobi-Wong, Edmondo M. Benetti. Enzymatically crosslinked poly(2-alkyl-2-oxazoline) networks for 3D cell culture. Journal of Materials Chemistry B 2018, 6 (46) , 7568-7572. DOI: 10.1039/C8TB02382D.
  20. Denis Jabaudon, Madeline Lancaster. Exploring landscapes of brain morphogenesis with organoids. Development 2018, 145 (22) , dev172049. DOI: 10.1242/dev.172049.
  21. Chien-Chi Lin, Murray Korc. Designer hydrogels: Shedding light on the physical chemistry of the pancreatic cancer microenvironment. Cancer Letters 2018, 436, 22-27. DOI: 10.1016/j.canlet.2018.08.008.
  22. Nicolas Broguiere, Florian A. Formica, Gonçalo Barreto, Marcy Zenobi-Wong. Sortase A as a cross-linking enzyme in tissue engineering. Acta Biomaterialia 2018, 77, 182-190. DOI: 10.1016/j.actbio.2018.07.020.
  23. Christopher M. Madl, Sarah C. Heilshorn. Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche. Annual Review of Biomedical Engineering 2018, 20 (1) , 21-47. DOI: 10.1146/annurev-bioeng-062117-120954.
  24. Christopher M. Madl, Sarah C. Heilshorn, Helen M. Blau. Bioengineering strategies to accelerate stem cell therapeutics. Nature 2018, 557 (7705) , 335-342. DOI: 10.1038/s41586-018-0089-z.
  25. Christopher M. Madl, Sarah C. Heilshorn. Bioorthogonal Strategies for Engineering Extracellular Matrices. Advanced Functional Materials 2018, 28 (11) , 1706046. DOI: 10.1002/adfm.201706046.
  26. Alexandra C. Braun, Marcus Gutmann, Tessa Lühmann, Lorenz Meinel. Bioorthogonal strategies for site-directed decoration of biomaterials with therapeutic proteins. Journal of Controlled Release 2018, 273, 68-85. DOI: 10.1016/j.jconrel.2018.01.018.
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  28. Chen Guo, Heejae Kim, Elisa M. Ovadia, Christine M. Mourafetis, Mingrui Yang, Wilfred Chen, April M. Kloxin. Bio-orthogonal conjugation and enzymatically triggered release of proteins within multi-layered hydrogels. Acta Biomaterialia 2017, 56, 80-90. DOI: 10.1016/j.actbio.2017.04.002.
  29. Jorge Valdez, Christi D. Cook, Caroline Chopko Ahrens, Alex J. Wang, Alexander Brown, Manu Kumar, Linda Stockdale, Daniel Rothenberg, Kasper Renggli, Elizabeth Gordon, Douglas Lauffenburger, Forest White, Linda Griffith. On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks. Biomaterials 2017, 130, 90-103. DOI: 10.1016/j.biomaterials.2017.03.030.
  30. Christi D. Cook, Abby S. Hill, Margaret Guo, Linda Stockdale, Julia P. Papps, Keith B. Isaacson, Douglas A. Lauffenburger, Linda G. Griffith. Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function. Integrative Biology 2017, 9 (4) , 271-289. DOI: 10.1039/c6ib00245e.
  31. S.J. Park, B.H. Lui, J.R. Cochran. 4.14 Rational and Combinatorial Methods to Create Designer Protein Interfaces ☆. 2017,,, 221-247. DOI: 10.1016/B978-0-12-803581-8.10152-3.
  32. V Milleret, P.S. Lienemann, M. Ehrbar. 4.31 Cell-Demanded Release of Growth Factors ☆. 2017,,, 571-588. DOI: 10.1016/B978-0-12-803581-8.10180-8.
  33. A. M. Clark, S. E. Wheeler, C. L. Young, L. Stockdale, J. Shepard Neiman, W. Zhao, D. B. Stolz, R. Venkataramanan, D. Lauffenburger, L. Griffith, A. Wells. A liver microphysiological system of tumor cell dormancy and inflammatory responsiveness is affected by scaffold properties. Lab on a Chip 2017, 17 (1) , 156-168. DOI: 10.1039/C6LC01171C.
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  35. Xiaozhong Cheng, Tao Zhu, Haofei Hong, Zhifang Zhou, Zhimeng Wu. Sortase A-mediated on-resin peptide cleavage and in situ ligation: an efficient one-pot strategy for the synthesis of functional peptides and proteins. Organic Chemistry Frontiers 2017, 4 (10) , 2058-2062. DOI: 10.1039/C7QO00481H.
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  37. Huiling Jiang, Victor Pan, Skanda Vivek, Eric R. Weeks, Yonggang Ke. Programmable DNA Hydrogels Assembled from Multidomain DNA Strands. ChemBioChem 2016, 17 (12) , 1156-1162. DOI: 10.1002/cbic.201500686.
  38. Takuya Matsumoto, Yuki Isogawa, Kosuke Minamihata, Tsutomu Tanaka, Akihiko Kondo. Twigged streptavidin polymer as a scaffold for protein assembly. Journal of Biotechnology 2016, 225, 61-66. DOI: 10.1016/j.jbiotec.2016.03.030.
  39. Hongyan He, Changsheng Liu. Stem Cell Differentiation Mediated by Biomaterials/Surfaces. 2016,,, 187-251. DOI: 10.1007/978-981-10-2293-7_8.
  40. Musammir Khan, Swen Schuster, Michael Zharnikov. Chemical derivatization and biofunctionalization of hydrogel nanomembranes for potential biomedical and biosensor applications. Phys. Chem. Chem. Phys. 2016, 18 (17) , 12035-12042. DOI: 10.1039/C5CP07840G.
  41. Julia Günter, Petra Wolint, Annina Bopp, Julia Steiger, Elena Cambria, Simon P. Hoerstrup, Maximilian Y. Emmert. Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment. Stem Cells International 2016, 2016, 1-20. DOI: 10.1155/2016/9098523.
  42. Morten Meldal, Sanne Schoffelen. Recent advances in covalent, site-specific protein immobilization. F1000Research 2016, 5, 2303. DOI: 10.12688/f1000research.9002.1.

  • Abstract

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    Scheme 1

    Scheme 1. Process Used to Tether GGG-EGF to the Hydrogela
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    aTop: Hydrogels are formed by Michael-type additions of 8-arm PEG-acrylate stars cross-linked with 4-arm PEG-thiol stars. Adhesion peptides (SynKRGD) and sortase motif peptides (LPRTG) can also be cross-linked into the hydrogel if they contain a cysteine residue to react with the acrylate of the 8-arm PEG star. Bottom: (A) Sortase-mediated ligation of GGG-EGF to pre-formed PEG hydrogels containing LPRTG peptide. (B) Sortase diffuses into the hydrogel, cleaves the peptide bond between the threonine and the glycine of the LPRTG peptide, releasing G, then the N-terminus of GGG-EGF, which has diffused into the hydrogel, is ligated to the C-terminus of the LPRT-motif (C). The same sequence of steps can be used to cleave EGF once it is ligated to the hydrogel, by adding sortase and the simple peptide GGG; GGG will displace tethered EGF, releasing it into solution.

    Figure 1

    Figure 1. Amount of GGG-EGF peptide nucleophile remaining in solution as a function of total LPRTG concentration in hydrogel. GGG-EGF is quantified by sandwich ELISA before and after sortase-mediated ligation of GGG-EGF to preformed hydrogels containing LPRTG, using nucleophile concentrations of 2 μM (A) or 20 μM (B). The amount of GGG-EGF remaining in solution after sortase-mediated ligation decreases with increasing LPRTG concentration in hydrogel. Error bars represent standard error of the mean (n = 2).

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    Figure 2

    Figure 2. Amount of reacted LPRTG as a function of total initial LPRTG amount in hydrogel. After correction for photobleaching, fluorescence intensities were converted to pmol LPRTG in the hydrogel and the amount of reacted LPRTG was calculated as the difference between the values before and after sortase-mediated ligation. Error bars represent standard error of the mean (n = 2).

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    Figure 3

    Figure 3. Amount of GGG-EGF released in solution as a function of total LPRTG concentration in hydrogel. GGG-EGF is quantified by sandwich ELISA after hydrogel washes and sortase-mediated hydrogel cleavage following tethering of GGG-EGF at either 2 μM (A) or 20 μM (B). The amount of GGG-EGF released in solution after sortase-mediated cleavage increases with increasing LPRTG concentration in hydrogel. Error bars represent standard error of the mean (n = 2).

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    Figure 4

    Figure 4. Nucleophile mass balance for sortase-mediated ligation of GGG-EGF to LPRTG-containing hydrogels as a function of LPRTG concentration in the hydrogel and for GGG-EGF concentrations of either 2 μM (A) or 20 μM (B). The amount of GGG-EGF present in initial tethering solution and in hydrogel supernatant after washes and after cleavage was quantified by sandwich ELISA. Error bars represent standard error of the mean (n = 2).

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    Figure 5

    Figure 5. Detection of GGG-EGF on hydrogels as a function of total LPRTG concentration in hydrogel after sortase-mediated ligation of 2 or 20 μM GGG-EGF. (A) GGG-EGF nonspecific binding was tested after incubation of the hydrogels with 2 or 20 μM GGG-EGF in the absence of sortase. (B) Measurements performed without sortase were subtracted from the ones performed in the presence of the enzyme. Error bars represent standard error of the mean (n = 2).

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    Figure 6

    Figure 6. Cell attachment and DNA synthesis of primary human hepatocytes (A, B) and endometrial epithelial cells (C, D). Cells were seeded on hydrogels containing 0 or 500 μM synKRGD and 250 μM LPRTG and on standard culture substrates. After 48 h (A, B) or 40 h in culture (C, D), cells were incubated with 10 μM EdU for 24 h. Tethered EGF stimulated DNA synthesis compared to unmodified hydrogels or soluble hEGF. Error bars represent standard error of the mean (n = 3).

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    This article references 49 other publications.

    1. 1
      Griffith, L. G.; Swartz, M. A. Nat. Rev. Mol. Cell Biol. 2006, 7, 211– 224
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      1
      Capturing complex 3D tissue physiology in vitro
      Griffith, Linda G.; Swartz, Melody A.
      Nature Reviews Molecular Cell Biology (2006), 7 (3), 211-224CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. The emergence of tissue engineering raises new possibilities for the study of complex physiol. and pathophysiol. processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. The authors discuss here some of the 'design principles' for recreating the interwoven set of biochem. and mech. cues in the cellular microenvironment, and the methods for implementing them. The authors emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xhs1Kisro%253D&md5=5a957aaf2581e5bcc5fc06690e8883ad
    2. 2
      Poly(ethylene glycol) Chemistry; Harris, J. M., Ed.; Springer US: Boston, MA, 1992.
      [ Crossref], Google Scholar
      There is no corresponding record for this reference.
    3. 3
      Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A. J. Controlled Release 2001, 76, 11– 25
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      3
      Protein delivery from materials formed by self-selective conjugate addition reactions
      Elbert, D. L.; Pratt, A. B.; Lutolf, M. P.; Halstenberg, S.; Hubbell, J. A.
      Journal of Controlled Release (2001), 76 (1-2), 11-25CODEN: JCREEC; ISSN:0168-3659. (Elsevier Science Ireland Ltd.)
      A new chem. crosslinking scheme was utilized for the formation of degradable poly(ethylene glycol) hydrogels suitable for the delivery of protein drugs. An aq. soln. contg. a PEG-multiacrylate and solid particles of albumin was mixed with an aq. soln. contg. a PEG-dithiol, rapidly producing a cross-linked hydrogel through a Michael-type addn. reaction. For some formulations, it was obsd. that about 65% of the incorporated protein was released with zero-order kinetics over a period of about 4 days. By changing the functionality of the cross-linker, the release of protein could even be delayed for about 4 days, followed by zero-order release. The mechanism for release appeared to be a combination of slow dissoln. of protein in the presence of PEG and hindered diffusion of protein through the gel. The crosslinking of the gels was studied rheometrically, and the hydrolytic degrdn. of the gels was characterized by measuring the swelling of the gels. Biochem. anal. of the released proteins demonstrated that the polymers reacted with each other, but not with proteins. Utilizing the Flory-Rehner and Peppas-Merrill equations, a framework for modeling the protein release from the gels is described.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXmt1Glsr4%253D&md5=d40f8c15efca8049cf93228dc3a31a71
    4. 4
      Lutolf, M. P.; Hubbell, J. A. Biomacromolecules 2003, 4, 713– 722
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      4
      Synthesis and Physicochemical Characterization of End-Linked Poly(ethylene glycol)-co-peptide Hydrogels Formed by Michael-Type Addition
      Lutolf, M. P.; Hubbell, J. A.
      Biomacromolecules (2003), 4 (3), 713-722CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The synthesis of novel hybrid hydrogels by stepwise copolymn. of multi-arm vinyl sulfone-terminated poly(ethylene glycol) macromers and α-ω cysteine oligopeptides via Michael-type addns. is described. Crosslinking kinetics, studied by in situ rheometry, can be controlled by pH and the presence of charged amino acid residues in close proximity to the Cys, which modulates the pKa of the thiol group. These end-linked networks were characterized by their equil. swelling in water, by their viscoelastic properties in the swollen state, and by their sol. fraction. Structure and properties are sensitive to the prepn. state including stoichiometry and precursor concn. and less sensitive to the pH during crosslinking. For each network the concn. of elastically active chains (ν) was calcd. from exptl. detd. sol fractions using Miller-Macosko theory and compared to values obtained from swelling and rheometry studies and by calcn. from Flory's classical network models. Hydrogels were also prepd. with varying macromer structures, and their properties were shown to respond to both macromer functionality and mol. wt.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhsVaqurw%253D&md5=1c7b5b8bb1a980b6489b06da10cdf5ad
    5. 5
      Lutolf, M. P.; Raeber, G. P.; Zisch, A. H.; Tirelli, N.; Hubbell, J. A. Adv. Mater. 2003, 15, 888– 892
      [ Crossref], [ CAS], Google Scholar
      5
      Cell-responsive synthetic hydrogels
      Lutolf, Matthias P.; Raeber, George P.; Zisch, Andreas H.; Tirelli, Nicola; Hubbell, Jeffrey A.
      Advanced Materials (Weinheim, Germany) (2003), 15 (11), 888-892CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Synthetic biomaterial matrixes have been formed in the presence of cells by a Michael-type addn. reaction between thiolate groups of cysteine-contg. peptides and a vinylsulfone functionalized multi-arm poly(ethylene glycol). These materials locally degrade in response to cell-surface proteases, allowing cells to create paths for 3D cell migration. Results demonstrate that synthetic materials can be responsive to cellular influences in vitro and in vivo, including here, as an example, inducing and responding to angiogenic invasion.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXkvFCmurg%253D&md5=de92fd194b9c82d5bc065f6f9850f94c
    6. 6
      Zhu, J. Biomaterials 2010, 31, 4639– 4656
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      6
      Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering
      Zhu, Junmin
      Biomaterials (2010), 31 (17), 4639-4656CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      In this review, the authors explore different approaches for introducing bioactivity into poly(ethylene glycol) (PEG) hydrogels. Hydrogels are excellent scaffolding materials for repairing and regenerating a variety of tissues because they can provide a highly swollen 3-dimensional (3D) environment similar to soft tissues. Synthetic hydrogels like PEG-based hydrogels have advantages over natural hydrogels, such as the ability for photopolymn., adjustable mech. properties, and easy control of scaffold architecture and chem. compns. However, PEG hydrogels alone cannot provide an ideal environment to support cell adhesion and tissue formation due to their bio-inert nature. The natural extracellular matrix (ECM) was an attractive model for the design and fabrication of bioactive scaffolds for tissue engineering. ECM-mimetic modification of PEG hydrogels has emerged as an important strategy to modulate specific cellular responses. To tether ECM-derived bioactive mols. (BMs) to PEG hydrogels, various strategies were developed for the incorporation of key ECM biofunctions, such as specific cell adhesion, proteolytic degrdn., and signal mol.-binding. A no. of cell types were immobilized on bioactive PEG hydrogels to provide fundamental knowledge of cell/scaffold interactions. This review addresses the recent progress in material designs and fabrication approaches leading to the development of bioactive hydrogels as tissue engineering scaffolds.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksV2ru7o%253D&md5=ea20a8041483618e93fe71d3642e5f21
    7. 7
      Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47– 55
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      7
      Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering
      Lutolf, M. P.; Hubbell, J. A.
      Nature Biotechnology (2005), 23 (1), 47-55CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      A review. New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrixes (ECMs) and ECM-bound growth factors, both for therapeutic applications and basic biol. studies. Recent advances include nanofibrillar networks formed by self-assembly of small building blocks, artificial ECM networks from protein polymers or peptide-conjugated synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable proteolytic remodeling. These materials have already found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis. Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biol. may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsFGmtg%253D%253D&md5=081fb3c462e35263fedf6690fc67ec07
    8. 8
      Burdick, J. A.; Murphy, W. L. Nat. Commun. 2012, 3, 1269
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      8
      Moving from static to dynamic complexity in hydrogel design
      Burdick Jason A; Murphy William L
      Nature communications (2012), 3 (), 1269 ISSN:.
      Hydrogels are water-swollen polymer networks that have found a range of applications from biological scaffolds to contact lenses. Historically, their design has consisted primarily of static systems and those that exhibit simple degradation. However, advances in polymer synthesis and processing have led to a new generation of dynamic systems that are capable of responding to artificial triggers and biological signals with spatial precision. These systems will open up new possibilities for the use of hydrogels as model biological structures and in tissue regeneration.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s3htlaguw%253D%253D&md5=e50e82f5a80b7732b248d882511c2a62
    9. 9
      Nimmo, C. M.; Shoichet, M. S. Bioconjugate Chem. 2011, 22, 2199– 2209
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      9
      Regenerative Biomaterials that "Click": Simple, Aqueous-Based Protocols for Hydrogel Synthesis, Surface Immobilization, and 3D Patterning
      Nimmo, Chelsea M.; Shoichet, Molly S.
      Bioconjugate Chemistry (2011), 22 (11), 2199-2209CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A review. The click chem. era has generated a library of versatile "spring-loaded" reactions that offer high yields, regio- and stereospecificity, and outstanding functional group tolerance. These powerful transformations are particularly advantageous for the design of sophisticated biomaterials that require high levels of precision and control, namely, materials that promote tissue regeneration such as hydrogels, 2D functionalized substrates, and 3D biomimetic scaffolds. In this review, the synthesis and application of regenerative biomaterials via click chem. are summarized. Particular emphasis is placed on the copper(I)-catalyzed alkyne-azide cycloaddn., Diels-Alder cycloaddns., and thiol-click coupling.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlKksLbF&md5=de16a6a21d6f007679541112e8fe28ab
    10. 10
      Drumheller, P. D.; Hubbell, J. A. Anal. Biochem. 1994, 222, 380– 388
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      10
      Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates
      Drumheller, Paul D.; Hubbell, Jeffrey A.
      Analytical Biochemistry (1994), 222 (2), 380-8CODEN: ANBCA2; ISSN:0003-2697. (Academic)
      Polymer networks of poly(ethylene glycol) (PEG) in densely cross-linked matrixes of acrylic acid (AA) and trimethylolpropane triacrylate were synthesized as biospecific cell adhesive substrates. Networks grafted with synthetic adhesion peptides produced substrates to investigate long-term, receptor-mediated cell/surface interactions, without nonspecific protein adsorption producing spurious adhesion signals. PEG rendered the networks very resistant to cell adhesion in vitro, and AA provided reactive carboxyl moieties for N-terminal grafting of peptides. Networks with higher mass fractions of AA had greater background cell adhesion, which diminished with higher mass fractions of PEG such that complete resistance to cell adhesion could be obtained. Networks grafted with inactive control peptides (GRGES or no peptide) remained completely cell nonadhesive in the presence of serum or even when preincubated with adhesion proteins, while networks grafted with bioadhesive peptides (GRGDS, GYIGSRY, or GREDVY) supported morphol. complete fibroblast adhesion. The amt. of AA in the network readily controlled the amt. of incorporated peptide. These networks may be suitable as anal. tools specifically to investigate long-term cell/substrate interactions in the presence of serum, yet without nonspecific protein adsorption producing adhesion signals other than those immobilized for study.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhvFWisro%253D&md5=5d8d291a7f9dfc809b54ce596f2ef6c4
    11. 11
      Hahn, M. S.; Miller, J. S.; West, J. L. Adv. Mater. 2005, 17, 2939– 2942
      [ Crossref], [ CAS], Google Scholar
      11
      Laser scanning lithography for surface micropatterning on hydrogels
      Hahn, Mariah S.; Miller, Jordan S.; West, Jennifer L.
      Advanced Materials (Weinheim, Germany) (2005), 17 (24), 2939-2942CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A confocal-based laser scanning lithog. method for controlled, high-fidelity two-dimensional and three-dimensional surface patterning of poly(ethylene glycol) hydrogels. The method can be applied to any optically clear, photoactive substrate. Laser scanning lithog. employs user-defined masks to spatially control laser irradn. during pattern generation, eliminating the need for photomasks and a clean-room. Also, pattern mols. are covalently immobilized onto the substrate surface providing mech. and phys. stable patterns. In this work, the surface properties of deformable, biocompatible poly(ethylene glycol)-diacrylate hydrogels were modified in a controlled manner using laser scanning lithog., with pattern feature sizes down to at least 5 μm. Human dermal fibroblast (HDF) were selectively bind to regions patterned with appropriate cell-adhesive peptides. Patterned substrates functionalized with gradients of different cell-adhesive ligands can be fabricated, providing a tool for investigating cell-biomaterial interactions and the structure-function relationships of tissues.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xlt1Wmsg%253D%253D&md5=b5daf05a6da8563bb3a16d5c1a919716
    12. 12
      Hahn, M. S.; Taite, L. J.; Moon, J. J.; Rowland, M. C.; Ruffino, K. A.; West, J. L. Biomaterials 2006, 27, 2519– 2524
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      12
      Photolithographic patterning of polyethylene glycol hydrogels
      Hahn, Mariah S.; Taite, Lakeshia J.; Moon, James J.; Rowland, Maude C.; Ruffino, Katie A.; West, Jennifer L.
      Biomaterials (2006), 27 (12), 2519-2524CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      A simple, inexpensive photolithog. method for surface patterning deformable, solvated substrates is demonstrated using photoactive poly(ethylene glycol) (PEG)-diacrylate hydrogels as model substrates. Photolithog. masks were prepd. by printing the desired patterns onto transparencies using a laser jet printer. Precursor solns. contg. monoacryloyl-PEG-peptide and photoinitiator were layered onto hydrogel surfaces. The acrylated moieties in the precursor soln. were then conjugated in monolayers to specific hydrogel regions by exposure to UV light through the transparency mask. The effects of UV irradn. time and precursor soln. concn. on the levels of immobilized peptide were characterized, demonstrating that bound peptide concn. can be controlled by tuning these parameters. Multiple peptides can be immobilized to a single hydrogel surface in distinct patterns by sequential application of this technique, opening up its potential use in co-cultures. In addn., 3D structures can be generated by incorporating PEG-diacrylate into the precursor soln. To evaluate the feasibility of using these patterned surfaces for guiding cell behavior, human dermal fibroblast adhesion on hydrogel surfaces patterned with acryloyl-PEG-RGDS was investigated. This patterning method may find use in tissue engineering, the elucidation of fundamental structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnol.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xls1Witg%253D%253D&md5=6fdeb9d68e857b2ffb116d83cdb91421
    13. 13
      Lee, W.; Lee, T. G.; Koh, W.-G. J. Ind. Eng. Chem. 2007, 13, 1195– 1200
      [ CAS], Google Scholar
      13
      Grafting of poly(acrylic acid) on the poly(ethylene glycol) hydrogel using surface-initiated photopolymerization for covalent immobilization of collagen
      Lee, Woojin; Lee, Tae Gyu; Koh, Won-Gun
      Journal of Industrial and Engineering Chemistry (Seoul, Republic of Korea) (2007), 13 (7), 1195-1200CODEN: JIECFI; ISSN:1226-086X. (Korean Society of Industrial and Engineering Chemistry)
      The modification of the protein-repellent poly(ethylene glycol) (PEG) hydrogel surface was achieved by a two-step process using immobilization of benzophenone on the PEG hydrogel as surface initiator and subsequent surface-initiated graft polymn. of acrylic acid by UV irradn. Formation of poly(acrylic acid) (PAA) layer on the PEG hydrogel was demonstrated by confirming the presence of carboxyl groups in the poly(acrylic acid) (PAA) with FTIR/ATR spectroscopy and measuring the height of PAA layers with alpha-step surface profiler. In the grafted region, PAA and PEG hydrogel formed an interpenetrating polymer network extending 200 μm into PEG hydrogel and homo PAA protruded 14∼17 μm above the PEG hydrogel surface. Activation of the carboxyl groups in PAA allowed covalent immobilization of collagen, a cell adhesion protein, on the PAA-grafted PEG hydrogel, which was demonstrated with FTIR/ATR spectroscopy by confirming the formation of a new amide bond. Surface-initiated graft polymn. combined with photolithog. produced well-defined PAA micropatterns on the PEG hydrogels and collagen was immobilized only on the PAA region due to the lack of adhesion for proteins to PEG, producing protein micropattern on the PEG hydrogel.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVWqsb8%253D&md5=e79f0c58c6aa6d3ae4063ad64a8e8b97
    14. 14
      Hynd, M. R.; Frampton, J. P.; Dowell-Mesfin, N.; Turner, J. N.; Shain, W. J. Neurosci. Methods 2007, 162, 255– 263
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      14
      Directed cell growth on protein-functionalized hydrogel surfaces
      Hynd, Matthew R.; Frampton, John P.; Dowell-Mesfin, Natalie; Turner, James N.; Shain, William
      Journal of Neuroscience Methods (2007), 162 (1-2), 255-263CODEN: JNMEDT; ISSN:0165-0270. (Elsevier Ltd.)
      Biochem. surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymd. in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labeled proteins. Soft protein lithog. (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biol. assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphol. on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72 h in vitro, and remained viable on protein-stamped areas for more than 4 wk. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjvFaksrg%253D&md5=35701075e65d107cda23c5f253d38511
    15. 15
      Mazmanian, S. K.; Liu, G.; Hung, T. T.; Schneewind, O. Science 1999, 285, 760– 763
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      15
      Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall
      Mazmanian, Sarkis K.; Liu, Gwen; Ton-That, Hung; Schneewind, Olaf
      Science (Washington, D. C.) (1999), 285 (5428), 760-763CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Surface proteins of Gram-pos. bacteria are linked to the bacterial cell wall by a mechanism that involves cleavage of a conserved Leu-Pro-X-Thr-Gly (LPXTG) motif and that occurs during assembly of the peptidoglycan cell wall. A Staphylococcus aureus mutant defective in the anchoring of surface proteins was isolated and shown to carry a mutation in the srtA gene. Overexpression of srtA increased the rate of surface protein anchoring, and homologs of srtA were found in other pathogenic Gram-pos. bacteria. The protein specified by srtA, sortase, may be a useful target for the development of new antimicrobial drugs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXltVehu7Y%253D&md5=cc26737979b307271ac69d6a630649eb
    16. 16
      Ton-That, H.; Mazmanian, S. K.; Faull, K. F.; Schneewind, O. J. Biol. Chem. 2000, 275, 9876– 9881
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      16
      Anchoring of surface proteins to the cell wall of Staphylococcus aureus.: Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH2-Gly3 substrates
      Ton-That, Hung; Mazmanian, Sarkis K.; Faull, Kym F.; Schneewind, Olaf
      Journal of Biological Chemistry (2000), 275 (13), 9876-9881CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. Purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH2-Gly3, sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH2-Gly3. In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXitlCqtr8%253D&md5=b438f87b461d02fb19e0021120a15b28
    17. 17
      Tsukiji, S.; Nagamune, T. ChemBioChem 2009, 10, 787– 798
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      17
      Sortase-mediated ligation: a gift from gram-positive bacteria to protein engineering
      Tsukiji, Shinya; Nagamune, Teruyuki
      ChemBioChem (2009), 10 (5), 787-798CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review on sortase-mediated ligation and current state of its applications. Topics discussed include: recombinant sol. sortase; transpeptidation mechanism; substrate specificity; introduction of new functionalities into proteins; covalent immobilization of proteins onto solid supports; protein-protein bioconjugation; protein circularization; and self-cleavable tag for one-step purifn. of recombinant proteins. It also tackles the prepn. of oligopeptide-nucleic acid hybrids; chemoenzymic synthesis of neoglycoconjugates; and cell-surface protein labeling/engineering.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXktVajt7o%253D&md5=121bdaba8de4db865d821683b7d968ff
    18. 18
      Ritzefeld, M. Chem.—Eur. J. 2014, 20, 8516– 8529
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      18
      Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy
      Ritzefeld, Markus
      Chemistry - A European Journal (2014), 20 (28), 8516-8529CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Bioorthogonal, chemoselective ligation methods are an essential part of the tools used to study biochem. pathways. Specifically enzymic approaches are valuable methods in this context due to the inherent specificity of the deployed enzymes and the mild conditions of the modification reactions. One of the most common strategies is based on the transpeptidation catalyzed by sortase A derived from Staphylococcus aureus. The procedure is well established and a wide variety of applications have been published to date. Here, implementations of sortase A, which range from protein labeling using fluorescence dyes and the prepn. of cyclic proteins to the modification of entire cells, are summarized. Furthermore, there is a focus on the optimization approaches established to solve the drawbacks of sortase-mediated transpeptidation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtVant7%252FK&md5=ef811f41468b796ba34df71822aa81a7
    19. 19
      Parthasarathy, R.; Subramanian, S.; Boder, E. T. Bioconjugate Chem. 2007, 18, 469– 476
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      19
      Sortase A as a Novel Molecular "Stapler" for Sequence-Specific Protein Conjugation
      Parthasarathy, Ranganath; Subramanian, Shyamsundar; Boder, Eric T.
      Bioconjugate Chemistry (2007), 18 (2), 469-476CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-pos. bacteria. Here, the authors describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chem. modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, the authors show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. The authors find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addn. to the protein immobilization and modification tool kit.
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    20. 20
      Chan, L.; Cross, H. F.; She, J. K.; Cavalli, G.; Martins, H. F. P.; Neylon, C. PLoS One 2007, 2, e1164
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      20
      Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation
      Chan Lilyan; Cross Hannah F; She Joseph K; Cavalli Gabriel; Martins Hugo F P; Neylon Cameron
      PloS one (2007), 2 (11), e1164 ISSN:.
      BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2snnslGjsg%253D%253D&md5=ac914b821bf0510d98644ae5a6c1143e
    21. 21
      Ito, T.; Sadamoto, R.; Naruchi, K.; Togame, H.; Takemoto, H.; Kondo, H.; Nishimura, S.-I. Biochemistry 2010, 49, 2604– 2614
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      21
      Highly Oriented Recombinant Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A
      Ito, Takaomi; Sadamoto, Reiko; Naruchi, Kentaro; Togame, Hiroko; Takemoto, Hiroshi; Kondo, Hirosato; Nishimura, Shin-Ichiro
      Biochemistry (2010), 49 (11), 2604-2614CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Recombinant glycosyltransferases are potential biocatalysts for the construction of a compd. library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chem. and enzymic synthetic procedures. The structurally defined glycan-related compd. library is a key resource both in the basic studies of their functional roles in various biol. processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile sepn. of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here, a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto com. available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A is described. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human β1,4-galactosyltranseferase or recombinant Helicobacter pylori α1,3-fucosyltransferase with simple aliph. amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the post-translational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of this strategy based on site-specific transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.
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    22. 22
      Matsumoto, T.; Tanaka, T.; Kondo, A. Langmuir 2012, 28, 3553– 3557
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      22
      Sortase A-Catalyzed Site-Specific Coimmobilization on Microparticles via Streptavidin
      Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko
      Langmuir (2012), 28 (7), 3553-3557CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      A microparticle surface was designed by the unique method incorporating streptavidin-biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG) was immobilized on the surface using streptavidin-biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin-FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-assocd. fluorescence was obsd. using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepd. using chem. modification. The authors' method provides a simple strategy for site-specific coimmobilization on mol. surfaces and expands the use of protein hybrid devices.
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    23. 23
      Sijbrandij, T.; Cukkemane, N.; Nazmi, K.; Veerman, E. C. I.; Bikker, F. J. Bioconjugate Chem. 2013, 24, 828– 831
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      23
      Sortase A as a Tool to Functionalize Surfaces
      Sijbrandij, Tjitske; Cukkemane, Nivedita; Nazmi, Kamran; Veerman, Enno C. I.; Bikker, Floris J.
      Bioconjugate Chemistry (2013), 24 (5), 828-831CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A widely accepted approach to combat surface fouling is based on the prevention of biofoulants to attach to a surface by the functionalization with poly(ethylene glycol) (PEG). The goal of this study was to generate a proof of concept for the enzymic coupling of PEG to a peptide precoated surface by using the enzyme Sortase A (SrtA). A hydrophobic polystyrene surface was primed with anchoring peptide P3 equipped with a pentaglycine acceptor motif for SrtA, to enable subsequent transpeptidation with either biotin or a PEG-tail contg. the sortase recognition motif LPETG. High levels of surface-bound biotin were detected only in cases with biotin-LPETG and SrtA. Little if any reactivity was detected in wells treated with the SrtA scrambled motif EGLTP, or in the absence of SrtA. Conjugation of PEG resulted in a significant decrease of bacterial adherence to the surface.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXksFKnu70%253D&md5=a39473b4daa3b0fb928bcc473a3b021b
    24. 24
      Sinisi, A.; Popp, M. W.-L.; Antos, J. M.; Pansegrau, W.; Savino, S.; Nissum, M.; Rappuoli, R.; Ploegh, H. L.; Buti, L. Bioconjugate Chem. 2012, 23, 1119– 1126
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      24
      Development of an influenza virus protein array using Sortagging technology
      Sinisi, Antonia; Popp, Maximilian Wei-Lin; Antos, John M.; Pansegrau, Werner; Savino, Silvana; Nissum, Mikkel; Rappuoli, Rino; Ploegh, Hidde L.; Buti, Ludovico
      Bioconjugate Chemistry (2012), 23 (6), 1119-1126CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Protein array technol. is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during a bacterial or viral infection. The authors developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as Sortagging. LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular exts. were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purifn. steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technol. has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes to develop protein arrays for high-throughput screening.
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    25. 25
      Jiang, R.; Weingart, J.; Zhang, H.; Ma, Y.; Sun, X.-L. Bioconjugate Chem. 2012, 23, 643– 649
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      25
      End-Point Immobilization of Recombinant Thrombomodulin via Sortase-Mediated Ligation
      Jiang, Rui; Weingart, Jacob; Zhang, Hailong; Ma, Yong; Sun, Xue-Long
      Bioconjugate Chemistry (2012), 23 (3), 643-649CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      We report an enzymic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM456) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM456 deriv. was site-specifically modified with N-terminal diglycine contg. mols. such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM456 was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM456 was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM456 was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was obsd. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chem. modification of the target protein. This site-specific covalent modification leads to mols. being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biol. activity.
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    26. 26
      Clow, F.; Fraser, J. D.; Proft, T. Biotechnol. Lett. 2008, 30, 1603– 1607
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      26
      Immobilization of proteins to biacore sensor chips using Staphylococcus aureus sortase A
      Clow, Fiona; Fraser, John D.; Proft, Thomas
      Biotechnology Letters (2008), 30 (9), 1603-1607CODEN: BILED3; ISSN:0141-5492. (Springer)
      The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purifn. protocols.
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    27. 27
      Leung, M. K. M.; Hagemeyer, C. E.; Johnston, A. P. R.; Gonzales, C.; Kamphuis, M. M. J.; Ardipradja, K.; Such, G. K.; Peter, K.; Caruso, F. Angew. Chem., Int. Ed. 2012, 51, 7132– 7136
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      27
      Bio-Click Chemistry: Enzymatic Functionalization of PEGylated Capsules for Targeting Applications
      Leung, Melissa K. M.; Hagemeyer, Christoph E.; Johnston, Angus P. R.; Gonzales, Catalina; Kamphuis, Marloes M. J.; Ardipradja, Katie; Such, Georgina K.; Peter, Karlheinz; Caruso, Frank
      Angewandte Chemie, International Edition (2012), 51 (29), 7132-7136, S7132/1-S7132/5CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The authors demonstrated an efficient method for the covalent and site-specific protein functionalization of polymer capsules by using Staphylococcus aureus enzyme Sortase A (Srt A). Owing to the precision of the ligation of genetically encoded substrates, this bioclick approach allows targeting ligands to be oriented with the antigen-binding sites available for binding, thus retaining their bioactivity. The GPIIb/IIIa-specific scFv-functionalized capsules showed a high level of specific targeting to thrombi, thus making them promising for anti-thrombotic and thrombolytic therapy. This SrtA mediated biofunctionalization technique is expected to find a range of applications in functionalizing materials for use in drug delivery as well as diagnostics and imaging.
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    28. 28
      Guo, X.; Wu, Z.; Guo, Z. Bioconjugate Chem. 2012, 23, 650– 655
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      28
      New Method for Site-Specific Modification of Liposomes with Proteins Using Sortase A-Mediated Transpeptidation
      Guo, Xueqing; Wu, Zhimeng; Guo, Zhongwu
      Bioconjugate Chemistry (2012), 23 (3), 650-655CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A new method was developed for site-specific modifications of liposomes by proteins via sortase A (SrtA)-mediated transpeptidation reactions. In this regard, the enhanced green fluorescent protein (eGFP) was biol. engineered to carry at its polypeptide C-terminus the LPATG motif recognized by SrtA and used as the protein donor for linking to liposomes that were decorated with phospholipids carrying a diglycine motif as the other SrtA substrate and the eGFP acceptor. Under the influence of SrtA, eGFP was efficiently attached to liposomes, as proved by analyzing the enzymic reaction products and the resultant fluorescent liposomes. Increasing the concn. and the distance of the diglycine motif on and from the liposome surface could significantly improve the efficiency of liposome modification by proteins. It is anticipated that this strategy can be widely useful for the modification of liposomes by other proteins.
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    29. 29
      Piluso, S.; Cassell, H. C.; Gibbons, J. L.; Waller, T. E.; Plant, N. J.; Miller, A. F.; Cavalli, G. Soft Matter 2013, 9, 6752– 6756
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      29
      Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool
      Piluso, Susanna; Cassell, Heather C.; Gibbons, Jonathan L.; Waller, Thomas E.; Plant, Nick J.; Miller, Aline F.; Cavalli, Gabriel
      Soft Matter (2013), 9 (29), 6752-6756CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheol. studies and TEM of the corresponding hydrogels (including PNIPAAm-contg. systems) showed no significant variation in properties and hydrogel morphol. compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.
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    30. 30
      Krueger, A. T.; Kroll, C.; Sanchez, E.; Griffith, L. G.; Imperiali, B. Angew. Chem., Int. Ed. 2014, 53, 2662– 2666
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      30
      Tailoring Chimeric Ligands for Studying and Biasing ErbB Receptor Family Interactions
      Krueger, Andrew T.; Kroll, Carsten; Sanchez, Edgar; Griffith, Linda G.; Imperiali, Barbara
      Angewandte Chemie, International Edition (2014), 53 (10), 2662-2666CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chem., which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was obsd. to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1ynu7k%253D&md5=77624f08fac56b88c4490abe5c2ae243
    31. 31
      Ehrbar, M.; Rizzi, S. C.; Hlushchuk, R.; Djonov, V.; Zisch, A. H.; Hubbell, J. A.; Weber, F. E.; Lutolf, M. P. Biomaterials 2007, 28, 3856– 3866
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      31
      Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering
      Ehrbar, Martin; Rizzi, Simone C.; Hlushchuk, Ruslan; Djonov, Valentin; Zisch, Andreas H.; Hubbell, Jeffrey A.; Weber, Franz E.; Lutolf, Matthias P.
      Biomaterials (2007), 28 (26), 3856-3866CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      The mol. engineering of cell-instructive artificial extracellular matrixes is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chem. and the biomol. characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quant. incorporated and released upon cell-derived proteolytic degrdn. of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochem. and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering.
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    32. 32
      Chen, I.; Dorr, B. M.; Liu, D. R. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11399– 11404
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      32
      A general strategy for the evolution of bond-forming enzymes using yeast display
      Chen, Irwin; Dorr, Brent M.; Liu, David R.
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (28), 11399-11404, S11399/1-S11399/29CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the mol. life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXptlartb4%253D&md5=5575b7d5bfc7962314b20eaf371737d8
    33. 33
      Swee, L. K.; Lourido, S.; Bell, G. W.; Ingram, J. R.; Ploegh, H. L. ACS Chem. Biol. 2015, 10, 460– 465
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      33
      One-Step Enzymatic Modification of the Cell Surface Redirects Cellular Cytotoxicity and Parasite Tropism
      Swee, Lee Kim; Lourido, Sebastian; Bell, George W.; Ingram, Jessica R.; Ploegh, Hidde L.
      ACS Chemical Biology (2015), 10 (2), 460-465CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. The authors demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. The authors successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymic approach enables engineering of the plasma membrane for research or therapy under physiol. reaction conditions that ensure the viability of the modified cells.
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    34. 34
      Osteen, K. G.; Hill, G. A.; Hargrove, J. T.; Gorstein, F. Fertil. Steril. 1989, 52, 965– 972
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      34
      Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens
      Osteen K G; Hill G A; Hargrove J T; Gorstein F
      Fertility and sterility (1989), 52 (6), 965-72 ISSN:0015-0282.
      Appropriate endometrial maturation is of paramount importance to achieve reproductive success. Practical and ethical considerations require that in vitro methods be available to evaluate regulation of human endometrial function. Additionally, tissue complexity requires separation of individual cell populations. This report describes an improved method for isolation of endometrial epithelial and stromal cells, using biopsy specimens as a tissue source. Separated cells were obtained using selective enzymatic digestion in conjunction with physical separation procedures. Isolated populations exhibited over 95% homogeneity, ascertained immunocytochemically. Using this system, isolated cells from normal endometrium can readily be obtained for in vitro studies. Within the defined conditions of a culture system, important areas of current concern in the endometrium such as ectopic endometrial growth and implantation can be addressed.
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    35. 35
      Huang, X.; Aulabaugh, A.; Ding, W.; Kapoor, B.; Alksne, L.; Tabei, K.; Ellestad, G. Biochemistry 2003, 42, 11307– 11315
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      35
      Kinetic mechanism of Staphylococcus aureus sortase SrtA
      Huang, Xinyi; Aulabaugh, Ann; Ding, Weidong; Kapoor, Bhupesh; Alksne, Lefa; Tabei, Keiko; Ellestad, George
      Biochemistry (2003), 42 (38), 11307-11315CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal contg. a LPXTG motif is cleaved between the Thr and Gly residues. The resulting Thr C-terminus of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-contg. peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. Here, the authors analyzed the steady-state kinetics of these 2 types of reactions catalyzed by sortase. The kinetic results fully supported a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction had a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. The authors also demonstrated that the nucleophile binding site of S. aureus sortase SrtA was specific for diglycine. Whereas the S1' and S2' sites of the enzyme both preferred a Gly residue, the S1' site was exclusively selective for Gly. The lengthening of the polyglycine acceptor nucleophile beyond diglycine did not further enhance the binding and catalysis.
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    36. 36
      Cruise, G. M.; Scharp, D. S.; Hubbell, J. A. Biomaterials 1998, 19, 1287– 1294
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      36
      Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels
      Cruise, Gregory M.; Scharp, David S.; Hubbell, Jeffrey A.
      Biomaterials (1998), 19 (14), 1287-1294CODEN: BIMADU; ISSN:0142-9612. (Elsevier Science Ltd.)
      Poly(ethylene glycol) (PEG) diacrylate hydrogels are being explored as permselective membranes for the transplantation of islets of Langerhans. Hydrogel membranes formed by interfacially photopolymg. PEG diacrylate precursor soln. were prepd. from PEG diacrylate of mol. wts. (MW) ranging from 2000 (2K) to 20,000 (20K) with concns. 10-30% wt./wt. The effects of PEG diacrylate MW and concn. in the membrane precursor soln. upon the diffusivities of vitamin B12, myoglobin, ovalbumin, albumin, and IgG were detd. Regardless of the concn. of the PEG diacrylate in the precursor soln., hydrogels prepd. with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepd. with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepd. with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K and 8K diacrylates were also seen in calcns. of the mol. wt. between crosslinks and the mesh size, with values in the range of 150-170 g/mol and 15-35°A, resp., depending on PEG diacrylate concn. In contrast, hydrogels formed from PEG 20K diacrylate had mol. wt. between crosslinks 1150-2000 g/mol and mesh sizes 45-70°A, with larger values being obsd. in membranes polymd. from more dil. PEG diacrylate precursor. The results indicate that PEG diacrylate hydrogels can prevent the diffusion of albumin and the components specific to an immune response (such as IgG, IgM, and C1q) while maintaining the rapid permeation of insulin and glucose.
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    37. 37
      S, J. J.; Griffith, L. G. Macromolecules 1997, 30, 5255– 5264
      [ ACS Full Text ACS Full Text], Google Scholar
      There is no corresponding record for this reference.
    38. 38
      Zustiak, S. P.; Boukari, H.; Leach, J. B. Soft Matter 2010, 6, 3609– 3618
      [ Crossref], [ CAS], Google Scholar
      38
      Solute diffusion and interactions in cross-linked poly(ethylene glycol) hydrogels studied by Fluorescence Correlation Spectroscopy
      Zustiak, Silviya P.; Boukari, Hacene; Leach, Jennie B.
      Soft Matter (2010), 6 (15), 3609-3618CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      Controlled diffusion and release of sol. mols. is one of the key challenges in developing 3-dimensional (3D) scaffolds for tissue engineering and drug delivery applications in part because current methods to measure dynamic transport properties are difficult to perform directly, are strongly affected by the exptl. setup, and therefore can be a subject to various artifacts. In this work the authors present a method for direct measurement of translational diffusion of solutes, namely Fluorescence Correlation Spectroscopy (FCS), by characterizing the diffusion of model proteins through a 3D cross-linked poly(ethylene glycol) (PEG) hydrogel scaffold. The authors examd. both the dynamics of hydrogel structure (e.g., crosslinking and swelling) as well as protein size and their effect on protein diffusivity. For example, the authors demonstrated that protein diffusivity was closely related to protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., γ-globulin or Ig). The authors validated the FCS protein diffusivity results by comparison to std. bulk diffusion assays. Addnl., due to the nature of FCS measurements, the authors were able to probe for hydrogel-protein interactions during crosslinking that may contribute to the obstructed protein diffusion in the 3D scaffold. The authors detd. that such interactions in this system were not covalent (i.e., were independent of the crosslinking chem.) but may be due to weaker hydrogen bonding or ionic interactions. Also, these interactions were protein specific and contributed up to 25% of the total decrease in protein diffusivity in the hydrogel as compared to diffusivity in water. Though interactions between various proteins and PEG were reported, this is the first study that has explored these effects in detail in cross-linked PEG hydrogels using FCS; the authors' findings question the assumption that PEG hydrogels are completely inert to protein interactions when applied as drug delivery matrixes and tissue engineering scaffolds.
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    39. 39
      Singh, B.; Coffey, R. J. Annu. Rev. Physiol. 2014, 76, 275– 300
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      39
      Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells
      Singh, Bhuminder; Coffey, Robert J.
      Annual Review of Physiology (2014), 76 (), 275-300CODEN: ARPHAD; ISSN:0066-4278. (Annual Reviews)
      A review. A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochem. and functionally sep. apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a crit. regulator of epithelial homeostasis and is perturbed in a no. of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the sol. form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biol. consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of sol. EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiol., and demonstrate the pathophysiol. consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes.
      >> More from SciFinder ®
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    40. 40
      Kuhl, P. R.; Griffith-Cima, L. G. Nat. Med. 1996, 2, 1022– 1027
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      40
      Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase
      Kuhl, Philip R.; Griffith-Cima, Linda G.
      Nature Medicine (New York) (1996), 2 (9), 1022-1027CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Co.)
      We have tethered epidermal growth factor (EGF) to a solid substrate in a manner permitting the factor to retain its biol. activity as assessed by both mitogenic and morphol. assays. Mouse EGF was covalently coupled to aminosilane-modified glass via star poly(ethylene oxide) (PEO), which allows the ligand to retain significant mobility and active conformation. Tethered EGF was as effective as sol. EGF in eliciting DNA synthesis and cell rounding responses of primary rat hepatocytes under different surface conditions. In contrast, phys. adsorbed EGF at comparable surface concns. showed no activity. Presentation of growth factors in this manner may help to expedite their clin. use by permitting greater control of temporal and spatial availability in the extracellular environment.
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    41. 41
      Mehta, G.; Williams, C. M.; Alvarez, L.; Lesniewski, M.; Kamm, R. D.; Griffith, L. G. Biomaterials 2010, 31, 4657– 4671
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      41
      Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels
      Mehta, Geeta; Williams, Courtney M.; Alvarez, Luis; Lesniewski, Martha; Kamm, Roger D.; Griffith, Linda G.
      Biomaterials (2010), 31 (17), 4657-4671CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)
      The compn., presentation, and spatial orientation of extracellular matrix mols. and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of sol. EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between sol. and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrixes reveal new facets of hepatocellular biol. in culture and may be more broadly useful in culture of other soft tissues.
      >> More from SciFinder ®
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    42. 42
      Williams, C. M.; Mehta, G.; Peyton, S. R.; Zeiger, A. S.; Van Vliet, K. J.; Griffith, L. G. Tissue Eng., Part A 2011, 17, 1055– 1068
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      42
      Autocrine-Controlled Formation and Function of Tissue-Like Aggregates by Primary Hepatocytes in Micropatterned Hydrogel Arrays
      Williams, Courtney M.; Mehta, Geeta; Peyton, Shelly R.; Zeiger, Adam S.; Van Vliet, Krystyn J.; Griffith, Linda G.
      Tissue Engineering, Part A (2011), 17 (7 and 8), 1055-1068CODEN: TEPAB9; ISSN:1937-3341. (Mary Ann Liebert, Inc.)
      The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concns. of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mech. compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5β1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsFOgtrc%253D&md5=661a3dd2a88f900756785452195ef534
    43. 43
      Chegini, N.; Rossi, M. J.; Masterson, B. J. Endocrinology 1992, 130, 2373– 2385
      [ PubMed], [ CAS], Google Scholar
      43
      Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF β-receptors in human endometrial tissue: localization and in vitro action
      Chegini, Nasser; Rossi, Michael J.; Masterson, Byron J.
      Endocrinology (1992), 130 (4), 2373-85CODEN: ENDOAO; ISSN:0013-7227.
      Human endometrial tissue and primary stromal cell culture contain immunoreactive EGF, platelet-derived growth factor (PDGF)-AB as well as EGF receptor, and PDGF β-receptors. The immunostaining for EGF, EGF receptor, and PDGF β-receptor was assocd. with endometrial luminal and glandular epithelial and stromal cells, whereas only the stromal cells contained immunoreactive PDGF-AB. The immunostaining intensity of EGF, EGF receptor, and PDGF-AB was similar in both phases of the menstrual cycle, whereas PDGF β-receptor immunostaining was highest in the proliferative phase and was considerably reduced, particularly in luminal and glandular epithelial cells in the secretory phase. In addn., primary stromal cell cultures express EGF, PDGF-AB, and contain EGF and PDGF-β receptors, and very low levels of PDGF-α receptor. [3H]thymidine incorporation indicated that after 48 h of incubation in serum-free medium approx. 75-80% of stromal cells are quiescent. Incubation of quiescent stromal cells with 10% fetal bovine serum stimulated [3H]thymidine incorporation in a time-dependent manner reaching maximal level after 30-48 h, with a doubling time of 38.2 h. EGF (1.5-15 ng/mL) stimulates [3H]thymidine incorporation by quiescent stromal cells. This effect was reduced at concns. >15 ng/mL. PDGF-AB (3-10 ng/mL) and PDGF-BB (0.5-10 ng/mL) also stimulate [3H]thymidine incorporation in quiescent stromal cells compared to controls. The action of EGF (15 ng/mL) and PDGF-AB (10 ng/mL) was time dependent, reaching maximal after 36 and 48 h of incubation. Addn. of PDGF-AB (10 ng/mL) to EGF (15 ng/mL) enhanced the action of EGF or PDGF-AB used individually. 17β-Estradiol or progesterone at 1μM did not stimulate [3H]thymidine incorporation; although they were stimulatory in combination; they did not alter the action of EGF or PDGF when added in combination. These observations provide further evidence that human endometrial tissue contains specific immunoreactive EGF receptors. They also demonstrate the presence of immunoreactive EGF, PDGF-AB, and PDGF-β receptors in endometrial tissue as well as stromal cells in primary culture. Both EGF and PDGF are mitogenic for endometrial stromal cells, suggesting an autocrine/paracrine role in modulation of endometrial cell growth and differentiation.
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    44. 44
      Klenkler, B. J.; Dwivedi, D.; West-Mays, J. A.; Sheardown, H. J. Biomed. Mater. Res. 2010, 93, 1043– 1049
      [ CAS], Google Scholar
      44
      Corneal epithelial cell adhesion and growth on EGF-modified aminated PDMS
      Klenkler, Bettina J.; Dwivedi, Dhruva; West-Mays, Judith A.; Sheardown, Heather
      Journal of Biomedical Materials Research, Part A (2010), 93A (3), 1043-1049CODEN: JBMRCH; ISSN:1549-3296. (John Wiley & Sons, Inc.)
      Growth factor tethering has significant potential to mediate cellular responses in biomaterials and tissue engineering. We have previously demonstrated that epidermal growth factor (EGF) can be tethered to polydimethylsiloxane (PDMS) substrates and that these surfaces promoted interactions with human corneal epithelial cells in vitro. The goal of the current work was to better understand the specific effects of the tethered growth factor on the cells. The EGF was reacted with a homobifunctional N-hydroxysuccinimide (NHS) polyethylene glycol (PEG) deriv., and then bound to allyamine plasma-modified PDMS. Human corneal epithelial cells were seeded on the surfaces and cultured in serum-free medium for periods of up to 5 days. Cell growth was monitored and quantified by trypsinization and counting with a Coulter counter. Expression of matrix proteins and α6-integrins was assessed by immunostaining and confocal microscopy. A centrifugation assay was used to det. cell adhesion under an applied detachment force. Binding of EGF was found to significantly increase cell nos. and coverage across the surfaces at 5 days of culture in vitro. Immunofluorescence expts. indicate increased expression of fibronectin, laminin, and α6-integrins on the EGF-modified surfaces, and expression is localized at the cell-material interface as obsd. by confocal microscopy. In accordance with these results, the highest quantity of adherent cells is found on the EGF-modified substrates at 5 days of culture. The results provide initial evidence that binding of EGF may be used to improve the epithelialization of and the adhesion of the cells on a polymeric artificial cornea device. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.
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    45. 45
      Marcantonio, N. A.; Boehm, C. A.; Rozic, R. J.; Au, A.; Wells, A.; Muschler, G. F.; Griffith, L. G. Biomaterials 2009, 1– 10
      Google Scholar
      There is no corresponding record for this reference.
    46. 46
      Brown, A. C.; Rowe, J. A.; Barker, T. H. Tissue Eng., Part A 2011, 17, 139– 150
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      46
      Guiding Epithelial Cell Phenotypes with Engineered Integrin-Specific Recombinant Fibronectin Fragments
      Brown, Ashley C.; Rowe, Jessica A.; Barker, Thomas H.
      Tissue Engineering, Part A (2011), 17 (1-2), 139-150CODEN: TEPAB9; ISSN:1937-3341. (Mary Ann Liebert, Inc.)
      The extracellular matrix (ECM) provides important cues for directing cell phenotype. Cells interact with underlying ECM through cell-surface receptors known as integrins, which bind to specific sequences on their ligands. During tissue development, repair, and regeneration of epithelial tissues, cells must interact with an interstitial fibronectin (Fn)-rich matrix, which has been shown to direct a more migratory/repair phenotype, presumably through interaction with Fn's cell binding domain comprised of both synergy Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) sequences. We hypothesized that the Fn synergy site is crit. to the regulation of epithelial cell phenotype by directing integrin specificity. Epithelial cells were cultured on Fn fragments displaying stabilized synergy and RGD (FnIII9'10), or RGD alone (FnIII10) and cell phenotype analyzed by cytoskeleton changes, epithelial cell-cell contacts, changes in gene expression of epithelial and mesenchymal markers, and wound healing assay. Data indicate that epithelial cells engage RGD only with αv integrins and display a significant shift toward a mesenchymal phenotype due, in part, to enhanced transforming growth factor-β activation and/or signaling compared with cells on the synergy contg. FnIII9'10. These studies demonstrate the importance of synergy in regulating epithelial cell phenotype relevant to tissue engineering as well as the utility of engineered integrin-specific ECM fragments in guiding cell phenotype.
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    47. 47
      Kuhlman, W.; Taniguchi, I.; Griffith, L. G.; Mayes, A. M. Biomacromolecules 2007, 8, 3206– 3213
      [ ACS Full Text ACS Full Text], [ CAS], Google Scholar
      47
      Interplay Between PEO Tether Length and Ligand Spacing Governs Cell Spreading on RGD-Modified PMMA-g-PEO Comb Copolymers
      Kuhlman, William; Taniguchi, Ikuo; Griffith, Linda G.; Mayes, Anne M.
      Biomacromolecules (2007), 8 (10), 3206-3213CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      The effects of tether length on cell adhesion to poly(Me methacrylate)-graft-poly(ethylene oxide), PMMA-g-PEO, comb copolymer films functionalized with the adhesion peptide RGD were investigated. Copolymers having PEO tether lengths of 10 and 22 EO segments were synthesized and coupled with a synthetic peptide that contained both RGD and the synergy sequence PHSRN. Cell spreading assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. Fluorescence resonance energy transfer (FRET) measurements indicated a mean sepn. between integrin-bound peptides of 15.6±1.4 nm for combs with long (22-mer) tethers, compared with 17.5±1.3 nm for short (10-mer) tethers, on films of comparable peptide d. (∼2500 peptides/μm2). The results suggest that the added mobility afforded by the more extensible tethers encouraged the formation of focal adhesions by allowing cells to reorganize tethered peptides on the nanometer length scale. In addn., adhesion peptides were selectively coupled to 10-mer or 22-mer PEO tethers within a bimodal brush to investigate stratification effects on cell adhesion. Peptides bound by short tethers in a bed of long unsubstituted chains resulted in surfaces that resisted, rather than promoted, cell adhesion. By contrast, when long peptide tethers were employed with short unsubstituted chains, cell attachment and spreading were comparable to that found on a monomodal brush of long chains at equiv. peptide d.
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    48. 48
      Sand, T. E.; Christoffersen, T. J. Cell. Physiol. 1987, 131, 141– 148
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      48
      Temporal requirement for epidermal growth factor and insulin in the stimulation of hepatocyte DNA synthesis
      Sand, Tor Erik; Christoffersen, Thoralf
      Journal of Cellular Physiology (1987), 131 (2), 141-8CODEN: JCLLAX; ISSN:0021-9541.
      Primary monolayer cultures of adult rat hepatocytes were used to study the temporal interaction of EGF and insulin in their stimulation of DNA synthesis. The hepatocytes were cultured both under defined conditions and with serum. EGF and insulin interacted synergistically. The entry into S phase (G1 exit) followed 1st-order kinetics both in untreated and hormone-stimulated cells. Addn. of EGF and insulin at the time of plating did not alter the lag period before the DNA synthesis started (25-26 h), but the rate const. for the S phase entry increased 5-6-fold. Expts. where the time of hormone addn. was varied indicated that insulin exerted its strongest effect at the time of plating, whereas the cells became more responsive to EGF after being cultured for up to 40-50 h. The responsiveness to EGF at these later stages required an early exposure of the hepatocytes to insulin. When the administration of EGF to insulin-pretreated hepatocytes was postponed for 44 h after plating in serum-free medium, the cellular sensitivity was increased as compared with EGF treatment at 0 h (a 1-log shift of the dose-effect curve), the rate of S phase entry was more rapid, and the lag period for the onset of the EGF effect (i.e., shift of rate const.) was shortened (6-7 vs. 26 h).
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    49. 49
      Ismail, T.; Howl, J.; Wheatley, M.; McMaster, P.; Neuberger, J. M.; Strain, A. J. Hepatology 1991, 14, 1076– 1082
      [ Crossref], [ PubMed], [ CAS], Google Scholar
      49
      Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis
      Ismail, Tariq; Howl, John; Wheatley, Mark; McMaster, Paul; Neuberger, James M.; Strain, Alastair J.
      Hepatology (Philadelphia, PA, United States) (1991), 14 (6), 1076-82CODEN: HPTLD9; ISSN:0270-9139.
      Human hepatocytes isolated from normal liver tissue were maintained in culture for up to 5 days, and DNA synthesis was detd. Hydroxyurea reduced [3H]thymidine incorporation into DNA by 95%, indicating replicative DNA synthesis. As previously found with rat hepatocytes, EGF and transforming growth factor-α stimulated DNA synthesis at low nanomolar concns.; transforming growth factor-α was slightly more potent. Although the overall rate of thymidine incorporation was lower than that for rodent cells, human hepatocytes were sensitive to lower concns. of these growth factors, and the degree of stimulation was similar. Transforming growth factor-β inhibited DNA synthesis at low picomolar levels. By contrast (unlike rat hepatocytes), AVP failed to initiate or potentiate DNA synthesis in human cells. Thus, normal human hepatocytes can respond to low concns. of growth promoters or inhibitors previously shown to have activity on rat hepatocytes. These factors may then play a role in control of human liver growth. However, important species differences are apparent, highlighting the limitations of extrapolating from animal studies to humans.
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    Protocol for primary human epithelial cells isolation and purification; (S1) standard curve for conversion of hydrogel fluorescence arbitrary units to amount of LPRTG in pmol; (S2) photobleaching controls; (S3) percentage of retained hydrogel fluorescence as a function of LPRTG in hydrogel after sortase-mediated hydrogel cleavage; (S4) micrographs of hepatocyte DNA synthesis on tethered EGF hydrogels; (S5) micrographs of endometrial epithelial cell DNA synthesis on tethered EGF hydrogels. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00549.

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