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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
*E-mail: [email protected]
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.

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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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