Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coli
- Nico J. Claassens*Nico J. Claassens*E-mail: [email protected]. Tel.: 0031612880653.Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsMax Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, GermanyMore by Nico J. Claassens
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- Max Finger-BouMax Finger-BouLaboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsMore by Max Finger-Bou
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- Bart ScholtenBart ScholtenLaboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsMore by Bart Scholten
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- Frederieke MuisFrederieke MuisLaboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsMore by Frederieke Muis
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- Jonas J. de GrootJonas J. de GrootLaboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsMore by Jonas J. de Groot
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- Jan-Willem de GierJan-Willem de GierDepartment of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91, Stockholm, SwedenMore by Jan-Willem de Gier
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- Willem M. de VosWillem M. de VosLaboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsHuman Microbiome Research Program, Faculty of Medicine, University of Helsinki, Haartmaninkatu 3, FI-00014, Helsinki, FinlandMore by Willem M. de Vos
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- John van der OostJohn van der OostLaboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The NetherlandsMore by John van der Oost
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Abstract
Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.
The high-level heterologous production of membrane proteins in E. coli and other hosts has proven challenging, especially due to oversaturation of the membrane protein biogenesis machinery. (1) Common systems for recombinant protein production, such as those based on the strong T7 promoter, often lead to the jamming of chaperones and membrane translocation systems, consequently making it impossible to produce correctly folded membrane proteins at high levels. (1,2)
Several E. coli strains and expression systems have been developed to improve the production of especially bacterial membrane proteins. Commonly used systems include the E. coli Walker strains (C41(DE3),C43(DE3)), (3)E. coli BL21-AI, (4) and the more recently developed E. coli Lemo21(DE3). (5−7) These systems rely on downregulating the levels of T7 RNA polymerase (T7RNAP), consequently reducing expression rates to better accommodate translocation and folding of membrane proteins. (8) Particularly, E. coli Lemo21(DE3) has been constructed to fine-tune transcription through an indirect control of T7RNAP activity through l-rhamnose-inducible production of its inhibitor, T7-lysozyme (LysY). (5−7) The system has proven successful and has been recently streamlined into a one-plasmid system named pReX, (9) but it requires the properly timed addition of two different inducer compounds: l-rhamnose and the expensive IPTG (isopropyl β-d-1-thiogalactopyranoside). Additionally, to date, neither Lemo21(DE3) nor any other currently available system has successfully demonstrated long-term (>24 h) continuous production of membrane proteins in E. coli. Realizing inducer-free, stable production remains a major challenge, which is relevant for many synthetic biology applications. This includes, for example, the heterologous production of transporter membrane proteins in microbial cell factories to be used in (continuous) production processes, or signal-transduction membrane proteins in strains that need to function as a biosensor over a long time.
Another limitation in heterologous protein production relates to ribosome binding site (RBS) accessibility. (10−12) Several proteins, including membrane proteins, have been categorized as “difficult-to-produce” due to strong mRNA secondary structures in the 5′ untranslated region (5′-UTR) and in the start of the coding sequence (CDS), which impede the proper translation initiation at the RBS. (13,14) Some efforts aimed at resolving such structures rely on fusing well-expressed short peptide tags to the N-terminus of membrane proteins. (13,15,16) However, fusion peptides can affect protein stability, structure, and function. (13) Furthermore, translation initiation has been successfully improved by randomly mutating nucleotides around the start codon, (13,14,17) which enhanced the production of several “difficult-to-produce” membrane proteins, but this method requires a high-throughput screening or selection approach.
To overcome the limitations of state-of-the-art membrane protein production, we explored an alternative method for protein expression: the so-called bicistronic design elements (BCDs). (18) The system is based on a constitutive promoter and tuning by using two RBSs that are translationally coupled. The first RBS mediates strong translation initiation of a short leader peptide, while the second RBS, which is located within the leader peptide’s CDS and drives the translation of the protein of interest, has a tunable strength (Figure 1). It is hypothesized that the intrinsic helicase activity of the ribosomes translating the leader peptide can unwind potential secondary structures of the mRNA, thereby eliminating translation initiation problems near the second RBS and the start of the target CDS. (19)
Figure 1
Figure 1. Expression vector design and assembly. (a) The standard expression vector contains a medium-strength constitutive promoter, RBS1, which allows for strong translation initiation of a leader peptide, and a translationally coupled, variable RBS2, mediating translation initiation of the coding sequence (CDS) of the membrane protein of interest. (18) (b) Vectors are assembled with different BCD elements. First, the vector is amplified by PCR, subsequently it is digested by type IIS restriction enzymes. The latter allows for seamless assembly with a library of annealed oligo pairs (encoding the different BCD variants), which have overhangs complementary to the digested vector.
In this work, we employ a library of translational coupling elements to tune and optimize the constitutive production of several bacterial membrane proteins. A medium-strength, constitutive promoter (P14) and 22 variable-strength translational coupling elements (BCDs) were selected from the work of Mutalik et al.; (18) the BCD elements can be inserted seamlessly in the expression vector using a simple golden gate-based cloning method (20) (Figure 1).
We tested our system for the expression of four different membrane proteins: YidC, AraH, and two rhodopsins. YidC is a membrane-translocation chaperone in E. coli, which has been frequently used as a model for studying membrane protein production. (6,21,22) AraH is the integral membrane component of the E. coli arabinose ABC transporter, which is considered a “difficult-to-produce” protein because of its translation initiation limitations in a typical pET vector; (13,14) GR is a proton-pumping rhodopsin photosystem from the cyanobacterium Gloeobacter violaceus, and TR is a thermophilic rhodopsin from Thermus thermophilus.
For the two first proteins, YidC and AraH, GFP was fused to their C-terminus, rendering YidC-GFP and AraH-GFP. All the 22 BCD variants were cloned separately into expression vectors carrying YidC-GFP and AraH-GFP. We then estimated levels of membrane inserted YidC and AraH by measuring fluorescence levels. C-terminal GFP only folds properly and results in fluorescence when membrane proteins fused to the GFP are integrated into the membrane and do not end up in inclusion bodies, hence providing a quantitative approximation for membrane-embedded expression. (23,24) The expression of YidC-GFP and AraH-GFP by different BCD elements was ranked in order of the translation initiation strengths previously observed for each BCD element in the work by Mutalik et al., (18) in order to allow for a systematic analysis of their fluorescence (Figure 2a,e).
Figure 2
Figure 2. Production of YidC-GFP, AraH-GFP, and rhodopsins by BCD elements and comparison to state-of-the art systems. (a) Volumetric YidC-GFP production based on whole-cell fluorescence measurements and final growth yields for the BCD constructs, and a comparison to Lemo21(DE3)-based production at optimized (2 mM l-rhamnose) and nonoptimized (0 mM l-rhamnose) conditions. BCD variants are ordered in the X-axis based on previously reported translation initiation strength. (18) (b) Western blots performed with antihis-tag antibody (upper panels) to visualize both inclusion body and well-folded YidC-GFP-his, and anti-IbpB (6,21) (lower panels) to visualize inclusion body binding protein B. (c) Single-cell production of YidC-GFP analyzed by flow cytometry for several increasing strength BCD elements and optimized Lemo21(DE3). (d) Single-cell production of YidC-GFP by BCD19 and BCD2 in a 72 h stability experiment. (e) Volumetric AraH-GFP production based on whole-cell fluorescence measurements and final growth yields for the BCD constructs, and a comparison to pET-opt-AraH-GFP. (14) BCD variants are ordered in the X-axis based on previously reported variant strength. (18) (f) Western blots performed with antihis-tag antibody (upper panels) to visualize both AraH-GFP-his, and anti-IbpB (6,21) (lower panels) to visualize inclusion body binding protein. (g) Single-cell production of AraH-GFP analyzed by flow cytometry for several increasing strength BCD elements and pET-opt-AraH-GFP. (h) Single-cell production of AraH-GFP by BCD19 and BCD2 in a 72 h stability experiment. (i) Volumetric Gloeobacter Rhodopsin (GR) and (j) Thermophilic Rhodopsin (TR) production determined by spectroscopy, and pictures of red-pigmented pellets. All cultivations were performed in 10 mL of medium in 50 mL tubes, for YidC-AraH and AraH-GFP at 30 °C, for rhodopsins GR and TR at 37 °C, and for pET-opt-AraH-GFP in E. coli BL21(DE3) pLysS at 25 °C (as optimized for in original work). BCD-based production was measured after 22 h of cultivation, while Lemo21(DE3) and pET based production was measured after 22 h of induction. Whole-cell fluorescence or rhodopsin quantification data are based on at least three biological replicates. For 72 h stability experiments E. coli BL21(DE3) harboring BCD vectors were reinoculated 1:50 into fresh LB kanamycin medium every 24 h. Notation: RFU, relative fluorescence units; OD600, optimal density of 600 nm.
For both fusion proteins, the tested BCD variants resulted in a range of GFP fluorescence-signals, suggesting different levels of functional membrane protein production. In the case of YidC-GFP, a rough pattern was observed considering the correlation between the fluorescence and the expected translation initiation strength of the different BCD constructs. BCD elements up to BCD19 generally resulted in increased levels of production, whereas elements stronger than BCD19 mostly resulted in lower production levels (Figure 2a). Some of the strongest translation initiation variants resulted in negligible and/or highly irreproducible production levels. For example, in some replicate cultures the strong BCD2 gave high expression but in several other cultures expression was completely absent (ranging from no expression to 70 000 RFU/mL). When the BCD-based expression was compared to the optimized Lemo21(DE3)-based expression of YidC-GFP, the latter gave rise to an emerging nonproducing subpopulation of cells after 22 h, as indicated by flow cytometry analysis, whereas the production by the highest-producing BCD19-YidC-GFP remained homogeneous (Figure 2c). In agreement with this observation, Western blot analysis revealed that the formation of YidC-GFP in inclusion bodies was reduced for the BCD19 versus optimized Lemo21(DE3)-expression (Figure 2b). Moreover, the medium-strength BCD19 yielded approximately twice as much production per cell than E. coli Lemo21(DE3), which was previously proven to be a superior production system for YidC-GFP over other commonly used systems such as E. coli C41(DE3) and C43(DE3). (6)
In the case of AraH-GFP, a similar trend was observed, that is, increasing levels of fluorescence were measured up to BCD19. However, unlike for YidC-GFP, no large decrease or unstable AraH-GFP production was observed for stronger BCD elements, and the strongest BCD2 produced at similar high levels as BCD19 (Figure 2e). AraH-GFP production by BCD19 and BCD2 was compared to the production by a previously optimized pET vector (pET-opt-AraH-GFP), which was obtained by screening a large library of vectors with mutations around the start codon. (14) The volumetric production by both BCDs was found to be higher than that of pET-opt-AraH-GFP under the originally optimized conditions, mainly due to a higher biomass yield (Figure 2e, Figure S2). Production by the BCDs also resulted in less inclusion body binding protein than pET-based production, while a nonfolded protein could not be clearly detected in Western blot for any construct (Figure 2f). Additionally, production by pET-opt-AraH-GFP resulted in a small emerging nonproducing population of cells after 22 h of cultivation, as revealed by flow cytometry analysis, while production by the BCD elements was still fully homogeneous (Figure 2g). Notably, by screening a library of only 22 BCD variants, we were able to find clones for which the production was comparable (per cell) or even better (per volume) than that of the previously optimized pET-opt-AraH-GFP, which required the high-throughput screening of a large library of variants (1.6 × 104) through fluorescence-activated cell sorting (FACS). (14)
To further estimate the production stability of the medium-strength, high-producing BCD19 and the strongest translation initiation element BCD2, we assessed production per cell by flow cytometry during longer serial cultivation experiments up to 72 h. Remarkably, BCD19-YidC-GFP, and for AraH both BCD19 and BCD2, result in stable homogeneously producing populations, even after 72 h (Figure 2d,h, Figure S3). For BCD2-YidC-GFP, however, only 2 out of 4 precultures prepared for the stability experiment maintained their initial production level, despite the fact that the colonies used for initial inoculation were selected on the basis of high fluorescence. This demonstrated again an unstable production phenotype for the strong BCD2 with YidC, as observed we observed in earlier experiments. The two stably producing precultures were further inoculated for the long-term experiment in fresh medium, and their production decreased over the course of time (Figure 2d, Figure S3). The strong translation initiation of YidC-GFP driven by BCD2 seemed to stress the cells, favoring the emergence of nonexpressing cells in the population (Figure 2d), which may have been caused by suppressing mutations in plasmids or the genome (not further characterized).
The BCD system was further employed to optimize the production of bacterial rhodopsin proteins. These simple membrane-bound light-harvesting energy systems can be employed for light-driven cell-factories (25) or optogenetic regulation. (26) When properly folded in the cytoplasmic membrane, rhodopsins are known to bind the retinal pigment, leading to red pigmentation of the host cells. (27,28) Rather than cloning all the BCDs in parallel, this time the 22 BCD variants were pooled, cloned into the vectors containing GR or TR, and transformed as a library. Clones of the resulting transformation were then randomly picked and grown in 96-well plates with retinal (Figure S4). For both GR and TR, 11 clearly red-pigmented pellets were identified out of 89 and 96 screened clones, respectively. For each of the rhodopsin proteins, three intensely red clones were selected for further characterization. In the case of GR, one of the selected clones contained BCD14 and the other two carried BCD15. For TR, BCD9, BCD12, and BCD21 were identified. All these variants are in the medium-strength range of the BCD system.
Production of GR and TR by the best rhodopsin-producing BCD variants was scaled up from deep-well plates to 10 mL cultures in 50 mL tubes. While all elements rendered very similar levels of GR expression (Figure 2i), the production of TR significantly differed from one BCD to another; BCD9 performed significantly better than BCD12, while BCD21 gave the lowest production (Figure 2j). These three variants performed very similar when grown in deep-96-well plates, while the results of the production assays in 50 mL tubes are significantly different. This indicates that results are not always comparable when scaling-up from deep-well plates (0.5 mL culture) to test tubes (10 mL culture). The previously discussed strains of YidC-GFP and AraH-GFP were initially grown directly in 10 mL cultures in 50 mL tubes, and now compared with their performance in deep-well plates (0.5 mL culture) (Figure S5), confirming differences in performance depending on culture conditions. This reflects the common challenge of optimizing and scaling up recombinant production. However, the optimization for the BCD system in scaled-up conditions is quite feasible given the limited number of tuning variants that need to be tested.
We then compared the production of GR and TR to the levels produced by the Lemo21(DE3) system, which was optimized in tubes. Compared to rhodopsin production by the Lemo21(DE3) system, the best performing BCD variants for GR and TR resulted in at least 2-fold and 3-fold higher volumetric rhodopsin production, respectively (Figure 2i,j).
The here employed BCD system outperforms the membrane protein productivities of previously established approaches. Moreover, the BCD system provides stable membrane protein production for at least 72 h, a stability never reported to date. While most current systems for membrane protein production are limited to specific E. coli strains, the applied P14 constitutive promoter allows our system to be generally applicable in all E. coli strains. By applying the principles of our method and potentially including different promoters and/or BCDs, our system is likely feasible for bacterial membrane protein production in other bacterial hosts as well. The BCD system may also be applicable for producing certain eukaryotic membrane proteins in E. coli, although expression of eukaryotic proteins in bacterial hosts may lead to issues that cannot be solved just by tuning expression strength and tackling RBS-accessibility, this includes issues as glycosylation or the requirement of eukaryotic-like membrane lipids. (1) Overall, it is anticipated that the here described approach for bacterial membrane protein production will be useful for many future studies, ranging from biochemical characterization to cell factory and biosensor engineering.
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00101.
Methods; Supplementary figures; Codon-adapted gene sequences; Supplementary references (PDF)
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Author Information
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- Nico J. Claassens - Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands; Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany;
http://orcid.org/0000-0003-1593-0377;
- Max Finger-Bou - Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands;http://orcid.org/0000-0002-9164-7082
- Jan-Willem de Gier - Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91, Stockholm, Sweden;http://orcid.org/0000-0001-5537-4358
Acknowledgments
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We thank Wendy van der Vliet and Snezana Gegic for their kind assistance with the flow cytometry experiments. We thank Daniel Daley for sharing pGFPe-AraH-his. All BIOFAB plasmids were a kind gift of Drew Endy. We gratefully acknowledge financial support from the Wageningen University IP/OP Systems Biology (Grant KB-17-003.02-024) and The Netherlands Organization for Scientific Research (NWO) via the Spinoza and SIAM Gravitation Grant (Project 024.002.002) to W.M.d.V., and a Rubicon Grant to N.C. (019.163LW.035), M.F.B and J.v.d.O are supported by the NWO Gravitation Grant BaSyC (024.003.019). The funders had no role in study design, data collection and analysis, or preparation of the manuscript.
References
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This article references 28 other publications.
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- 3Miroux, B. and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289– 98, DOI: 10.1006/jmbi.1996.0399[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XksVygtLs%253D&md5=6377b33b2d3bd0eec0047038253b481dOver-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levelsMiroux, Bruno; Walker, John E.Journal of Molecular Biology (1996), 260 (3), 289-298CODEN: JMOBAK; ISSN:0022-2836. (Academic)We have investigated the over-prodn. of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expressing of the target membrane protein was induced, most of the BL21 (DE3) host cells died. Similar effects were also obsd. with expression vectors for ten globular proteins. Therefore, protein over-prodn. in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high satn. cell d., and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior t BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells contg. the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H+ symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit B was lower in C41(DE3) and C43(DE3), resp., than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts to overcome other toxic effects assocd. with over-expression.
- 4Narayanan, A., Ridilla, M., and Yernool, D. A. (2011) Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator – repressor interactions. Protein Sci. 20, 51– 61, DOI: 10.1002/pro.535[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVKjsLY%253D&md5=449847f3c90bea7240ba20c7ea075611Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactionsNarayanan, Anoop; Ridilla, Marc; Yernool, Dinesh A.Protein Science (2011), 20 (1), 51-61CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)A major rate-limiting step in detg. structures of membrane proteins is heterologous protein prodn. Toxicity often assocd. with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled redn. in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer iso-Pr β-D-1-thiogalactopyranoside. In addn., prodn. of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI. Using this approach, we can achieve a 200-fold range of green fluorescent protein expression levels. Application to members of a family of ion pumps results in 5- to 25-fold increases in expression over the benchmark BL21(DE3) host strain. A viral ion channel highly toxic to E. coli can also be overexpressed. In comparative analyses, restrained expression outperforms commonly used E. coli expression strategies. The mechanism underlying improved target protein yield arises from minimization of protein aggregation and proteolysis that reduce membrane integrity and cell viability. This study establishes a method to overexpress toxic proteins.
- 5Schlegel, S., Löfblom, J., Lee, C., Hjelm, A., Klepsch, M., Strous, M., Drew, D., Slotboom, D. J., and De Gier, J.-W. (2012) Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3). J. Mol. Biol. 423, 648– 659, DOI: 10.1016/j.jmb.2012.07.019[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1emsb3M&md5=6d7f1771bfb51b805365be782f82861aOptimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)Schlegel, Susan; Loefblom, John; Lee, Chiara; Hjelm, Anna; Klepsch, Mirjam; Strous, Marc; Drew, David; Slotboom, Dirk Jan; de Gier, Jan-WillemJournal of Molecular Biology (2012), 423 (4), 648-659CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concns. of rhamnose indicated that this strain may be very suitable for optimizing the prodn. of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by redn. of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.
- 6Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Hogbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O., and de Gier, J.-W. (2008) Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. U. S. A. 105, 14371– 14376, DOI: 10.1073/pnas.0804090105[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1SgtrnI&md5=104e412bd6933a8085ed11ebfac7edbbTuning Escherichia coli for membrane protein overexpressionWagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Hogbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-WillemProceedings of the National Academy of Sciences of the United States of America (2008), 105 (38), 14371-14376,S14371/1-S14371/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiol. response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiol., proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a deriv. strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
- 7Hjelm, A., Schlegel, S., Baumgarten, T., Klepsch, M., Wickström, D., Drew, D., and De Gier, J.-W. (2013) Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions. Methods Mol. Biol. 1033, 381– 400, DOI: 10.1007/978-1-62703-487-6_24[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVais7%252FK&md5=0cba38627ec36897ded0a1aebcfdf74bOptimizing E. coli-based membrane protein production using Lemo21 (DE3) and GFP-fusionsHjelm, Anna; Schlegel, Susan; Baumgarten, Thomas; Klepsch, Mirjam; Wickstroem, David; Drew, David; de Gier, Jan-WillemMethods in Molecular Biology (New York, NY, United States) (2013), 1033 (Membrane Biogenesis), 381-400CODEN: MMBIED; ISSN:1064-3745. (Springer)Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purifn. is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the prodn. of high-quality membrane protein material for functional and structural studies.
- 8Schlegel, S., Genevaux, P., and de Gier, J.-W. (2015) De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields. Cell Rep. 10, 1758– 1766, DOI: 10.1016/j.celrep.2015.02.029[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVaisbs%253D&md5=2a8cb01f343a9a0d6aeeca4f9bd12677De-convoluting the genetic adaptations of E. coli C41(DE3) in real time reveals how alleviating protein production stress improves yieldsSchlegel, Susan; Genevaux, Pierre; de Gier, Jan-WillemCell Reports (2015), 10 (10), 1758-1766CODEN: CREED8; ISSN:2211-1247. (Cell Press)The well-established E. coli protein prodn. strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it acquired multiple mutations during its isolation. Std. allelic replacement and competition expts. were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein prodn. stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein prodn. stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the prodn. of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein prodn. stress and can be harnessed to enhance prodn.
- 9Kuipers, G., Karyolaimos, A., Zhang, Z., Ismail, N., Trinco, G., Vikström, D., Slotboom, D. J., and de Gier, J.-W. (2017) The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli. Microb. Cell Fact. 16, 226, DOI: 10.1186/s12934-017-0840-4[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXis1Knurw%253D&md5=a8cd3f8bd9046b1f047fea8fe400c432The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coliKuipers, Grietje; Karyolaimos, Alexandros; Zhang, Zhe; Ismail, Nurzian; Trinco, Gianluca; Vikstroem, David; Slotboom, Dirk Jan; de Gier, Jan-WillemMicrobial Cell Factories (2017), 16 (), 226/1-226/11CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)Background: To optimize the prodn. of membrane and secretory proteins in Escherichia coli, it is crit. to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein prodn. strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-prodn. of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a std. T7-based expression vector. Results: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a std. T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized prodn. yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a std. T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the prodn. of this heme c protein. Conclusions: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the prodn. of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).
- 10Kudla, G., Murray, A. W., Tollervey, D., and Plotkin, J. B. (2009) Coding-Sequence Determinants of Gene Expression in Escherichia coli. Science (Washington, DC, U. S.) 324, 255– 258, DOI: 10.1126/science.1170160[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXktFals7o%253D&md5=7fc7f47cb747fcee5585c775bb20bda9Coding-Sequence Determinants of Gene Expression in Escherichia coliKudla, Grzegorz; Murray, Andrew W.; Tollervey, David; Plotkin, Joshua B.Science (Washington, DC, United States) (2009), 324 (5924), 255-258CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP mRNA levels, mRNA degrdn. patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our anal., mRNA folding and assocd. rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.
- 11Cambray, G., Guimaraes, J. C., and Arkin, A. P. (2018) Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coli. Nat. Biotechnol. 36, 1005– 1015, DOI: 10.1038/nbt.4238[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslOhtbnM&md5=891f6e737ef0b4e66ee975a42489e5e4Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coliCambray, Guillaume; Guimaraes, Joao C.; Arkin, Adam PaulNature Biotechnology (2018), 36 (10), 1005-1015CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Comparative analyses of natural and mutated sequences have been used to probe mechanisms of gene expression, but small sample sizes may produce biased outcomes. We applied an unbiased design-of-expts. approach to disentangle factors suspected to affect translation efficiency in E. coli. We precisely designed 244,000 DNA sequences implementing 56 replicates of a full factorial design to evaluate nucleotide, secondary structure, codon and amino acid properties in combination. For each sequence, we measured reporter transcript abundance and decay, polysome profiles, protein prodn. and growth rates. Assocns. between designed sequences properties and these consequent phenotypes were dominated by secondary structures and their interactions within transcripts. We confirmed that transcript structure generally limits translation initiation and demonstrated its physiol. cost using an epigenetic assay. Codon compn. has a sizable impact on translatability, but only in comparatively rare elongation-limited transcripts. We propose a set of design principles to improve translation efficiency that would benefit from more accurate prediction of secondary structures in vivo.
- 12Nieuwkoop, T., Claassens, N. J., and van der Oost, J. (2019) Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design. Microb. Biotechnol. 12, 173– 179, DOI: 10.1111/1751-7915.13332[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1SrurbK&md5=584cd5afade3b129645226334b54f1b5Improved protein production and codon optimization analyses in Escherichia coli by bicistronic designNieuwkoop, Thijs; Claassens, Nico J.; van der Oost, JohnMicrobial Biotechnology (2019), 12 (1), 173-179CODEN: MBIIB2; ISSN:1751-7915. (Wiley-Blackwell)Different codon optimization algorithms are available that aim at improving protein prodn. by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5'-UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, esp. near the start of a gene, may strongly influence translation initiation. In this study, we aim to reduce the effect of codon usage on translation initiation by applying a bicistronic design (BCD) element. Protein prodn. of several codon-optimized gene variants is tested in parallel for a BCD and a std. monocistronic design (MCD). We demonstrate that these distinct architectures can drastically change the relative performance of different codon optimization algorithms. We conclude that a BCD is indispensable in future studies that aim to reveal the impact of codon optimization and codon usage correlations. Furthermore, irresp. of the algorithm used, using a BCD does improve protein prodn. compared with an MCD. The overall highest expression from BCDs for both GFP and RFP is at least twofold higher than the highest levels found for the MCDs, while for codon variants having very low expression from the MCD, even 10-fold to 100-fold increases in expression were achieved by the BCD. This shows the great potential of the BCD element for recombinant protein prodn.
- 13Nørholm, M. H. H., Toddo, S., Virkki, M. T. I., Light, S., von Heijne, G., and Daley, D. O. (2013) Improved production of membrane proteins in Escherichia coli by selective codon substitutions. FEBS Lett. 587, 2352– 8, DOI: 10.1016/j.febslet.2013.05.063[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3sjivV2rsQ%253D%253D&md5=13b32139bef8eb04dda21b0df9327647Improved production of membrane proteins in Escherichia coli by selective codon substitutionsNorholm Morten H H; Toddo Stephen; Virkki Minttu T I; Light Sara; von Heijne Gunnar; Daley Daniel OFEBS letters (2013), 587 (15), 2352-8 ISSN:.Membrane proteins are extremely challenging to produce in sufficient quantities for biochemical and structural analysis and there is a growing demand for solutions to this problem. In this study we attempted to improve expression of two difficult-to-express coding sequences (araH and narK) for membrane transporters. For both coding sequences, synonymous codon substitutions in the region adjacent to the AUG start led to significant improvements in expression, whereas multi-parameter sequence optimization of codons throughout the coding sequence failed. We conclude that coding sequences can be re-wired for high-level protein expression by selective engineering of the 5' coding sequence with synonymous codons, thus circumventing the need to consider whole sequence optimization.
- 14Mirzadeh, K., Martinez, V., Toddo, S., Guntur, S., Herrgard, M., Elofsson, A., Nørholm, M. H. H., and Daley, D. O. (2015) Enhanced protein production in Escherichia coli by optimization of cloning scars at the vector:coding sequence junction. ACS Synth. Biol. 4, 959– 965, DOI: 10.1021/acssynbio.5b00033[ACS Full Text
], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnvFaku7w%253D&md5=39320d5e716303be59ae804db233801fEnhanced protein production in Escherichia coli by optimization of cloning scars at the vector-coding sequence junctionMirzadeh, Kiavash; Martinez, Virginia; Toddo, Stephen; Guntur, Suchithra; Herrgaard, Markus J.; Elofsson, Arne; Noerholm, Morten H. H.; Daley, Daniel O.ACS Synthetic Biology (2015), 4 (9), 959-965CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Protein prodn. in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research labs. Maximum prodn. is usually sought, as this reduces costs and facilitates downstream purifn. steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to 1000-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (ΔG) in this region were important, but not the only, determinants for high expression. - 15Kim, H. S., Ernst, J. a, Brown, C., Bostrom, J., Fuh, G., Lee, C. V., Huang, A., Vandlen, R. L., and Yansura, D. G. (2012) Translation levels control multi-spanning membrane protein expression. PLoS One 7, e35844, DOI: 10.1371/journal.pone.0035844[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntFWks74%253D&md5=6fd28efcd597e7e0b74a1b373c3ac9a5Translation levels control multi-spanning membrane protein expressionKim, Hok Seon; Ernst, James A.; Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.PLoS One (2012), 7 (4), e35844CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the crit. role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane.
- 16Vazquez-Albacete, D., Cavaleiro, A. M., Christensen, U., Seppälä, S., Møller, B. L., and Nørholm, M. H. H. (2017) An expression tag toolbox for microbial production of membrane bound plant cytochromes P450. Biotechnol. Bioeng. 114, 751– 760, DOI: 10.1002/bit.26203[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFelurzF&md5=bd92a78fbdb5cf518e3641fe40504905An expression tag toolbox for microbial production of membrane bound plant cytochromes P450Vazquez-Albacete, Dario; Cavaleiro, Ana Mafalda; Christensen, Ulla; Seppaelae, Susanna; Moller, Birger Lindberg; Norholm, Morten H. H.Biotechnology and Bioengineering (2017), 114 (4), 751-760CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Membrane-assocd. Cytochromes P 450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here, we explore a small library of N-terminal expression tag chimeras of the model plant P 450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Anal. of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalyzed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than twofold. The developed toolbox serves as a platform to tune P 450 performance in microbial cells, thereby facilitating recombinant prodn. of complex plant P 450-derived biochems. Biotechnol. Bioeng. 2016;9999: 1-10. © 2016 Wiley Periodicals, Inc.
- 17Rennig, M., Martinez, V., Mirzadeh, K., Dunas, F., Röjsäter, B., Daley, D. O. O., and Nørholm, M. H. H. H. H. (2018) TARSyn: Tuneable antibiotic resistance devices enabling bacterial synthetic evolution and protein production. ACS Synth. Biol. 7, 432– 442, DOI: 10.1021/acssynbio.7b00200[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvF2mtLjK&md5=80912a1017505d532cc2ae68811e3a48TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein ProductionRennig, Maja; Martinez, Virginia; Mirzadeh, Kiavash; Dunas, Finn; Rojsater, Belinda; Daley, Daniel O.; Noerholm, Morten H. H.ACS Synthetic Biology (2018), 7 (2), 432-442CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Evolution can be harnessed to optimize synthetic biol. designs. A prominent example is recombinant protein prodn.-a dominating theme in biotechnol. for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory. A major bottleneck during prodn. is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concns. adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the resp. antibiotic. The power of the approach is demonstrated by substantially increasing prodn. of two com. interesting proteins, a Nanobody and an Affibody. The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any lab.
- 18Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., Endy, D., Hoynes-O’Connor, A., and Moon, T. S. (2013) Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354– 360, DOI: 10.1038/nmeth.2404[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjs1Chur0%253D&md5=bbaa07c9dfc127bd8e5d3e0a7748d2aaPrecise and reliable gene expression via standard transcription and translation initiation elementsMutalik, Vivek K.; Guimaraes, Joao C.; Cambray, Guillaume; Lam, Colin; Christoffersen, Marc Juul; Mai, Quynh-Anh; Tran, Andrew B.; Paull, Morgan; Keasling, Jay D.; Arkin, Adam P.; Endy, DrewNature Methods (2013), 10 (4), 354-360CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)An inability to reliably predict quant. behaviors for novel combinations of genetic elements limits the rational engineering of biol. systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1000-fold obsd. dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain std. biol. parts that support reliable forward engineering of gene expression at genome scales.
- 19Takyar, S., Hickerson, R. P., and Noller, H. F. (2005) mRNA helicase activity of the ribosome. Cell 120, 49– 58, DOI: 10.1016/j.cell.2004.11.042[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXot1ChtA%253D%253D&md5=339ad7c565648fa0eb6dadc67cf755f7mRNA helicase activity of the ribosomeTakyar, Seyedtaghi; Hickerson, Robyn P.; Noller, Harry F.Cell (Cambridge, MA, United States) (2005), 120 (1), 49-58CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro translation system made up of purified components, the authors show that ribosomes are able to disrupt downstream helixes, including a perfect 27 base pair helix of predicted Tm = 70°. Using helixes of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. The authors conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.
- 20Engler, C., Kandzia, R., and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647, DOI: 10.1371/journal.pone.0003647[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cjhvV2ltA%253D%253D&md5=8910183a67aa28a0cc69522dcf7050b0A one pot, one step, precision cloning method with high throughput capabilityEngler Carola; Kandzia Romy; Marillonnet SylvestrePloS one (2008), 3 (11), e3647 ISSN:.Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.
- 21Schlegel, S., Löfblom, J., Lee, C., Hjelm, A., Klepsch, M., Strous, M., Drew, D., Slotboom, D. J., and de Gier, J.-W. (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J. Mol. Biol. 423, 648– 59, DOI: 10.1016/j.jmb.2012.07.019[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1emsb3M&md5=6d7f1771bfb51b805365be782f82861aOptimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)Schlegel, Susan; Loefblom, John; Lee, Chiara; Hjelm, Anna; Klepsch, Mirjam; Strous, Marc; Drew, David; Slotboom, Dirk Jan; de Gier, Jan-WillemJournal of Molecular Biology (2012), 423 (4), 648-659CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concns. of rhamnose indicated that this strain may be very suitable for optimizing the prodn. of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by redn. of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.
- 22Gialama, D., Kostelidou, K., Michou, M., Delivoria, D. C., Kolisis, F. N., and Skretas, G. (2017) Development of Escherichia coli strains that withstand membrane protein-induced toxicity and achieve high-level recombinant membrane protein production. ACS Synth. Biol. 6, 284– 300, DOI: 10.1021/acssynbio.6b00174[ACS Full Text], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslGku7zP&md5=dfe2de5711b7290706db542765d0fd87Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein ProductionGialama, Dimitra; Kostelidou, Kalliopi; Michou, Myrsini; Delivoria, Dafni Chrysanthi; Kolisis, Fragiskos N.; Skretas, GeorgiosACS Synthetic Biology (2017), 6 (2), 284-300CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Membrane proteins perform crit. cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochem./structural studies. Bacterial prodn. of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein prodn. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E. coli RNase E, RraA. E. coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, resp., accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of com. strains frequently utilized for recombinant membrane protein prodn. purposes.
- 23Drew, D., Slotboom, D.-J., Friso, G., Reda, T., Genevaux, P., Rapp, M., Meindl-Beinker, N. M., Lambert, W., Lerch, M., Daley, D. O., Van Wijk, K.-J., Hirst, J., Kunji, E., and De Gier, J.-W. (2005) A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci. 14, 2011– 2017, DOI: 10.1110/ps.051466205[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXntVejtr0%253D&md5=1f8888e6ff671a6b47cc228f62783ea2A scalable, GFP-based pipeline for membrane protein overexpression screening and purificationDrew, David; Slotboom, Dirk-Jan; Friso, Giulia; Reda, Torsten; Genevaux, Pierre; Rapp, Mikaela; Meindl-Beinker, Nadja M.; Lambert, Wietske; Lerch, Mirjam; Daley, Daniel O.; Van Wijk, Klaas-Jan; Hirst, Judy; Kunji, Edmund; De Gier, Jan-WillemProtein Science (2005), 14 (8), 2011-2017CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)The authors describe a generic, GFP-based pipeline for membrane protein overexpression and purifn. in Escherichia coli. The authors exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important ref. for the characterization of other membrane proteomes.
- 24Drew, D. E, von Heijne, G., Nordlund, P., and de Gier, J.-W. L (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett. 507, 220– 224, DOI: 10.1016/S0014-5793(01)02980-5[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXnvFCrtrc%253D&md5=d13802658359626a0a2bd013dd5ee69bGreen fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coliDrew, David E.; von Heijne, Gunnar; Nordlund, Par; de Gier, Jan-Willem L.FEBS Letters (2001), 507 (2), 220-224CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)E. coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favored to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion vs. inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease. This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.
- 25Claassens, N. J., Volpers, M., Martins dos Santos, V. A. P., van der Oost, J., and de Vos, W. M. (2013) Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms. Trends Biotechnol. 31, 633– 642, DOI: 10.1016/j.tibtech.2013.08.006[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1SktrnF&md5=a3ed814f5ee698a8a5c0e956f03749e0Potential of proton-pumping rhodopsins: engineering photosystems into microorganismsClaassens, Nico J.; Volpers, Michael; Martins dos Santos, Vitor A. P.; van der Oost, John; de Vos, Willem M.Trends in Biotechnology (2013), 31 (11), 633-642CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)A review. A wide range of proton-pumping rhodopsins (PPRs) have been discovered in recent years. Using a synthetic biol. approach, PPR photosystems with different features can be easily introduced in nonphotosynthetic microbial hosts. PPRs can provide hosts with the ability to harvest light and drive the sustainable prodn. of biochems. or biofuels. PPRs use light energy to generate an outward proton flux, and the resulting proton motive force can subsequently power cellular processes. Recently, the introduction of PPRs in microbial prodn. hosts has successfully led to light-driven biotechnol. conversions. In this review, we discuss relevant features of natural PPRs, evaluate reported biotechnol. applications of microbial prodn. hosts equipped with PPRs, and provide an outlook on future developments.
- 26Deisseroth, K. (2015) Nat. Neurosci. 18, 1213– 1225, DOI: 10.1038/nn.4091[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVWmtb7M&md5=c2e3010ffa02a7a8cfc1ad434b160461Optogenetics: 10 years of microbial opsins in neuroscienceDeisseroth, KarlNature Neuroscience (2015), 18 (9), 1213-1225CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)A review with commentary. Over the past 10 years, the development and convergence of microbial opsin engineering, modular genetic methods for cell-type targeting and optical strategies for guiding light through tissue have enabled versatile optical control of defined cells in living systems, defining modern optogenetics. Despite widespread recognition of the importance of spatiotemporally precise causal control over cellular signaling, for nearly the first half (2005-2009) of this 10-yr period, as optogenetics was being created, there were difficulties in implementation, few publications and limited biol. findings. In contrast, the ensuing years have witnessed a substantial acceleration in the application domain, with the publication of thousands of discoveries and insights into the function of nervous systems and beyond. This Historical Commentary reflects on the scientific landscape of this decade-long transition.
- 27Gourdon, P., Alfredsson, A., Pedersen, A., Malmerberg, E., Nyblom, M., Widell, M., Berntsson, R., Pinhassi, J., Braiman, M., Hansson, Ö., Bonander, N., Karlsson, G., and Neutze, R. (2008) Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump. Protein Expression Purif. 58, 103– 113, DOI: 10.1016/j.pep.2007.10.017[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVygtbY%253D&md5=6635650433efc5c4c3e0246db2eb3debOptimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pumpGourdon, Pontus; Alfredsson, Anna; Pedersen, Anders; Malmerberg, Erik; Nyblom, Maria; Widell, Mikael; Berntsson, Ronnie; Pinhassi, Jarone; Braiman, Marc; Hansson, Oerjan; Bonander, Nicklas; Karlsson, Goeran; Neutze, RichardProtein Expression and Purification (2008), 58 (1), 103-113CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier B.V.)Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro prodn. of pR using a com. cell-free synthesis system yielding 1.0 mg purified protein per mL of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purifn. yielding 5 mg of essentially pure functional protein per L of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for com. biotechnol. applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural detn. of pR are discussed.
- 28Martinez, A., Bradley, A. S., Waldbauer, J. R., Summons, R. E., and DeLong, E. F. (2007) Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host. Proc. Natl. Acad. Sci. U. S. A. 104, 5590– 5595, DOI: 10.1073/pnas.0611470104[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkt1emtbs%253D&md5=aea9d01354a321f7e98d08dbc42acb49Proteorhodopsin photosyst gene expression enables photophosphorylation in a heterologous hostMartinez, A.; Bradley, A. S.; Waldbauer, J. R.; Summons, R. E.; DeLong, E. F.Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (13), 5590-5595CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Proteorhodopsins (PRs) are retinal-contg. proteins that catalyze light-activated proton efflux across the cell membrane. These photoproteins are known to be globally distributed in the ocean's photic zone, and they are found in a diverse array of Bacteria and Archaea. Recently, light-enhanced growth rates and yields have been reported in at least one PR-contg. marine bacterium, but the physiol. basis of light-activated growth stimulation has not yet been detd. To describe more fully PR photosystem genetics and biochem., we functionally surveyed a marine picoplankton large-insert genomic library for recombinant clones expressing PR photosystems in vivo. Our screening approach exploited transient increases in vector copy no. that significantly enhanced the sensitivity of phenotypic detection. Two genetically distinct recombinants, initially identified by their orange pigmentation, expressed a small cluster of genes encoding a complete PR-based photosystem. Genetic and biochem. analyses of transposon mutants verified the function of gene products in the photopigment and opsin biosynthetic pathways. Heterologous expression of six genes, five encoding photopigment biosynthetic proteins and one encoding a PR, generated a fully functional PR photosystem that enabled photophosphorylation in recombinant Escherichia col cells exposed to light. Our results demonstrate that a single genetic event can result in the acquisition of phototrophic capabilities in an otherwise chemoorganotrophic microorganism, and they explain in part the ubiquity of PR photosystems among diverse microbial taxa.
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Abstract
Figure 1
Figure 1. Expression vector design and assembly. (a) The standard expression vector contains a medium-strength constitutive promoter, RBS1, which allows for strong translation initiation of a leader peptide, and a translationally coupled, variable RBS2, mediating translation initiation of the coding sequence (CDS) of the membrane protein of interest. (18) (b) Vectors are assembled with different BCD elements. First, the vector is amplified by PCR, subsequently it is digested by type IIS restriction enzymes. The latter allows for seamless assembly with a library of annealed oligo pairs (encoding the different BCD variants), which have overhangs complementary to the digested vector.
Figure 2
Figure 2. Production of YidC-GFP, AraH-GFP, and rhodopsins by BCD elements and comparison to state-of-the art systems. (a) Volumetric YidC-GFP production based on whole-cell fluorescence measurements and final growth yields for the BCD constructs, and a comparison to Lemo21(DE3)-based production at optimized (2 mM l-rhamnose) and nonoptimized (0 mM l-rhamnose) conditions. BCD variants are ordered in the X-axis based on previously reported translation initiation strength. (18) (b) Western blots performed with antihis-tag antibody (upper panels) to visualize both inclusion body and well-folded YidC-GFP-his, and anti-IbpB (6,21) (lower panels) to visualize inclusion body binding protein B. (c) Single-cell production of YidC-GFP analyzed by flow cytometry for several increasing strength BCD elements and optimized Lemo21(DE3). (d) Single-cell production of YidC-GFP by BCD19 and BCD2 in a 72 h stability experiment. (e) Volumetric AraH-GFP production based on whole-cell fluorescence measurements and final growth yields for the BCD constructs, and a comparison to pET-opt-AraH-GFP. (14) BCD variants are ordered in the X-axis based on previously reported variant strength. (18) (f) Western blots performed with antihis-tag antibody (upper panels) to visualize both AraH-GFP-his, and anti-IbpB (6,21) (lower panels) to visualize inclusion body binding protein. (g) Single-cell production of AraH-GFP analyzed by flow cytometry for several increasing strength BCD elements and pET-opt-AraH-GFP. (h) Single-cell production of AraH-GFP by BCD19 and BCD2 in a 72 h stability experiment. (i) Volumetric Gloeobacter Rhodopsin (GR) and (j) Thermophilic Rhodopsin (TR) production determined by spectroscopy, and pictures of red-pigmented pellets. All cultivations were performed in 10 mL of medium in 50 mL tubes, for YidC-AraH and AraH-GFP at 30 °C, for rhodopsins GR and TR at 37 °C, and for pET-opt-AraH-GFP in E. coli BL21(DE3) pLysS at 25 °C (as optimized for in original work). BCD-based production was measured after 22 h of cultivation, while Lemo21(DE3) and pET based production was measured after 22 h of induction. Whole-cell fluorescence or rhodopsin quantification data are based on at least three biological replicates. For 72 h stability experiments E. coli BL21(DE3) harboring BCD vectors were reinoculated 1:50 into fresh LB kanamycin medium every 24 h. Notation: RFU, relative fluorescence units; OD600, optimal density of 600 nm.
References
ARTICLE SECTIONSThis article references 28 other publications.
- 1Schlegel, S., Hjelm, A., Baumgarten, T., Vikström, D., and de Gier, J.-W. (2014) Bacterial-based membrane protein production. Biochim. Biophys. Acta, Mol. Cell Res. 1843, 1739– 49, DOI: 10.1016/j.bbamcr.2013.10.023[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOgs7bF&md5=2177f95fef21516b68ab7929cbd70ee6Bacterial-based membrane protein productionSchlegel, Susan; Hjelm, Anna; Baumgarten, Thomas; Vikstroem, David; de Gier, Jan-WillemBiochimica et Biophysica Acta, Molecular Cell Research (2014), 1843 (8), 1739-1749CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)A review. Escherichia coli is by far the most widely used bacterial host for the prodn. of membrane proteins. Usually, different strains, culture conditions and prodn. regimes are screened for to design the optimal prodn. process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein prodn. yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein prodn. characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient prodn. of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
- 2Wagner, S., Baars, L., Ytterberg, A. J., Klussmeier, A., Wagner, C. S., Nord, O., Nygren, P.-A., van Wijk, K. J., and de Gier, J.-W. (2007) Consequences of Membrane Protein Overexpression in Escherichia coli. Mol. Cell. Proteomics 6, 1527– 1550, DOI: 10.1074/mcp.M600431-MCP200[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVGqsrzI&md5=cd2d2aa77d971faa7b44278cf4503598Consequences of membrane protein overexpression in Escherichia coliWagner, Samuel; Baars, Louise; Ytterberg, A. Jimmy; Klussmeier, Anja; Wagner, Claudia S.; Nord, Olof; Nygren, Per-Aake; van Wijk, Klaas J.; de Gier, Jan-WillemMolecular and Cellular Proteomics (2007), 6 (9), 1527-1550CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiol. response to overexpression is needed to improve such yields. Therefore, the authors analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a sol. protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling expts. Compn. and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not sol. GST-GFP, resulted in accumulation of cytoplasmic aggregates contg. the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and sol. proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of satn. of the cytoplasmic membrane protein translocation machinery. Importantly, accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP prodn. and a downregulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.
- 3Miroux, B. and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289– 98, DOI: 10.1006/jmbi.1996.0399[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XksVygtLs%253D&md5=6377b33b2d3bd0eec0047038253b481dOver-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levelsMiroux, Bruno; Walker, John E.Journal of Molecular Biology (1996), 260 (3), 289-298CODEN: JMOBAK; ISSN:0022-2836. (Academic)We have investigated the over-prodn. of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expressing of the target membrane protein was induced, most of the BL21 (DE3) host cells died. Similar effects were also obsd. with expression vectors for ten globular proteins. Therefore, protein over-prodn. in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high satn. cell d., and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior t BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells contg. the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H+ symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit B was lower in C41(DE3) and C43(DE3), resp., than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts to overcome other toxic effects assocd. with over-expression.
- 4Narayanan, A., Ridilla, M., and Yernool, D. A. (2011) Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator – repressor interactions. Protein Sci. 20, 51– 61, DOI: 10.1002/pro.535[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVKjsLY%253D&md5=449847f3c90bea7240ba20c7ea075611Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactionsNarayanan, Anoop; Ridilla, Marc; Yernool, Dinesh A.Protein Science (2011), 20 (1), 51-61CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)A major rate-limiting step in detg. structures of membrane proteins is heterologous protein prodn. Toxicity often assocd. with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled redn. in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer iso-Pr β-D-1-thiogalactopyranoside. In addn., prodn. of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI. Using this approach, we can achieve a 200-fold range of green fluorescent protein expression levels. Application to members of a family of ion pumps results in 5- to 25-fold increases in expression over the benchmark BL21(DE3) host strain. A viral ion channel highly toxic to E. coli can also be overexpressed. In comparative analyses, restrained expression outperforms commonly used E. coli expression strategies. The mechanism underlying improved target protein yield arises from minimization of protein aggregation and proteolysis that reduce membrane integrity and cell viability. This study establishes a method to overexpress toxic proteins.
- 5Schlegel, S., Löfblom, J., Lee, C., Hjelm, A., Klepsch, M., Strous, M., Drew, D., Slotboom, D. J., and De Gier, J.-W. (2012) Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3). J. Mol. Biol. 423, 648– 659, DOI: 10.1016/j.jmb.2012.07.019[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1emsb3M&md5=6d7f1771bfb51b805365be782f82861aOptimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)Schlegel, Susan; Loefblom, John; Lee, Chiara; Hjelm, Anna; Klepsch, Mirjam; Strous, Marc; Drew, David; Slotboom, Dirk Jan; de Gier, Jan-WillemJournal of Molecular Biology (2012), 423 (4), 648-659CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concns. of rhamnose indicated that this strain may be very suitable for optimizing the prodn. of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by redn. of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.
- 6Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Hogbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O., and de Gier, J.-W. (2008) Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. U. S. A. 105, 14371– 14376, DOI: 10.1073/pnas.0804090105[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1SgtrnI&md5=104e412bd6933a8085ed11ebfac7edbbTuning Escherichia coli for membrane protein overexpressionWagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Hogbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-WillemProceedings of the National Academy of Sciences of the United States of America (2008), 105 (38), 14371-14376,S14371/1-S14371/13CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiol. response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiol., proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a deriv. strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
- 7Hjelm, A., Schlegel, S., Baumgarten, T., Klepsch, M., Wickström, D., Drew, D., and De Gier, J.-W. (2013) Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions. Methods Mol. Biol. 1033, 381– 400, DOI: 10.1007/978-1-62703-487-6_24[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVais7%252FK&md5=0cba38627ec36897ded0a1aebcfdf74bOptimizing E. coli-based membrane protein production using Lemo21 (DE3) and GFP-fusionsHjelm, Anna; Schlegel, Susan; Baumgarten, Thomas; Klepsch, Mirjam; Wickstroem, David; Drew, David; de Gier, Jan-WillemMethods in Molecular Biology (New York, NY, United States) (2013), 1033 (Membrane Biogenesis), 381-400CODEN: MMBIED; ISSN:1064-3745. (Springer)Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purifn. is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the prodn. of high-quality membrane protein material for functional and structural studies.
- 8Schlegel, S., Genevaux, P., and de Gier, J.-W. (2015) De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields. Cell Rep. 10, 1758– 1766, DOI: 10.1016/j.celrep.2015.02.029[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVaisbs%253D&md5=2a8cb01f343a9a0d6aeeca4f9bd12677De-convoluting the genetic adaptations of E. coli C41(DE3) in real time reveals how alleviating protein production stress improves yieldsSchlegel, Susan; Genevaux, Pierre; de Gier, Jan-WillemCell Reports (2015), 10 (10), 1758-1766CODEN: CREED8; ISSN:2211-1247. (Cell Press)The well-established E. coli protein prodn. strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it acquired multiple mutations during its isolation. Std. allelic replacement and competition expts. were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein prodn. stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein prodn. stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the prodn. of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein prodn. stress and can be harnessed to enhance prodn.
- 9Kuipers, G., Karyolaimos, A., Zhang, Z., Ismail, N., Trinco, G., Vikström, D., Slotboom, D. J., and de Gier, J.-W. (2017) The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli. Microb. Cell Fact. 16, 226, DOI: 10.1186/s12934-017-0840-4[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXis1Knurw%253D&md5=a8cd3f8bd9046b1f047fea8fe400c432The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coliKuipers, Grietje; Karyolaimos, Alexandros; Zhang, Zhe; Ismail, Nurzian; Trinco, Gianluca; Vikstroem, David; Slotboom, Dirk Jan; de Gier, Jan-WillemMicrobial Cell Factories (2017), 16 (), 226/1-226/11CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)Background: To optimize the prodn. of membrane and secretory proteins in Escherichia coli, it is crit. to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein prodn. strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-prodn. of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a std. T7-based expression vector. Results: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a std. T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized prodn. yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a std. T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the prodn. of this heme c protein. Conclusions: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the prodn. of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).
- 10Kudla, G., Murray, A. W., Tollervey, D., and Plotkin, J. B. (2009) Coding-Sequence Determinants of Gene Expression in Escherichia coli. Science (Washington, DC, U. S.) 324, 255– 258, DOI: 10.1126/science.1170160[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXktFals7o%253D&md5=7fc7f47cb747fcee5585c775bb20bda9Coding-Sequence Determinants of Gene Expression in Escherichia coliKudla, Grzegorz; Murray, Andrew W.; Tollervey, David; Plotkin, Joshua B.Science (Washington, DC, United States) (2009), 324 (5924), 255-258CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP mRNA levels, mRNA degrdn. patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our anal., mRNA folding and assocd. rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.
- 11Cambray, G., Guimaraes, J. C., and Arkin, A. P. (2018) Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coli. Nat. Biotechnol. 36, 1005– 1015, DOI: 10.1038/nbt.4238[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslOhtbnM&md5=891f6e737ef0b4e66ee975a42489e5e4Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coliCambray, Guillaume; Guimaraes, Joao C.; Arkin, Adam PaulNature Biotechnology (2018), 36 (10), 1005-1015CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Comparative analyses of natural and mutated sequences have been used to probe mechanisms of gene expression, but small sample sizes may produce biased outcomes. We applied an unbiased design-of-expts. approach to disentangle factors suspected to affect translation efficiency in E. coli. We precisely designed 244,000 DNA sequences implementing 56 replicates of a full factorial design to evaluate nucleotide, secondary structure, codon and amino acid properties in combination. For each sequence, we measured reporter transcript abundance and decay, polysome profiles, protein prodn. and growth rates. Assocns. between designed sequences properties and these consequent phenotypes were dominated by secondary structures and their interactions within transcripts. We confirmed that transcript structure generally limits translation initiation and demonstrated its physiol. cost using an epigenetic assay. Codon compn. has a sizable impact on translatability, but only in comparatively rare elongation-limited transcripts. We propose a set of design principles to improve translation efficiency that would benefit from more accurate prediction of secondary structures in vivo.
- 12Nieuwkoop, T., Claassens, N. J., and van der Oost, J. (2019) Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design. Microb. Biotechnol. 12, 173– 179, DOI: 10.1111/1751-7915.13332[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1SrurbK&md5=584cd5afade3b129645226334b54f1b5Improved protein production and codon optimization analyses in Escherichia coli by bicistronic designNieuwkoop, Thijs; Claassens, Nico J.; van der Oost, JohnMicrobial Biotechnology (2019), 12 (1), 173-179CODEN: MBIIB2; ISSN:1751-7915. (Wiley-Blackwell)Different codon optimization algorithms are available that aim at improving protein prodn. by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5'-UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, esp. near the start of a gene, may strongly influence translation initiation. In this study, we aim to reduce the effect of codon usage on translation initiation by applying a bicistronic design (BCD) element. Protein prodn. of several codon-optimized gene variants is tested in parallel for a BCD and a std. monocistronic design (MCD). We demonstrate that these distinct architectures can drastically change the relative performance of different codon optimization algorithms. We conclude that a BCD is indispensable in future studies that aim to reveal the impact of codon optimization and codon usage correlations. Furthermore, irresp. of the algorithm used, using a BCD does improve protein prodn. compared with an MCD. The overall highest expression from BCDs for both GFP and RFP is at least twofold higher than the highest levels found for the MCDs, while for codon variants having very low expression from the MCD, even 10-fold to 100-fold increases in expression were achieved by the BCD. This shows the great potential of the BCD element for recombinant protein prodn.
- 13Nørholm, M. H. H., Toddo, S., Virkki, M. T. I., Light, S., von Heijne, G., and Daley, D. O. (2013) Improved production of membrane proteins in Escherichia coli by selective codon substitutions. FEBS Lett. 587, 2352– 8, DOI: 10.1016/j.febslet.2013.05.063[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3sjivV2rsQ%253D%253D&md5=13b32139bef8eb04dda21b0df9327647Improved production of membrane proteins in Escherichia coli by selective codon substitutionsNorholm Morten H H; Toddo Stephen; Virkki Minttu T I; Light Sara; von Heijne Gunnar; Daley Daniel OFEBS letters (2013), 587 (15), 2352-8 ISSN:.Membrane proteins are extremely challenging to produce in sufficient quantities for biochemical and structural analysis and there is a growing demand for solutions to this problem. In this study we attempted to improve expression of two difficult-to-express coding sequences (araH and narK) for membrane transporters. For both coding sequences, synonymous codon substitutions in the region adjacent to the AUG start led to significant improvements in expression, whereas multi-parameter sequence optimization of codons throughout the coding sequence failed. We conclude that coding sequences can be re-wired for high-level protein expression by selective engineering of the 5' coding sequence with synonymous codons, thus circumventing the need to consider whole sequence optimization.
- 14Mirzadeh, K., Martinez, V., Toddo, S., Guntur, S., Herrgard, M., Elofsson, A., Nørholm, M. H. H., and Daley, D. O. (2015) Enhanced protein production in Escherichia coli by optimization of cloning scars at the vector:coding sequence junction. ACS Synth. Biol. 4, 959– 965, DOI: 10.1021/acssynbio.5b00033[ACS Full Text], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXnvFaku7w%253D&md5=39320d5e716303be59ae804db233801fEnhanced protein production in Escherichia coli by optimization of cloning scars at the vector-coding sequence junctionMirzadeh, Kiavash; Martinez, Virginia; Toddo, Stephen; Guntur, Suchithra; Herrgaard, Markus J.; Elofsson, Arne; Noerholm, Morten H. H.; Daley, Daniel O.ACS Synthetic Biology (2015), 4 (9), 959-965CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Protein prodn. in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research labs. Maximum prodn. is usually sought, as this reduces costs and facilitates downstream purifn. steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to 1000-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (ΔG) in this region were important, but not the only, determinants for high expression.
- 15Kim, H. S., Ernst, J. a, Brown, C., Bostrom, J., Fuh, G., Lee, C. V., Huang, A., Vandlen, R. L., and Yansura, D. G. (2012) Translation levels control multi-spanning membrane protein expression. PLoS One 7, e35844, DOI: 10.1371/journal.pone.0035844[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XntFWks74%253D&md5=6fd28efcd597e7e0b74a1b373c3ac9a5Translation levels control multi-spanning membrane protein expressionKim, Hok Seon; Ernst, James A.; Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.PLoS One (2012), 7 (4), e35844CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the crit. role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane.
- 16Vazquez-Albacete, D., Cavaleiro, A. M., Christensen, U., Seppälä, S., Møller, B. L., and Nørholm, M. H. H. (2017) An expression tag toolbox for microbial production of membrane bound plant cytochromes P450. Biotechnol. Bioeng. 114, 751– 760, DOI: 10.1002/bit.26203[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFelurzF&md5=bd92a78fbdb5cf518e3641fe40504905An expression tag toolbox for microbial production of membrane bound plant cytochromes P450Vazquez-Albacete, Dario; Cavaleiro, Ana Mafalda; Christensen, Ulla; Seppaelae, Susanna; Moller, Birger Lindberg; Norholm, Morten H. H.Biotechnology and Bioengineering (2017), 114 (4), 751-760CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Membrane-assocd. Cytochromes P 450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here, we explore a small library of N-terminal expression tag chimeras of the model plant P 450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Anal. of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalyzed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than twofold. The developed toolbox serves as a platform to tune P 450 performance in microbial cells, thereby facilitating recombinant prodn. of complex plant P 450-derived biochems. Biotechnol. Bioeng. 2016;9999: 1-10. © 2016 Wiley Periodicals, Inc.
- 17Rennig, M., Martinez, V., Mirzadeh, K., Dunas, F., Röjsäter, B., Daley, D. O. O., and Nørholm, M. H. H. H. H. (2018) TARSyn: Tuneable antibiotic resistance devices enabling bacterial synthetic evolution and protein production. ACS Synth. Biol. 7, 432– 442, DOI: 10.1021/acssynbio.7b00200[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvF2mtLjK&md5=80912a1017505d532cc2ae68811e3a48TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein ProductionRennig, Maja; Martinez, Virginia; Mirzadeh, Kiavash; Dunas, Finn; Rojsater, Belinda; Daley, Daniel O.; Noerholm, Morten H. H.ACS Synthetic Biology (2018), 7 (2), 432-442CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Evolution can be harnessed to optimize synthetic biol. designs. A prominent example is recombinant protein prodn.-a dominating theme in biotechnol. for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory. A major bottleneck during prodn. is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concns. adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the resp. antibiotic. The power of the approach is demonstrated by substantially increasing prodn. of two com. interesting proteins, a Nanobody and an Affibody. The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any lab.
- 18Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., Endy, D., Hoynes-O’Connor, A., and Moon, T. S. (2013) Precise and reliable gene expression via standard transcription and translation initiation elements. Nat. Methods 10, 354– 360, DOI: 10.1038/nmeth.2404[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjs1Chur0%253D&md5=bbaa07c9dfc127bd8e5d3e0a7748d2aaPrecise and reliable gene expression via standard transcription and translation initiation elementsMutalik, Vivek K.; Guimaraes, Joao C.; Cambray, Guillaume; Lam, Colin; Christoffersen, Marc Juul; Mai, Quynh-Anh; Tran, Andrew B.; Paull, Morgan; Keasling, Jay D.; Arkin, Adam P.; Endy, DrewNature Methods (2013), 10 (4), 354-360CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)An inability to reliably predict quant. behaviors for novel combinations of genetic elements limits the rational engineering of biol. systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1000-fold obsd. dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain std. biol. parts that support reliable forward engineering of gene expression at genome scales.
- 19Takyar, S., Hickerson, R. P., and Noller, H. F. (2005) mRNA helicase activity of the ribosome. Cell 120, 49– 58, DOI: 10.1016/j.cell.2004.11.042[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXot1ChtA%253D%253D&md5=339ad7c565648fa0eb6dadc67cf755f7mRNA helicase activity of the ribosomeTakyar, Seyedtaghi; Hickerson, Robyn P.; Noller, Harry F.Cell (Cambridge, MA, United States) (2005), 120 (1), 49-58CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro translation system made up of purified components, the authors show that ribosomes are able to disrupt downstream helixes, including a perfect 27 base pair helix of predicted Tm = 70°. Using helixes of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. The authors conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.
- 20Engler, C., Kandzia, R., and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647, DOI: 10.1371/journal.pone.0003647[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cjhvV2ltA%253D%253D&md5=8910183a67aa28a0cc69522dcf7050b0A one pot, one step, precision cloning method with high throughput capabilityEngler Carola; Kandzia Romy; Marillonnet SylvestrePloS one (2008), 3 (11), e3647 ISSN:.Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.
- 21Schlegel, S., Löfblom, J., Lee, C., Hjelm, A., Klepsch, M., Strous, M., Drew, D., Slotboom, D. J., and de Gier, J.-W. (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J. Mol. Biol. 423, 648– 59, DOI: 10.1016/j.jmb.2012.07.019[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1emsb3M&md5=6d7f1771bfb51b805365be782f82861aOptimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3)Schlegel, Susan; Loefblom, John; Lee, Chiara; Hjelm, Anna; Klepsch, Mirjam; Strous, Marc; Drew, David; Slotboom, Dirk Jan; de Gier, Jan-WillemJournal of Molecular Biology (2012), 423 (4), 648-659CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concns. of rhamnose indicated that this strain may be very suitable for optimizing the prodn. of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by redn. of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.
- 22Gialama, D., Kostelidou, K., Michou, M., Delivoria, D. C., Kolisis, F. N., and Skretas, G. (2017) Development of Escherichia coli strains that withstand membrane protein-induced toxicity and achieve high-level recombinant membrane protein production. ACS Synth. Biol. 6, 284– 300, DOI: 10.1021/acssynbio.6b00174[ACS Full Text], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslGku7zP&md5=dfe2de5711b7290706db542765d0fd87Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein ProductionGialama, Dimitra; Kostelidou, Kalliopi; Michou, Myrsini; Delivoria, Dafni Chrysanthi; Kolisis, Fragiskos N.; Skretas, GeorgiosACS Synthetic Biology (2017), 6 (2), 284-300CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Membrane proteins perform crit. cellular functions in all living organisms and constitute major targets for drug discovery. Escherichia coli has been the most popular overexpression host for membrane protein biochem./structural studies. Bacterial prodn. of recombinant membrane proteins, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low final biomass and minute volumetric yields. In this work, we aimed to rewire the E. coli protein-producing machinery to withstand the toxicity caused by membrane protein overexpression in order to generate engineered bacterial strains with the ability to achieve high-level membrane protein prodn. To achieve this, we searched for bacterial genes whose coexpression can suppress membrane protein-induced toxicity and identified two highly potent effectors: the membrane-bound DnaK cochaperone DjlA, and the inhibitor of the mRNA-degrading activity of the E. coli RNase E, RraA. E. coli strains coexpressing either djlA or rraA, termed SuptoxD and SuptoxR, resp., accumulated markedly higher levels of final biomass and produced dramatically enhanced yields for a variety of prokaryotic and eukaryotic recombinant membrane proteins. In all tested cases, either SuptoxD, or SuptoxR, or both, outperformed the capabilities of com. strains frequently utilized for recombinant membrane protein prodn. purposes.
- 23Drew, D., Slotboom, D.-J., Friso, G., Reda, T., Genevaux, P., Rapp, M., Meindl-Beinker, N. M., Lambert, W., Lerch, M., Daley, D. O., Van Wijk, K.-J., Hirst, J., Kunji, E., and De Gier, J.-W. (2005) A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci. 14, 2011– 2017, DOI: 10.1110/ps.051466205[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXntVejtr0%253D&md5=1f8888e6ff671a6b47cc228f62783ea2A scalable, GFP-based pipeline for membrane protein overexpression screening and purificationDrew, David; Slotboom, Dirk-Jan; Friso, Giulia; Reda, Torsten; Genevaux, Pierre; Rapp, Mikaela; Meindl-Beinker, Nadja M.; Lambert, Wietske; Lerch, Mirjam; Daley, Daniel O.; Van Wijk, Klaas-Jan; Hirst, Judy; Kunji, Edmund; De Gier, Jan-WillemProtein Science (2005), 14 (8), 2011-2017CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)The authors describe a generic, GFP-based pipeline for membrane protein overexpression and purifn. in Escherichia coli. The authors exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important ref. for the characterization of other membrane proteomes.
- 24Drew, D. E, von Heijne, G., Nordlund, P., and de Gier, J.-W. L (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett. 507, 220– 224, DOI: 10.1016/S0014-5793(01)02980-5[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXnvFCrtrc%253D&md5=d13802658359626a0a2bd013dd5ee69bGreen fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coliDrew, David E.; von Heijne, Gunnar; Nordlund, Par; de Gier, Jan-Willem L.FEBS Letters (2001), 507 (2), 220-224CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)E. coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favored to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion vs. inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease. This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.
- 25Claassens, N. J., Volpers, M., Martins dos Santos, V. A. P., van der Oost, J., and de Vos, W. M. (2013) Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms. Trends Biotechnol. 31, 633– 642, DOI: 10.1016/j.tibtech.2013.08.006[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1SktrnF&md5=a3ed814f5ee698a8a5c0e956f03749e0Potential of proton-pumping rhodopsins: engineering photosystems into microorganismsClaassens, Nico J.; Volpers, Michael; Martins dos Santos, Vitor A. P.; van der Oost, John; de Vos, Willem M.Trends in Biotechnology (2013), 31 (11), 633-642CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)A review. A wide range of proton-pumping rhodopsins (PPRs) have been discovered in recent years. Using a synthetic biol. approach, PPR photosystems with different features can be easily introduced in nonphotosynthetic microbial hosts. PPRs can provide hosts with the ability to harvest light and drive the sustainable prodn. of biochems. or biofuels. PPRs use light energy to generate an outward proton flux, and the resulting proton motive force can subsequently power cellular processes. Recently, the introduction of PPRs in microbial prodn. hosts has successfully led to light-driven biotechnol. conversions. In this review, we discuss relevant features of natural PPRs, evaluate reported biotechnol. applications of microbial prodn. hosts equipped with PPRs, and provide an outlook on future developments.
- 26Deisseroth, K. (2015) Nat. Neurosci. 18, 1213– 1225, DOI: 10.1038/nn.4091[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVWmtb7M&md5=c2e3010ffa02a7a8cfc1ad434b160461Optogenetics: 10 years of microbial opsins in neuroscienceDeisseroth, KarlNature Neuroscience (2015), 18 (9), 1213-1225CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)A review with commentary. Over the past 10 years, the development and convergence of microbial opsin engineering, modular genetic methods for cell-type targeting and optical strategies for guiding light through tissue have enabled versatile optical control of defined cells in living systems, defining modern optogenetics. Despite widespread recognition of the importance of spatiotemporally precise causal control over cellular signaling, for nearly the first half (2005-2009) of this 10-yr period, as optogenetics was being created, there were difficulties in implementation, few publications and limited biol. findings. In contrast, the ensuing years have witnessed a substantial acceleration in the application domain, with the publication of thousands of discoveries and insights into the function of nervous systems and beyond. This Historical Commentary reflects on the scientific landscape of this decade-long transition.
- 27Gourdon, P., Alfredsson, A., Pedersen, A., Malmerberg, E., Nyblom, M., Widell, M., Berntsson, R., Pinhassi, J., Braiman, M., Hansson, Ö., Bonander, N., Karlsson, G., and Neutze, R. (2008) Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump. Protein Expression Purif. 58, 103– 113, DOI: 10.1016/j.pep.2007.10.017[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVygtbY%253D&md5=6635650433efc5c4c3e0246db2eb3debOptimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pumpGourdon, Pontus; Alfredsson, Anna; Pedersen, Anders; Malmerberg, Erik; Nyblom, Maria; Widell, Mikael; Berntsson, Ronnie; Pinhassi, Jarone; Braiman, Marc; Hansson, Oerjan; Bonander, Nicklas; Karlsson, Goeran; Neutze, RichardProtein Expression and Purification (2008), 58 (1), 103-113CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier B.V.)Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro prodn. of pR using a com. cell-free synthesis system yielding 1.0 mg purified protein per mL of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purifn. yielding 5 mg of essentially pure functional protein per L of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for com. biotechnol. applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural detn. of pR are discussed.
- 28Martinez, A., Bradley, A. S., Waldbauer, J. R., Summons, R. E., and DeLong, E. F. (2007) Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host. Proc. Natl. Acad. Sci. U. S. A. 104, 5590– 5595, DOI: 10.1073/pnas.0611470104[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkt1emtbs%253D&md5=aea9d01354a321f7e98d08dbc42acb49Proteorhodopsin photosyst gene expression enables photophosphorylation in a heterologous hostMartinez, A.; Bradley, A. S.; Waldbauer, J. R.; Summons, R. E.; DeLong, E. F.Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (13), 5590-5595CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Proteorhodopsins (PRs) are retinal-contg. proteins that catalyze light-activated proton efflux across the cell membrane. These photoproteins are known to be globally distributed in the ocean's photic zone, and they are found in a diverse array of Bacteria and Archaea. Recently, light-enhanced growth rates and yields have been reported in at least one PR-contg. marine bacterium, but the physiol. basis of light-activated growth stimulation has not yet been detd. To describe more fully PR photosystem genetics and biochem., we functionally surveyed a marine picoplankton large-insert genomic library for recombinant clones expressing PR photosystems in vivo. Our screening approach exploited transient increases in vector copy no. that significantly enhanced the sensitivity of phenotypic detection. Two genetically distinct recombinants, initially identified by their orange pigmentation, expressed a small cluster of genes encoding a complete PR-based photosystem. Genetic and biochem. analyses of transposon mutants verified the function of gene products in the photopigment and opsin biosynthetic pathways. Heterologous expression of six genes, five encoding photopigment biosynthetic proteins and one encoding a PR, generated a fully functional PR photosystem that enabled photophosphorylation in recombinant Escherichia col cells exposed to light. Our results demonstrate that a single genetic event can result in the acquisition of phototrophic capabilities in an otherwise chemoorganotrophic microorganism, and they explain in part the ubiquity of PR photosystems among diverse microbial taxa.
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ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00101.
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