Tuning of Recombinant Protein Expression in <i>Escherichia coli</i> by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids

Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias L.; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens H.; Alfonta, Lital

doi:10.1021/acssynbio.7b00019

Cited by 25 publications

(28 citation statements)

References 35 publications

(68 reference statements)

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“…However, it is important to discuss two sources of this variability, which could be a common impediment in the employment of this system. First, the context and location of the site chosen for mutated to facilitate Uaa incorporation have a significant effect on the efficiency of the system [39,40], but notably, from our experience, the context, and locations could be different between in-vivo and cell-free systems [data not shown]. This subject warrants a systematic investigation that is outside the scope of this study.…”

Section: Resultsmentioning

confidence: 99%

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Chemla

Ozer

Shaferman

et al. 2019

Synthetic and Systems Biotechnology

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Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. Besides, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which could not be achieved in a living cell.

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Section: Resultsmentioning

confidence: 99%

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Chemla

Ozer

Shaferman

et al. 2019

Synthetic and Systems Biotechnology

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“…Most likely, a combination of all these factors is responsible for the impaired functionality of membrane proteins when overexpressed from high levels of transcription. The fine-tuning of transcription, mRNA stability and translation is often required for optimal protein synthesis 73,75,76 . Although our study only focused here on two membrane proteins, our data suggested that the Lemo21(DE3) strain did not overexpress well at 25 °C, as compared to the C41(DE3) strain.…”

Section: Discussionmentioning

confidence: 99%

Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain

Mathieu

Javed

Vallet

et al. 2019

Sci Rep

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Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli . While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae , when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.

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“…The functioning of TEF2 down-regulation in particular may be associated with the proteome mass reallocation from translation elongation factors (∼0.81%, SI Appendix, Fig. S22) and/or coordinated translation initiation and elongation vital for efficient protein translation (39)(40)(41). Down-regulation of the essential gene CDC39 involved in the cell cycle, conditional mutation of which resulted in G1 arrest (42), also enhanced the protein yield, confirming the universal applications of sustaining G0/G1 phase (43) and attenuating cell growth for improved protein production (44).…”

Section: Discussionmentioning

confidence: 99%

RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae

Wang

Björk

Huang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

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Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids

Cited by 25 publications

References 35 publications

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain

RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae

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