Cell-free protein synthesis from non-growing, stressed Escherichia coli

Abstract

Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

Elucidating the potential of crude cell extracts for producing pyruvate from glucose

“…The 2xYPTG condition, collected in early-log phase growth, is commonly used for CFPS (22). Cells collected early in log phase growth have the greatest specific growth rate, a parameter that is suggested to influence CFPS capabilities and may affect the abundance of glycolytic enzymes (23,24). These growth conditions were chosen based on variables with the potential to enrich for glycolytic enzymes and for their frequent use for bacterial growth and related experiments that employ crude cell extracts.…”

“…Here, it is evident that growth on a minimal medium results in the expression of many sink pathways for glycolytic intermediates. Moreover, it appears specific growth rate, which has been previously examined as a key variable in CFPS extract preparation, plays a role in reducing sinks due to the Krebs cycle, but at the price of directing flux towards undetermined biomass accumulation pathways (23). Proteomic analysis is a robust technique for determining candidates for genetic manipulation and can guide in vivo protein overexpression or knockdowns in source strains that will affect the flux of small molecules after extract preparation.…”

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

“…Although several CFPS systems are described and have their own advantages, the E. coli-based system is the most commonly used due to high protein synthesis rates with high protein yields, streamlined and cost-effective cultivation and extract preparation, and the ability to fold complex proteins. Additionally, this system has been well studied and optimized during recent years [22,23]. E. coli is also one of the most used expression strains for cell-based protein synthesis.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…are routinely obtained from growing cells in exponential phase. However, intact active translation systems are also demonstrated to be acquirable from cells harvested at stressed, nongrowth conditions where the protein synthesis rate is surprisingly comparable to that of fast‐growing cells (Failmezger, Rauter, Nitschel, Kraml, & Siemann‐Herzberg, 2017).…”