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.
Highlights
Cell-free transcription and translation systems have emerged as powerful toolboxes for systems and synthetic biology approaches[1,2,3]
More-recent approaches demonstrate the use of endogenous E. coli RNA polymerase and “housekeeping” σ70 as a strong transcription unit to produce proteins in vitro[15]
We demonstrate that cell-free extracts derived from non-growing and stressed E. coli cells cultivated over night are active, which was previously considered impossible
Summary
Cell-free transcription and translation systems have emerged as powerful toolboxes for systems and synthetic biology approaches[1,2,3]. Recent examples are (i) the directed incorporation of non-canonical amino acids into proteins at multiple sites[6], (ii) the construction and characterization of multiple genetic circuits[2], and (iii) the engineering of artificial minimal cell systems[10,11,12] such as phospholipid vesicles containing the entire translation machinery These artificial environments are designed to potentially perform multifaceted biological tasks such as controlled exchange of nutrients[3]. More-recent approaches demonstrate the use of endogenous E. coli RNA polymerase and “housekeeping” σ70 as a strong transcription unit to produce proteins in vitro[15] This setup allows for an expansion of transcription regulatory parts and has proven to be extremely suitable, with its application ranging from highly efficient protein production[16] to prototyping of gene circuits[17].
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