Cell-free expression is a powerful tool for machine learning assisted design of new biologics, however there are cost, productivity, and assay compatibility constraints with commercial extracts. Our group has made progress for this field through 1) E. coli extracts for disulfide bond formation, 2) optimized B. subtilis extracts for expression of biotherapeutics that could then be used in downstream cell assays, and 3) anaerobically conditioned E. coli extract that outperforms traditional aerobic extract. In the first work, we use the commercial SHuffle T7 strain to express a luciferase from Gaussia Princeps with disulfide bonds from PCR products. Protecting linear templates while improving extract processing resulted in a 14x increase in luminescent signal compared to when the methods are not implemented. In the second work we prepared extract from the protease deficient KO7A strain of Bacillus subtilis. We demonstrate expression of sfGFP and other active proteins from both constitutive and T7 promoters while protecting linear DNA templates from endogenous nucleases. This optimized extract represents a systematic process that can be used to optimize expression using new organisms. In the third work, we grow BL21 DE3 star E. coli under nitrogen, allowing them to acclimate to anerobic respiration prior to lysing. Using sfGFP as a reporter, we observe 1.5x more protein at 4 times the rate of aerobic extract. We confirm this increase in productivity by expressing subtilisin BPN’ and chitinase as well1; this extract will have great utility in cell-free reactions with limited surface area exposure to air, such as microtiter well plates in screening campaigns. Finally, we will describe next steps our group is taking to use these lysates for efficient design of biologic therapies.

Improved E. coli and B. subtilis extracts to enable high-throughput design of biologics via cell-free expression

ACS Fall 2021

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