Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins Biological Engineering Biotechnology Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are e?ective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we con?rmed our previous ?nding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our ?ndings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform. Sponsorship: NIH R15AI130988 MDPI Jin, Xing Kightlinger, Weston Hong, Seok Hoon 2019 Article islandora:1012022 http://hdl.handle.net/10560/islandora:1012022 ChBE / Chemical and Biological Engineering Illinois Institute of Technology en Creative Commons Attribution (CC BY) http://creativecommons.org/licenses/by/4.0/ Open Access