(627e) Optimizing Cell-Free Protein Synthesis for Increased Yield, Solubility and Activity of Antimicrobial Protein Colicins

Colicins are antimicrobial proteins produced by Escherichia coli and have a great potential to be developed as a viable alternative of antibiotics. Cell-free protein synthesis (CFPS) is an excellent platform to produce toxic proteins as it eliminates cell viability concern which is a frequent problem in cell-based protein production. Previously, we demonstrated that colicins produced by CFPS are active in eradicating antibiotic-tolerant bacteria known as persisters, but some colicins exhibited poor solubility or cell-killing activity. In this study, we improved solubility of colicin M from 16% to nearly 100% by supplementing chaperone-enriched E. coli extracts. Cytotoxicity of colicin E3 was increased by adding or co-producing the E3 immunity protein during CFPS reaction, implying that the E3 immunity protein is necessary to enhance the colicin E3 activity in addition to protecting the host strain. We also identified that the colicins are synthesized rapidly; colicin E1 within three hours of incubation was produced to the similar level of activity and solubility compared to the colicin incubated up to 20 hours. Taken together, colicin production could be easily optimized using CFPS platform for improving the solubility and activity of colicins. In addition, we will discuss modular design of colicins for making fusion proteins for improving and redirecting cell-killing activities.