Optimizing Cell-Free Protein Synthesis for Antimicrobial Protein Production

Cell-free protein synthesis provides a flexible platform for the production of difficult-to-express proteins, because maintaining cell viability is unnecessary. The antimicrobial proteins known as bacteriocins have great potential for development as antibiotic alternatives. Here, we describe detailed protocols for producing and characterizing colicins—antimicrobial proteins that are produced by Escherichia coli hosts and inactivate nonhost E. coli strains. Active colicins can be produced with lysates containing molecular chaperones and coproduction of immunity proteins in cell-free protein synthesis reactions.