A sequential expression system for identifying effectors of in vitro protein synthesis and folding

Cell-free protein synthesis (CFPS) platforms based on cell extracts derived from Escherichia coli take advantage of the last 40 years of knowledge and experience in high-level recombinant protein expression in this model organism. They offer attractive alternatives to conventional fermentation methods for protein production. Identifying key reactions that enhance or inhibit in vitro protein synthesis and folding has led to improvements in CFPS energetics and reaction conditions. Continued development of CFPS as a platform for rapid and economical production of therapeutic proteins will benefit from our ability to identify factors that can improve in vitro expression and folding.; We have developed a method that uses sequential rounds of CFPS to identify proteins that influence in vitro transcription, translation, and protein folding. The first round of CFPS creates an array of cell extracts that are individually enriched with a single target protein. These proteins are expressed from linear DNA templates constructed in parallel using a PCR procedure wherein the overlap-extension reaction for the addition of transcription regulatory elements is separated from a GC-rich single-primer amplification of the full-length product. The expression templates are used to direct the first round of CFPS using a cell extract engineered to enhance linear DNA stability and using reaction conditions conducive for protein activation. After the first round of CFPS, the linear DNA templates are specifically targeted for degradation and a plasmid-based template for a reporter protein is added to initiate a subsequent round of protein expression. In this manner, the array is screened to identify targets that enhance or inhibit the expression and folding of the reporter protein. The culmination of this work has allowed us to identify proteins and biochemical activities that could improve the productivity of CFPS through rational metabolic engineering. This approach promises to broaden our understanding of cell-free biology and is also expected to provide a valuable platform technology for functional genomics.