(567be) Simplifying and Streamlining E. Coli-Based Cell-Free Protein Synthesis

E. coli cell-free protein synthesis (CFPS) uses E. coli catalytic machinery to make active proteins in vitro. CFPS is an attractive protein expression system because its open environment has enabled the synthesis of proteins that were otherwise difficult to produce using traditional in vivo methods. These proteins include complex fusion proteins, membrane proteins, antibody fragments, and transcription factors. CFPS can serve as both a production platform as well as an evaluative, screening platform. In this poster presentation, we will discuss advances our lab has made toward simplifying reagent preparation for small scale, evaluative reactions.

The basic CFPS reaction mixture is comprised of four main reagent components: (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. We have streamlined the protocols for preparing CFPS chemicals, cell extract, and T7 RNAP. In the past, CFPS chemicals were added individually to the CFPS reaction mixture on the day of experiment. By combining the CFPS reagents into a single storable, 4X-concentrated Premix, we were able to reduce the number of pipetting steps on the day of experiment which decreased both setup time and yield variability. For extract, we replaced controlled fermentation protocols with simple shake flask cultures and also truncated the preparation protocol. Finally, we discovered that adding impure T7 RNAP to CFPS in lieu of pure T7 RNAP does not affect yields. The products of these streamlined procedures still enable high yield CFPS. In addition, these streamlined procedures both save time and enable greater accessibility to our lab's CFPS technology.