Improving Cell-Free Protein Synthesis for Rapid Screening Applications

Cell-free protein synthesis (CFPS) is a versatile tool for protein research and biomanufacturing. This presentation details a unit operations approach to simplifying extract preparation, as well as a novel method for DNA amplification. E. coli cell growth was optimized using a face centered cubic designed experiment that provided an IPTG induction time of 201 min and a harvest time of 255 min. These times correspond to 1 L of growth culture in a 2.5 L shake flask. Experiments were then conducted to determine the optimal number passes through a French press homogenizer (1) as well as the best time and temperature combination for lyophilization (4 hr and 15 Celsius). The resulting extract was more effective than that of commercial kits. This can be used in conjunction with a novel DNA amplification method that modifies a minimal linear template for use in rolling circle amplification. This minimalist template showed identical expression levels to traditional plasmid-based expression using sfGFP. This template was used to successfully express multiple proteins from various classes: sfGFP, mVenus, mCherry, chloramphenicol acetyl transferase, a chitinase catalytic domain, subtilisin, an anti-GFP nanobody, BP100, and CA(1-7)M(2-9). Future directions for the use of CFPS will also be discussed.