Improving cell-free protein synthesis for rapid screening applications

Abstract

<p>Cell-free protein synthesis (CFPS) is a versatile tool for protein research and biomanufacturing. This work goes into great detail explaining the history and biochemical utility of supplements used in various energy mixes. The biological role of each component is discussed in a way that is easily understood by newcomers to the field. This work also takes 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, four previously uncharacterized GFP variants, 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 in developing fusion proteins and nanobodies for therapeutics will also be discussed.</p>