Abstract
Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.
Highlights
Cell-free protein synthesis (CFPS) first emerged in the 1960s as part of research uncovering the genetic code.[1]
We considered three major components of system: plasmid selection, the concentrated reaction mixture composition, and the E. coli strain of the cell extract
We trialed two plasmids: pJL1, a CFPS-optimized plasmid created by the Jewett Lab at Northwestern University for superfolder GFP (sfGFP) production, and pET14bGFP, a plasmid design for in vivo expression of GFP+ with a 6×histidine tag originally developed by Martin Warren's group at the University of Kent
Summary
Cell-free protein synthesis (CFPS) first emerged in the 1960s as part of research uncovering the genetic code.[1] Subsequently, this in vitro production method has been adapted to produce a variety of products including antibodies, biotherapeutic peptides, fusion proteins, vaccine candidates, membrane proteins, toxic proteins, and bacteriophages.[2] There are broadly two types of CFPS: reconstituted CFPS and crude lysate CFPS. In reconstituted CFPS, known as the Protein synthesis Using Recombinant Elements (PURE) system, purified recombinant proteins and ribosomes are added to the reaction in a bottom-up approach.[3] In crude lysate CFPS, a top-down approach is used in which. Cells are lysed and the extract clarified In both systems, this extract is combined with a concentrated reaction mix containing nucleotides, amino acids, energy substrates, salts, molecular crowding agents, polymerases, and genetic material for the expression of the product of interest. Cell extracts have been generated from a variety of host organisms: archaea, E. coli, yeast like Saccharomyces cerevisiae and Pichia pastoris, mammalian cells like Chinese hamster ovary, HEK293, and HeLa, wheat germ, and tobacco.[4,5,6] In this study, we used E. coli-based CFPS; in addition to being one of the more economical options, it is the most wellstudied cell extract with dozens of publications to date and several commercial kits available on the market
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