A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems

Abstract

Cell‐free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell‐free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re‐examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell‐free system with a 95% reduction in reagent costs (excluding cell‐extract, plasmid, and T7 RNA polymerase made in‐house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze‐thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high‐throughput screening, and biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:823–831, 2015

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

“…164 This lead to a systematic re-examination of the optimal concentration of the essential components of the cell-free protein synthesis reaction mixture, which resulted in a 95% reduction in reagent costs. 165 These results, while obtained using an antibody as a model, are likely to be applicable to other recombinant immunotherapeutic molecules and vaccine antigens.…”

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

“…The cost of protein prototyping from this in vitro method is competitive especially when one uses scalable extract preparation techniques as noted above (Dopp & Reuel, ) and minimal reagent recipes (Cai et al, ; Calhoun & Swartz, ; Jewett & Swartz, ; Kim et al, ). When analyzing the cost per gram of sfGFP produced (Supporting Information Figures S10), we found a minimalist reagent mix, like those used in previous works (Cai et al, ; Dopp & Reuel, ), produced sfGFP at 110 µg/ml (found by fluorescence calibration curve Figure S12) after 4 hr of expression which resulted in $61/mg sfGFP. When using a vendor kit, such as Promega T7 S30 High‐Yield with the minimalist, circular templates a 64% decrease in protein yield (40 µg/ml) was observed (Supporting Information Figure S11) which resulted in a 135‐fold increase in cost ($8,265/mg sfGFP; Supporting Information Figure S13).…”

“…Cell‐free expression with E. coli extract cannot form disulfide bonds between cysteine residues without changing the redox potential. Disulfide bonding is commonly achieved through the pretreatment of extract with iodoacetamide, the addition of glutathione, and the addition of disulfide bond isomerase C (DsbC; Cai et al, ). However, this limitation can be overcome by mutating cysteines involved with disulfide bonding as long as the bonds are not necessary for protein structure and function (Lu, Welsh, & Swartz, ).…”

Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

“…Cell-free energy mixes can also be simplified through the rational testing and removal of unnecessary components that upon re-examination are largely historical artefacts from protocols that have now been superseded by improved cell-free methodologies. Such approaches have been used to develop minimal energy mixes that maintain cell-free performance but with seven fewer energy components than widely reported cell-free protocols (Cai et al, 2015).…”

“…Upon consideration of several cell-free methodologies, a fairly simplified cell extract preparation workflow was used in this study adapted from our previous cell-free work and coupled with a recently reported glutamate-based minimal energy mix (Cai et al, 2015). Using these cell-free methods several batches of E. coli MG1655 TX-TL cell extracts and minimal energy mix were generated (see materials and methods 2.3 and 2.4).…”