Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments

Abstract

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.

A Robust Fractionation Method for Protein Subcellular Localization Studies in Escherichia Coli

“…To assess the extraction of a recombinant secreted protein from periplasm using PureFrac, the periplasmic PhoA (fused to a TorA-signal peptide) was co-expressed with PDI and Erv1p in WT and Tat-null strains. Such cells were cultivated in varying settings (Table2) thatare commonly used to overexpress proteins in the literature [18,19,25,26,32,33,38] to test whether growth conditions affect cross-contamination propensity.…”

“…Gene expression plasmids were constructed using the Golden Gate technology [37]. Initially, the vector pDPH340 [38] was modified by PCR mutagenesis to remove the TypeIIS restriction site, BsaI in the vector backbone; subsequently BsaI sites were strategically inserted at specific points downstream from the IPTG-inducible tac promoter, to accept either one or two compatible-gene cassettes for in-frame single or polycistronic gene expression.…”

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

“…In the last decades, numerous E. coli strains engineered to improve their efficiency in the production of recombinant proteins have been developed [49][50][51][52][53] and have been also largely used for the preparation of recombinant antibody-like fragments [54][55][56]. Some of them have also become the gold standard for biopharmaceutical applications while others have remained only tools for basic research [44].…”

“…Stress minimization results in the increased viability of cells and process robustness [70,94]. In the so-called Design of Experiments (DoE) setting [95] the optimization of the expression of recombinant Fab and scFv fragments in stress minimization conditions has been successfully achieved through the selection of media [70,88,96,97], screening of signal peptide sequences [67,68,75,98] and optimization of co-expressing chaperone proteins [56,88,[99][100][101][102]. At the transcriptional level, the concept of "codon harmonization", a sophisticated version of the codon usage optimization, has largely improved the expression of antibodies fragments [60,103].…”

Extraction of recombinant periplasmic proteins under industrially relevant process conditions: Selectivity and yield strongly depend on protein titer and methodology

“…Much scientific research has been performed to learn how to drive periplasmic expression of complex recombinant proteins in gramnegative bacteria. [1][2][3][4] The oxidative environment of the periplasm favors disulfide bridge formation, 5,6 and the presence of specific chaperones can enable correct protein folding. 7,8 Notably, of the 25 known cellular proteases in Escherichia coli, only seven are present in the periplasm.…”