Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli

Abstract

Background Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. Results We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173–2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. Conclusions Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

“…Co-producing LepB, which cleaves off signal peptides upon protein translocation, can also increase periplasmic protein production yields ( van Dijl et al, 1991 ). It has been shown that increasing the synthesis of protease IV (SppA) by modifying its TIR also results in enhanced periplasmic protein production ( Gawin et al, 2020 ). SppA is a membrane-bound signal peptide peptidase that is required for maintaining proper protein secretion ( Novak and Dev, 1988 ; Dalbey et al, 2012 ).…”

“…Rather than deleting the gene encoding DegP or inactivating its protease function, periplasmic protein production yields have also been improved by lowering DegP levels. This was done by replacing the ribosome binding site in the TIR controlling degP expression with a weaker one ( Gawin et al, 2020 ).…”

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity

“…Among them, SecB-dependent and SRP-mediated pathways both complete the translocation process by binding to SecA, and genetic fusion of signal peptides to RPs can enable them to utilize these pathways to translocate. Commonly used signal peptides include pelB, OmpA and DsbA [89,90], but each signal peptide triggers a different mechanism that greatly affects the effectiveness of RP transport. In contrast to SRP-mediated DsbA, SecB-dependent OmpA drives the synthesis of endogenous secreted and membrane proteins, preventing Sec translocator saturation [89].…”

Harnessing the potential of bacterial oxidative folding to aid protein production

“…For proteins that require expression in defined genetic backgrounds, or when periplasmic expression is preferred, cell envelope folding catalysts can be overexpressed by means of modifying their native RBS strength (Gawin et al, 2020), or by introducing an exogenous gene copy on a plasmid. pTUM4 is such as plasmid;…”

“…For proteins that require expression in defined genetic backgrounds, or when periplasmic expression is preferred, cell envelope folding catalysts can be overexpressed by means of modifying their native RBS strength (Gawin et al., 2020), or by introducing an exogenous gene copy on a plasmid. pTUM4 is such as plasmid; it constitutively overexpresses both DsbA and DsbC, as well as the folding chaperones SurA and FkpA, to aid expression of difficult proteins (Schlapschy et al., 2006; Shriver‐Lake et al., 2017).…”