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

Agnieszka Gawin,Sine Alise Hartvigsen Hansen,Jostein Malmo,Trygve Brautaset,Helga Ertesvåg

doi:10.1186/s12896-020-00615-0

Agnieszka Gawin, Sine Alise Hartvigsen Hansen + Show 3 more

Open Access

https://doi.org/10.1186/s12896-020-00615-0

Copy DOI

Abstract

BackgroundAdvantages 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.ResultsWe 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.ConclusionsOur 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.

Highlights

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
In order to determine desired translation initiation rates (TIRs) of the synthetic ribosome binding sites (RBS) variants, we investigated the previously reported levels of translation efficiency that can be achieved by co-expressing a gene of interest from a plasmid
It is worth noting that Browning et al [34] recently developed the TatExpress strains, where transcriptional control of the chromosomal tatABCD operon allowed for improved recombinant periplasmic production of human growth hormone and a Single-chain variable antibody fragment (scFv)

Summary

Introduction

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. 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. Basic strategies employed to overcome the bottlenecks in folding of translocated proteins rely on the co-expression of periplasmic chaperones, signal peptide peptidases, and thiol-disulfide oxidoreductases, or on deletions of protease genes [3,4,5]. It has previously been demonstrated that simultaneous over-expression of DsbA and DsbB from a helper plasmid gives positive effects on production of active horseradish peroxidase (HRP) [10] and on soluble expression of a single-chain variable antibody fragment (scFv) [11]

Methods

Results

Discussion

Conclusion