Tuned Escherichia coli as a host for the expression of disulfide‐rich proteins

Abstract

Disulfide-bond formation is a major post-translational modification and is essential for protein folding, stability, and function. This is especially true for secreted proteins, many of which possess great potential for biotechnological applications. Focusing on the use of Escherichia coli for the production of this class of proteins, we describe the mechanisms that maintain redox compartmentalization in the cell, with an emphasis on those that promote the formation and isomerization of disulfide bonds in the bacterial periplasm, while presenting parallel pathways in the eukaryotic endoplasmic reticulum. Based on these concepts, we review the use of E. coli as a cell factory for the production of heterologous disulfide-containing proteins using either peri- or cytoplasmic expression and, in particular, how these compartments can be tuned to improve the yield of correctly folded recombinant proteins. Finally, we describe a few examples of the production of small disulfide-rich proteins (protease inhibitors) to illustrate how soluble, active, and fully oxidized recombinants may be successfully obtained upon peri- or cytoplasmic expression in E. coli.

Production and Preclinical Evaluation of Plasmodium falciparum MSP-1 19 and MSP-3 11 Chimeric Protein, PfMSP-Fu 24

“…In order to improve the yield and purity of the chimera protein, an alternative E. coli expression system was used to express PfMSPFu 24 in a 10-liter fermentation. We were able to significantly scale up the production of PfMSP-Fu 24 using E. coli SHuffle cells that express a signal-truncated disulfide bond isomerase (DsbC) in the cytoplasm and rearrange incorrect disulfide bonds (45). A 10-liter fermentation batch yielded ϳ230 mg of PfMSP-Fu 24 which was highly pure and homogeneous, as indicated by SDS-PAGE, reverse-phase chromatography, and gel permeation chromatography.…”

“…To resolve the problem of low yield, we expressed PfMSP-Fu 24 in the E. coli SHuffle strain, which has disulfide isomerase (Dsbc) and allows improved cytoplasmic disulfide bond formation (45). We transformed E. coli SHuffle cells using the same plasmid (pET28a-MSP-Fu 24 ) construct.…”

Soluble Expression of Recombinant Nerve Growth Factor in Cytoplasm of Escherichia coli

“…However, the cytoplasm of E. coli can be genetically engineered to provide a suitable situation for successful expression of proteins containing disulfide bonds. In compare to periplasmic expression level, protein yields will be significantly higher when correct folding occurs in cytoplasm (19). As mentioned previously, the reducing environment of the cytoplasm is maintained by Trx and GSH systems; therefore, the mutation of these pathways leads to an oxidizing cytoplasm.…”

“…The cytoplasm of E. coli can be genetically engineered to provide a suitable situation for successful expression of proteins containing disulfide bonds. Correct cytoplasmic expression of proteins results in significantly higher protein yields relative to periplasmic expression of disulfide-rich proteins (19).…”

Editorial: In vivo protein folding – at the crossroad between basic research and biotechnology

“…This is especially true for secreted proteins, many of which possess great potential for biotechnological applications. Salinas et al [9] review information regarding the use of E. coli for the production of disulfide-rich proteins. The authors describe mechanisms that maintain redox compartmentalization in the cell, with an emphasis on those promoting the formation of disulfide bonds in the bacterial periplasm, and how these mechanisms can be tuned to improve the yield of correctly folded recombinant proteins.…”