Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

Abstract

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.

DsbC activation by the N-terminal domain of DsbD

“…DsbD should be able to distinguish between the active-site disulfide bonds of substrates such as DsbC and DsbG and other disulfides within the periplasm. By contrast, the substrate-binding interactions of DsbC are sufficiently nonspecific to allow the successful periplasmic expression of recombinant nonbacterial proteins containing multiple disulfide bonds (40)(41)(42)(43)(44). We are interested in learning how DsbC participates in these very different types of interaction.…”

Production of Brain-Derived Neurotrophic Factor inEscherichia coliby Coexpression of Dsb Proteins

“…15,16) In addition to PDI, overexpression of Dsb proteins has been shown to increase the yield of various eukaryotic proteins in bacteria. [8][9][10][11][12] Soluble BDNF production was estimated from the biological activity for neurite outgrowth of embryonic chick DRG neurons. Soluble BDNF fractions (Fig.…”

“…9) Horseradish peroxidase (HRP) was used as a recombinant protein to investigate the eŠects of coexpressing HRP with molecular chaperons and Dsb proteins. The total HRP production was increased several-fold by overexpression of DsbC 10) and the active HRP production was increased signiˆcantly by coexpression of DsbAB or DsbCD. 11) Human nerve growth factor (NGF) belongs to a neurotrophic factor family.…”

Differential protein expression in pancreatic islets after treatment with an imidazoline compound

“…PDI, in addition to inhibiting protein aggregation, may accelerate posttranslational processing of proteins necessary to facilitate insulin uptake [26] . Furthermore, PDI may have a beneficial effect on the secretion of insulin, since overexpression of PDI stabilizes secretory proteins [27] . Misfolded proteins are translocated from the endoplasmatic reticulum, polyubiquinated and transported to cytosolic proteasomes for degradation [23] , but we found that proteasome subunits a and b are down-regulated.…”