Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media

Abstract

Background: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm.

Results: Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA₁ antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source.

Conclusions: Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Keywords: Avidin; Cytoplasm; Disulfide bonds; Escherichia coli; Fed-batch; Fermentation; Growth hormone; Interleukin 6; scFv.

Fig. 1

Production of GH1. a, b Representative growth profiles of K-12 strain BW25113 co-expressing coPDI, coErv1p and GH1; a constant temperature growth, b growth at 37 °C to increase cell mass, then shift to 30 °C pre-induction. Error bars represent the standard deviation from 3 samples. F indicates feeding started, I indicates induction. Cells were harvested and protein purified from the last time point shown. c Representative elution profile from IMAC purification of GH1. d Coomassie stained reducing SDS-PAGE analysis of produced proteins: molecular marker, total E. coli lysate (T), soluble protein fraction (S) and IMAC purified GH1. e rpHPLC analysis of purified GH1: comparison of GH1 produced in fed-batch fermentation (red) and in shake flask (black)

Fig. 2

Production of scFv IgA₁. a Representative growth profile of K-12 strain BW25113 co-expressing coPDI, coErv1p and scFv IgA₁. Error bars represent the standard deviation from 3 samples. F indicates feeding started, I indicates induction. Cells were harvested and protein purified from the last time point shown. b Coomassie stained reducing SDS-PAGE analysis of produced proteins: molecular marker, total E. coli lysate (T), soluble protein fraction (S) and IMAC purified scFv. c Bradford analysis of sheared (+) and non-sheared (−) cells from two fermentations of scFv IgA₁ (n = 6). There was no significant difference in protein concentration between sheared and non-sheared cells indicating the E. coli strains with CyDisCo components are robust