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Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations. Here we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active. Our results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.

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. 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. 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.

The majority of secreted and outer membrane eukaryotic proteins contain disulfide bonds, formed by complex interdependent pathways in the endoplasmic reticulum. The current model for the major route of disulfide formation is the regulated flow of oxidizing equivalents from molecular oxygen to the membrane-associated enzyme Ero1 to protein disulfide isomerase, and hence to substrate proteins. One molecule of hydrogen peroxide is produced by Ero1 per disulfide bond made. This peroxide is usually considered to be a dangerous by-product. Here we show that peroxide, added to a refolding buffer or generated enzymatically in situ, results in the efficient refolding of a model protein to the native state. At pH 7.0, the kinetics of obtaining the native folded state are more efficient using peroxide than by the use of a glutathione redox buffer. Disulfide bond formation by peroxide is kinetically favored over oxidation of cysteine to cysteine sulfinic acid and over the oxidation of other amino acids in the proteins such as methionine. Hence, unless peroxides are added in excess, oxidative damage to the folding protein is minimal. Our results offer insights into potential mechanisms for disulfide bond formation in vivo. Antioxid. Redox Signal. 11, 963–970.