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BackgroundDisulfide 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.ResultsHere 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS-linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.

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.