The production of disulfide bond-containing recombinant proteins in has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of : i) engineered strains with deletions in the disulfide reduction pathways, SHuffle, and ii) the co-expression of oxidative folding catalysts, CyDisCo.
View Article and Find Full Text PDFIn the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG.
View Article and Find Full Text PDFFragment of antigen-binding region (Fab) of antibodies are important biomolecules, with a broad spectrum of functionality in the biomedical field. While full length antibodies are usually produced in mammalian cells, the smaller size, lack of N-glycosylation and less complex structure of Fabs make production in microbial cell factories feasible. Since Fabs contain disulfide bonds, such production is often done in the periplasm, but there the formation of the inter-molecular disulfide bond between light and heavy chains can be problematic.
View Article and Find Full Text PDFOxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae.
View Article and Find Full Text PDFHigh-value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins.
View Article and Find Full Text PDFProteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione.
View Article and Find Full Text PDFRecessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.
View Article and Find Full Text PDFThe folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility.
View Article and Find Full Text PDFis the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation.
View Article and Find Full Text PDFDisulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization.
View Article and Find Full Text PDFOxidative protein folding in the ER is driven mainly by oxidases of the endoplasmic reticulum oxidoreductin 1 (Ero1) family. Their action is regulated to avoid cell stress, including hyperoxidation. Previously published regulatory mechanisms are based on the rearrangement of active site and regulatory disulfides.
View Article and Find Full Text PDFCoronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels.
View Article and Find Full Text PDFBackground: 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.
View Article and Find Full Text PDFDisturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process.
View Article and Find Full Text PDFBackground: 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.
View Article and Find Full Text PDFSignificance: Disulfide bond formation is critical for biogenesis of many proteins. While most studies in this field are aimed at elucidating the mechanisms in the endoplasmic reticulum, intermembrane space of mitochondria, and prokaryotic periplasm, structural disulfide bond formation also occurs in other compartments including the cytoplasm. Such disulfide bond formation is essential for biogenesis of some viruses, correct epidermis biosynthesis, thermal adaptation of some extremophiles, and efficient recombinant protein production.
View Article and Find Full Text PDFDisulfide bond formation in the endoplasmic reticulum by the sulfhydryl oxidase Ero1 family is thought to be accompanied by the concomitant formation of hydrogen peroxide. Since secretory cells can make substantial amounts of proteins that contain disulfide bonds, the production of this reactive oxygen species could have potentially lethal consequences. Here, we show that two human proteins, GPx7 and GPx8, labeled as secreted glutathione peroxidases, are actually endoplasmic reticulum-resident protein disulfide isomerase peroxidases.
View Article and Find Full Text PDFDehydroascorbate (DHA) is a higher oxidation state of ascorbate formed through its action as an intracellular antioxidant. The recycling of DHA back to ascorbate is thought to be catalyzed by a variety of enzymes, including protein disulfide isomerase (PDI), linking ascorbate metabolism to oxidative protein folding in the endoplasmic reticulum (ER). Here we examine the possible role of PDI as a dehydroascorbate reductase.
View Article and Find Full Text PDFGlutaredoxins are oxidoreductases specialized in reducing glutathione-protein mixed disulfides. In the first step of deglutathionylation, glutaredoxins form a mixed disulfide with glutathione, releasing reduced peptide. The specificity of this reaction is based on the unusual amide linkage formed between the gamma-carboxylate of the N-terminal glutamic acid and the alpha-amino group of the cysteine present in glutathione.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFProtein disulphide isomerase (PDI) is a key multi-domain protein folding catalyst in the endoplasmic reticulum. The b' domain of PDI is essential for the non-covalent binding of incompletely folded protein substrates. Earlier, we defined the substrate binding site in the b' domain of human PDI by modelling and mutagenesis studies.
View Article and Find Full Text PDF