A5018427084- Endoplasmic Reticulum Stress and Disease
- Protein purification and stability
- Redox biology and oxidative stress
- Bacterial Genetics and Biotechnology
- Monoclonal and Polyclonal Antibodies Research
- Lipid metabolism and disorders
- Viral Infectious Diseases and Gene Expression in Insects
- Bacteriophages and microbial interactions
- Mitochondrial Function and Pathology
- Cellular transport and secretion
- Autophagy in Disease and Therapy
- Glycosylation and Glycoproteins Research
- Biomarkers in Disease Mechanisms
- Neurogenetic and Muscular Disorders Research
- Heat shock proteins research
- Amyotrophic Lateral Sclerosis Research
- Heme Oxygenase-1 and Carbon Monoxide
- Escherichia coli research studies
- Galectins and Cancer Biology
- Fungal and yeast genetics research
- Apelin-related biomedical research
- Adipokines, Inflammation, and Metabolic Diseases
- Porphyrin Metabolism and Disorders
- Sulfur Compounds in Biology
- Transgenic Plants and Applications
University of Oulu
2016-2025
Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide depending on type, with scFv fragments containing two disulfides and Fab five or six bonds. The production of antibody that native can be challenging, especially a large scale. protein needs targeted prokaryotic periplasm eukaryotic endoplasmic reticulum. These compartments specialised for bond formation, but both have limitations. Here we show introduction...
The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein interest needs to be either targeted oxidizing periplasm or expressed cytoplasm form inclusion bodies, then solubilized and re-folded vitro. Both these approaches have limitations. Previously we showed that soluble expression bonded E. possible at shake flask scale with a system, known as CyDisCo, which based on co-expression along sulfhydryl oxidase bond isomerase....
The majority of secreted and outer membrane eukaryotic proteins contain disulfide bonds, formed by complex interdependent pathways in the endoplasmic reticulum. current model for major route formation is regulated flow oxidizing equivalents from molecular oxygen to membrane-associated enzyme Ero1 protein isomerase, hence substrate proteins. One molecule hydrogen peroxide produced per bond made. This usually considered be a dangerous by-product. Here we show that peroxide, added refolding...
Coronary artery disease is the most common cause of death globally and linked to a number risk factors including serum low density lipoprotein, high triglycerides lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 4, have emerged from genetic studies as being that significantly modulate plasma triglyceride levels coronary disease. The exact function mechanism action both proteins remains be elucidated, however, mutations in these results up 34% reduction inhibition reduced...
Dehydroascorbate (DHA) is a higher oxidation state of ascorbate formed through its action as an intracellular antioxidant. The recycling DHA back to thought be catalyzed by variety enzymes, including protein disulfide isomerase (PDI), linking metabolism oxidative folding in the endoplasmic reticulum (ER). Here we examine possible role PDI dehydroascorbate reductase. We find reaction too slow major route for reduction ER, with second-order rate constant reduced only 12.5 M(-1)s(-1). Rates...
Bacillus subtilis is a bacterial cell factory with outstanding protein secretion capabilities that has been deployed as workhorse for the production of industrial enzymes more than century. Nevertheless, other proteins B. subtilis, such antibody formats, thus far challenging due to specific requirements relate correct folding and disulfide bond formation upon export from cytoplasm. In present study, we explored possibility producing functional scFvs scFabs, using genome-reduced Midi-...
Oxidative protein folding in the ER is driven mainly by oxidases of endoplasmic reticulum oxidoreductin 1 (Ero1) family. Their action regulated to avoid cell stress, including hyperoxidation. Previously published regulatory mechanisms are based on rearrangement active site and disulfides. In this study, we identify two novel mechanisms. First, both human Ero1 isoforms exist a dynamic mixed disulfide complex with isomerase, which involves cysteines (Cys166 Ero1α Cys165 Ero1β) that have...
Escherichia coli is the most widely used protein production host in academia and a major for industrial production. However, recombinant of eukaryotic proteins prokaryotes has challenges. One these post-translational modifications, including native disulfide bond formation. Proteins containing bonds have traditionally been made by targeting to periplasm or vitro refolding as inclusion bodies. More recently, systems disulfide-containing cytoplasm introduced. it unclear if capacity...
Abstract High‐value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via Sec pathway as it, among other advantages, enables bond formation and simplifies downstream processing. However, system cannot transport complex or rapidly folding proteins, it only transports an unfolded state. The Tat also periplasm, has significant potential alternative means of recombinant protein production because fully folded proteins....
In the methylotrophic yeast Komagataella phaffii, we identified an ER-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 motifs (a and a'), followed by alpha-helical ERp29c C-terminal domain (c domain). However, while typical PDI-like motif (CGHC), a' instead CGYC, glutaredoxin-like confers to exceptional affinity for GSH/GSSG. This so far been unreported in...
Glutaredoxins are oxidoreductases specialized in reducing glutathione-protein mixed disulfides. In the first step of deglutathionylation, glutaredoxins form a disulfide with glutathione, releasing reduced peptide. The specificity this reaction is based on unusual amide linkage formed between gamma-carboxylate N-terminal glutamic acid and alpha-amino group cysteine present glutathione. second glutathione reduces glutaredoxin-glutathione disulfide. Here we show that for Escherichia coli Grx1,...
The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein factors, some which are protein-specific. ER resident saposin-like pERp1 (MZB1, CNPY5) crucial for correct IgA, IgM and integrins. also plays role calcium homeostasis plasma cell mobility. As an important factor proper maturation hence immune function, upregulated many auto-immune diseases. This makes it potential therapeutic target. belongs to CNPY family proteins....
Abstract Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by disulfide isomerase PDI and oxidoreductin Ero1. Their activity tightly regulated interconnected with unfolded response (UPR). The mechanisms of bond formation have been studied human or yeast Saccharomyces cerevisiae. Here we analyze kinetics non-conventional Komagataella phaffii , a common host for production recombinant secretory proteins. Surprisingly, found significant differences both S. cerevisiae...
Proteins in the thioredoxin superfamily share a similar fold, contain -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- sites. For vitro studies, oxidation/reduction of PDI during catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for also vivo, but at least oxidation reactions, GSSG probably not major acceptor have evolved to react glutathione high...
Fragment of antigen-binding region (Fab) antibodies are important biomolecules, with a broad spectrum functionality in the biomedical field. While full length usually produced mammalian cells, smaller size, lack N-glycosylation and less complex structure Fabs make production microbial cell factories feasible. Since contain disulfide bonds, such is often done periplasm, but there formation inter-molecular bond between light heavy chains can be problematic. Here we studied use CyDisCo system...