Biophysical and structural studies of fibulin-2.

Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer.

Cytoplasmic Expression of Disulfide-Bonded Proteins: Side-by-Side Comparison between Two Competing Strategies.

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.

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In 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.

Bulky hydrophobic side chains in the β1-sandwich of microsomal triglyceride transfer protein are critical for the transfer of both triglycerides and phospholipids.

Hyperlipidemia predisposes individuals to cardiometabolic diseases, the most common cause of global mortality. Microsomal triglyceride transfer protein (MTP) transfers multiple lipids and is essential for the assembly of apolipoprotein B-containing lipoproteins. MTP inhibition lowers plasma lipids but causes lipid retention in the liver and intestine.

Cytoplasmic production of Fabs in chemically defined media in fed-batch fermentation.

Fragment 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.

Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

Oxidative 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.

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli.

With increased accessibility and tissue penetration, smaller antibody formats such as antibody fragments (Fab) and single chain variable fragments (scFv) show potential as effective and low-cost choices to full-length antibodies. These formats derived from the modular architecture of antibodies could prove to be game changers for certain therapeutic and diagnostic applications. Microbial hosts have shown tremendous promise as production hosts for antibody fragment formats.

Mechanistic insights into the ROS-mediated inactivation of human aldehyde oxidase.

Human aldehyde oxidase (hAOX1) is a molybdoenzyme that oxidizes aldehydes and N-heterocyclic compounds, thereby generating hydrogen peroxide (H O ) and superoxide during turnover. hAOX1 has been shown previously to be inactivated under turnover conditions by H O . Here, we investigated the effect of exogenously added H O on the activity of hAOX1.

Highly efficient export of a disulfide-bonded protein to the periplasm and medium by the Tat pathway using CyDisCo in Escherichia coli.

High-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.

Inactivation of Human Aldehyde Oxidase by Small Sulfhydryl-Containing Reducing Agents.

Human aldehyde oxidase (hAOX1) is a molybdoflavoenzyme that belongs to the xanthine oxidase (XO) family. hAOX1 is involved in phase I drug metabolism, but its physiologic role is not fully understood to date, and preclinical studies consistently underestimated hAOX1 clearance. In the present work, we report an unexpected effect of the common sulfhydryl-containing reducing agents, e.

Production of neutralizing antibody fragment variants in the cytoplasm of E. coli for rapid screening: SARS-CoV-2 a case study.

Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges.

Efficient Production of Fc Fusion Proteins in the Cytoplasm of : Dissecting and Mitigating Redox Heterogeneity.

Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, .

Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding.

Proteins 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.

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H αβ tetramer.

Collagen prolyl 4-hydroxylases (C-P4H) are αβ tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown.

Mutation in protein disulfide isomerase A3 causes neurodevelopmental defects by disturbing endoplasmic reticulum proteostasis.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.

So you want to express your protein in Escherichia coli?

Recombinant proteins have been extensively employed as therapeutics for the treatment of various critical and life-threatening diseases and as industrial enzymes in high-value industrial processes. Advances in genetic engineering and synthetic biology have broadened the horizon of heterologous protein production using multiple expression platforms. Selection of a suitable expression system depends on a variety of factors ranging from the physicochemical properties of the target protein to economic considerations.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

The 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.

Efficient soluble production of folded cat allergen Fel d 1 in Escherichia coli.

The major cat allergen Fel d 1 is one of the most common and potent causes of animal related allergy. Medical treatment of cat allergy has relied on immunotherapy carried out with cat dander extract. This approach has been problematic, mainly due to inconsistent levels of the major allergen in the produced extracts.

The Amino-Terminal Oligomerization Domain of Angiopoietin-2 Affects Vascular Remodeling, Mammary Gland Tumor Growth, and Lung Metastasis in Mice.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer.

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase.

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail.

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism.

Exome Sequencing Reveals a Phenotype Modifying Variant in ZNF528 in Primary Osteoporosis With a COL1A2 Deletion.

We studied a family with severe primary osteoporosis carrying a heterozygous p.Arg8Phefs*14 deletion in COL1A2, leading to haploinsufficiency. Three affected individuals carried the mutation and presented nearly identical spinal fractures but lacked other typical features of either osteogenesis imperfecta or Ehlers-Danlos syndrome.

Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)-protein disulfide-isomerase relay when protein folding capacity is exceeded.

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD).

Production of Extracellular Matrix Proteins in the Cytoplasm of : Making Giants in Tiny Factories.

is 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.

Applications of catalyzed cytoplasmic disulfide bond formation.

Disulfide 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.