Browse
Submit Data
Databases
API
Help

Dataset Information

83 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural characterization of recombinant human CD81 produced in Pichia pastoris.

ABSTRACT: Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

SUBMITTER: Jamshad M

PROVIDER: S-EPMC2635529 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris.

Project description:The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.

| S-EPMC3989067 | biostudies-literature

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris.

Project description:Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4?°C, while the activity was markedly reduced at 37 and 45?°C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.

| S-EPMC4932491 | biostudies-literature

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris.

Project description:The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.

| S-EPMC2206967 | biostudies-literature

Human recombinant lysosomal β-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts.

Project description:GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the β-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αβ or ββ subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant β-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant β-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

| S-EPMC8045741 | biostudies-literature

rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization.

Project description:A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast alpha-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN-, Br- and Cl-. On the basis of the estimated K(m) and kcat values it is evident that the pseudohalide SCN- is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

| S-EPMC1422775 | biostudies-other

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris.

Project description:In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris, was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g-1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi-Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L-1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.

| S-EPMC6999280 | biostudies-literature

Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris.

Project description:Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.

| S-EPMC3838785 | biostudies-literature

Production of recombinant human epidermal growth factor in Pichia pastoris.

Project description:This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27μg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.

| S-EPMC5470459 | biostudies-literature

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris.

Project description:Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

| S-EPMC5826528 | biostudies-literature

Characterization and high-yield production of non-N-glycosylated recombinant human BCMA-Fc in Pichia pastoris.

Project description:B-cell maturation antigen (BCMA) fused at the C-terminus to the Fc portion of human IgG1 (BCMA-Fc) blocks B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL)-mediated B-cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA-Fc (rhBCMA-Fc) in Pichia pastoris under the control of the AOX1 methanol-inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N-linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed-batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA-Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA-Fc possessed high-binding affinity to APRIL and dose-dependent inhibition of APRIL-induced proliferative activity in vitro through three-step purification. Thus, this yeast-derived expression method could be a low-cost and effective alternative to the production of rhBCMA-Fc in mammalian cell lines.

| S-EPMC6999299 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data