Characterization of a Gene Encoding a Pichia pastoris Protein Disulfide Isomerase

Warsame, Abdirashid; Vad, Randi; Kristensen, Tom; Øyen, Tordis B.

doi:10.1006/bbrc.2001.4479

Cited by 17 publications

(11 citation statements)

References 36 publications

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“…The sequence confirmed that 57-kDa band indeed was PDI protein, and processed as expected. The first five amino acid of the N-terminus of the 57-kDa protein was SDQEA, the expected sequence for PDI as found in the literature (Warsame et al, 2001). …”

Section: Overexpression Of Protein Disulfide Isomerase In Na-asp1 Mulsupporting

confidence: 59%

“…The PDI gene was amplified with Pfu DNA polymerase (Stratagene, La Jolla, CA) from chromosomal DNA of wild type P. pastoris X33 using forward primer 5 0 -ATGCAATT-CAA-CTGGGATATTAAAACTG-3 0 and reverse primer 5 0 -TTAAAGCTGTCGTGAGCGTC-3 0 based on published sequence of P. pastoris PDI gene (EMBL AJ302014) (Warsame et al, 2001).…”

Section: Construction Of Pdi Expression Plasmid Ppic35k-pdimentioning

confidence: 99%

“…Therefore, we have over-expressed homologous PDI protein in multiple (2-4) Na-ASP1 copy clones. The endogenous PDI gene was amplified from P. pastoris X-33 chromosome based on a published gene sequence (Warsame et al, 2001). The amplified PDI gene was subcloned into pPIC3.5K vector under the control of AOX1 promoter.…”

Section: Overexpression Of Protein Disulfide Isomerase In Na-asp1 Mulmentioning

confidence: 99%

See 2 more Smart Citations

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

İnan

Aryasomayajula

Sinha

et al. 2006

Biotechnol. Bioeng.

158

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A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.

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Section: Overexpression Of Protein Disulfide Isomerase In Na-asp1 Mulsupporting

confidence: 59%

Section: Construction Of Pdi Expression Plasmid Ppic35k-pdimentioning

confidence: 99%

Section: Overexpression Of Protein Disulfide Isomerase In Na-asp1 Mulmentioning

confidence: 99%

See 1 more Smart Citation

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

İnan

Aryasomayajula

Sinha

et al. 2006

Biotechnol. Bioeng.

158

View full text Add to dashboard Cite

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“…According to the 5-domain model that has been proposed for PDI activity, there are 2 protein-thiol oxidoreductase active sites with a CGHC sequence in both the C-and the N-terminal regions, and an ER retention signal with a KDEL sequence in the C-terminal. The latter finding is evidence that PDI is located and retained in the ER lumen and functions as an ER chaperone (Chivers et al 1996;Noiva 1999;Ciaffi et al 2001;Warsame et al 2001).…”

Section: Introductionmentioning

confidence: 88%

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI)

Goo

Yun²,

Hwang³

et al. 2002

Cell Stress Chaper

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We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.

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“…The PpPDI fragment, lacking a signal peptide, was amplified from the P. pastoris genomic DNA using forward primer 5′-CATGGAATTCATGCAATTCAACTGGGATATT and reverse primer 5′-CGCCTCGAGATTAAAGCTCGTCGTGAGCG based on a published sequence [19]. The PCR products of PpPDI was digested with EcoRI and XhoI and inserted into the vector pGAPZA.…”

Section: Construction Of Expression Plasmidsmentioning

confidence: 99%

Co-overexpression of PpPDI Enhances Secretion of Ancrod in Pichia pastoris

Zhang

Fang

Zhao

et al. 2011

Appl Biochem Biotechnol

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Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni²⁺ affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.

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Characterization of a Gene Encoding a Pichia pastoris Protein Disulfide Isomerase

Cited by 17 publications

References 36 publications

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

Enhancement of protein secretion inPichia pastoris by overexpression of protein disulfide isomerase

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI)

Co-overexpression of PpPDI Enhances Secretion of Ancrod in Pichia pastoris

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