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Pichia pastoris

About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.


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Journal ArticleDOI
TL;DR: Cloned and expressed the metalloprotease domain of human ADAM 9 in Pichia pastoris and activity was detected against gelatin, beta-casein, and fibronectin.

48 citations

Journal ArticleDOI
TL;DR: The results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.
Abstract: Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.

48 citations

Journal ArticleDOI
TL;DR: Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that the authors expressed a single PLE isoenzyme which showed a high preference for proline‐β‐naphthylamide.
Abstract: The N-terminal amino acid sequence of pig liver esterase (PLE) from a commercial sample was determined and shown to match closely to a published sequence encoding a proline-beta-naphthylamidase from pig liver. Next, mRNA isolated from pig liver was transcribed into cDNA and primers deduced from the N-terminal sequence were used to clone the 1698 base pairs of PLE cDNA. Initial attempts to express the cDNA in Escherichia coli and Pichia pastoris with different expression vectors and secretion signal sequences failed. Only after deletion of the putative C-terminal sequence His-Ala-Glu-Leu, usually considered as an endoplasmic reticulum retention signal, could heterologous expression of PLE be readily achieved in the methylotrophic yeast P. pastoris. Recombinant PLE (rPLE) was secreted into the medium and exhibited a specific activity of approximately 600 Umg(-1) and a Vmax/Km value of 139 micromolmin(-1)mM(-1) with p-nitrophenyl acetate as a substrate. Activity staining of renatured sodium dodecylsulfate-polyacrylamide gels gave a single band with esterolytic activity for rPLE, whereas several bands are visible in crude commercial PLE preparations. This was confirmed by native gels, which also show that rPLE is active as a trimer. Biochemical characterization of the recombinant enzyme and comparison with properties of commercial PLE preparations as well as with published data confirmed that we expressed a single PLE isoenzyme which showed a high preference for proline-beta-naphthylamide. This is a substrate specificity for the so-called gamma subunit of PLE. The optimum pH value and temperature for the recombinant PLE were 8.0 and 60 degrees C, respectively. The determined molecular weight of the secreted enzyme was approximately 61-62 kDa, which closely matches the calculated value of 62.419 kDa. The active site residues are located at Ser203, His448, and Asp97, and the typical consensus sequence motif for hydrolases was found around the active site serine (Gly-Glu-Ser-Ala-Gly).

48 citations

Patent
31 Jul 1998
TL;DR: In this article, regulatory nucleotide sequences for a novel Pichia pastoris gene, designated PpSEC10 gene, and respective amino acid sequences for the secretion leader and mature Sec10p protein components of the precursor polypeptide encoded by this novel gene are provided.
Abstract: Regulatory nucleotide sequences for a novel Pichia pastoris gene, designated PpSEC10 gene, and the nucleotide sequences and respective amino acid sequences for the secretion leader and the mature Sec10p protein components of the precursor polypeptide encoded by this novel gene are provided. These compositions are useful in methods for expression and secretion of proteins when assembled in proper reading frame, individually or in combination, within a DNA construct that further comprises a nucleotide sequence encoding a protein of interest. Vectors comprising the DNA constructs of the invention can be used to transform a yeast host cell, which can then be cultured to obtain the secreted protein of interest. Kits useful in this method and in methods of detection of the Sec10p protein using antibodies are also disclosed.

48 citations

Journal ArticleDOI
TL;DR: A new acidic family 7 endo-β-1,3- 1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp.
Abstract: Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg–1) was higher than toward carboxymethyl cellulose sodium (395 U mg–1), which was different from other family 7 endo-β-glucanases.

48 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023150
2022340
2021255
2020303
2019374
2018401