| Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71. | |
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MedLine Citation:
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PMID: 20472730 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 microg/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination. |
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Authors:
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Karine Salamin; Dev Sriranganadane; Barbara Léchenne; Olivier Jousson; Michel Monod |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-05-14 |
Journal Detail:
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Title: Applied and environmental microbiology Volume: 76 ISSN: 1098-5336 ISO Abbreviation: Appl. Environ. Microbiol. Publication Date: 2010 Jul |
Date Detail:
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Created Date: 2010-06-24 Completed Date: 2010-09-07 Revised Date: 2011-07-19 |
Medline Journal Info:
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Nlm Unique ID: 7605801 Medline TA: Appl Environ Microbiol Country: United States |
Other Details:
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Languages: eng Pagination: 4269-76 Citation Subset: IM |
Affiliation:
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Service de Dermatologie et Vénéréologie, Centre Hospitalier Universitaire Vaudois, BT422, 1011 Lausanne, Switzerland. |
| Data Bank Information | |
Bank Name/Acc. No.:
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GENBANK/UNKNOWN |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Biotechnology
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methods Computational Biology / methods Culture Media Fermentation Methanol / metabolism Molecular Sequence Data Phenylmethylsulfonyl Fluoride / pharmacology Pichia / enzymology*, growth & development, metabolism Recombinant Proteins / genetics, metabolism Sequence Analysis, DNA Serine Proteases / metabolism Subtilisin / antagonists & inhibitors, metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Culture Media; 0/Recombinant Proteins; 329-98-6/Phenylmethylsulfonyl Fluoride; 67-56-1/Methanol; EC 3.4.-/Serine Proteases; EC 3.4.21.62/Subtilisin |
| Comments/Corrections | |
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