Secretory expression and characterization of insulin in Pichia pastoris
The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic pr...
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| Veröffentlicht in: | Biotechnology and applied biochemistry 1999-02, Vol.29 (1), p.79-86 |
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| creator | Kjeldsen, Thomas Pettersson, Annette Frost Hach, Morten |
| description | The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro‐leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated α‐factor pro‐leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro‐leader, but not an α‐factor prepro‐leader lacking N‐glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin. |
| doi_str_mv | 10.1111/j.1470-8744.1999.tb01151.x |
| format | Article |
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The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro‐leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated α‐factor pro‐leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro‐leader, but not an α‐factor prepro‐leader lacking N‐glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin.</description><identifier>ISSN: 0885-4513</identifier><identifier>EISSN: 1470-8744</identifier><identifier>DOI: 10.1111/j.1470-8744.1999.tb01151.x</identifier><identifier>PMID: 9889087</identifier><identifier>CODEN: BABIEC</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Amino Acid Sequence ; Biological and medical sciences ; Biotechnology ; Chromatography, High Pressure Liquid - methods ; Fundamental and applied biological sciences. Psychology ; Genetic engineering ; Genetic technics ; Humans ; Insulin - genetics ; Insulin - isolation & purification ; Insulin - metabolism ; Insulin Secretion ; Mass Spectrometry ; Methods. Procedures. Technologies ; Modification of gene expression level ; Molecular Sequence Data ; Molecular Weight ; Peptide Mapping ; Pichia - genetics ; Pichia - metabolism ; Proinsulin - genetics ; Proinsulin - isolation & purification ; Proinsulin - metabolism ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae - metabolism</subject><ispartof>Biotechnology and applied biochemistry, 1999-02, Vol.29 (1), p.79-86</ispartof><rights>1999 International Union of Biochemistry and Molecular Biology</rights><rights>1999 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5051-346fc17782f5c421eec0962e9eba4b922222edf5ed96eff1d06cd9f359c113ac3</citedby><cites>FETCH-LOGICAL-c5051-346fc17782f5c421eec0962e9eba4b922222edf5ed96eff1d06cd9f359c113ac3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1470-8744.1999.tb01151.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1470-8744.1999.tb01151.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27903,27904,45553,45554</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1741803$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9889087$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kjeldsen, Thomas</creatorcontrib><creatorcontrib>Pettersson, Annette Frost</creatorcontrib><creatorcontrib>Hach, Morten</creatorcontrib><title>Secretory expression and characterization of insulin in Pichia pastoris</title><title>Biotechnology and applied biochemistry</title><addtitle>Biotechnol Appl Biochem</addtitle><description>The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro‐leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated α‐factor pro‐leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro‐leader, but not an α‐factor prepro‐leader lacking N‐glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin.</description><subject>Amino Acid Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>Humans</subject><subject>Insulin - genetics</subject><subject>Insulin - isolation & purification</subject><subject>Insulin - metabolism</subject><subject>Insulin Secretion</subject><subject>Mass Spectrometry</subject><subject>Methods. Procedures. Technologies</subject><subject>Modification of gene expression level</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Peptide Mapping</subject><subject>Pichia - genetics</subject><subject>Pichia - metabolism</subject><subject>Proinsulin - genetics</subject><subject>Proinsulin - isolation & purification</subject><subject>Proinsulin - metabolism</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae - metabolism</subject><issn>0885-4513</issn><issn>1470-8744</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVUN1r2zAQF6MjS7v9CQMzSt_s6SzLlvZS0rBmg-4DtpG9CUU-UWWOnUoOS_rXVyYmeyy7lzvu93HHj5B3QDOI9X6dQVHRVFRFkYGUMutXFIBDtn9BpifojEypEDwtOLBX5DyENaVUVCKfkIkUQsZ5ShY_0HjsO39IcL_1GILr2kS3dWLutdemR-8edT8sO5u4Nuwa18aefHfm3ulkq0MUu_CavLS6Cfhm7Bfk1-3Hn_NP6d23xef57C41nHJIWVFaA1V8wnJT5IBoqCxzlLjSxUrmQ2FtOdayRGuhpqWppWVcGgCmDbsgV0ffre8edhh6tXHBYNPoFrtdUKUsoYz0Z4lQgcwFHYgfjkTjuxA8WrX1bqP9QQFVQ9xqrYZM1ZCpGuJWY9xqH8Vvxyu71Qbrk3TMN-KXI66D0Y31ujUu_LtQFSAoi7TrI-2va_DwHw-om9nNMEWH9OjgQo_7k4P2f1RZsYqr5deF4vMvcslYrn6zJ8eUrEI</recordid><startdate>199902</startdate><enddate>199902</enddate><creator>Kjeldsen, Thomas</creator><creator>Pettersson, Annette Frost</creator><creator>Hach, Morten</creator><general>Blackwell Publishing Ltd</general><general>Portland Press</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>199902</creationdate><title>Secretory expression and characterization of insulin in Pichia pastoris</title><author>Kjeldsen, Thomas ; Pettersson, Annette Frost ; Hach, Morten</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5051-346fc17782f5c421eec0962e9eba4b922222edf5ed96eff1d06cd9f359c113ac3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Amino Acid Sequence</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>Humans</topic><topic>Insulin - genetics</topic><topic>Insulin - isolation & purification</topic><topic>Insulin - metabolism</topic><topic>Insulin Secretion</topic><topic>Mass Spectrometry</topic><topic>Methods. Procedures. Technologies</topic><topic>Modification of gene expression level</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Peptide Mapping</topic><topic>Pichia - genetics</topic><topic>Pichia - metabolism</topic><topic>Proinsulin - genetics</topic><topic>Proinsulin - isolation & purification</topic><topic>Proinsulin - metabolism</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kjeldsen, Thomas</creatorcontrib><creatorcontrib>Pettersson, Annette Frost</creatorcontrib><creatorcontrib>Hach, Morten</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology and applied biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kjeldsen, Thomas</au><au>Pettersson, Annette Frost</au><au>Hach, Morten</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Secretory expression and characterization of insulin in Pichia pastoris</atitle><jtitle>Biotechnology and applied biochemistry</jtitle><addtitle>Biotechnol Appl Biochem</addtitle><date>1999-02</date><risdate>1999</risdate><volume>29</volume><issue>1</issue><spage>79</spage><epage>86</epage><pages>79-86</pages><issn>0885-4513</issn><eissn>1470-8744</eissn><coden>BABIEC</coden><abstract>The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro‐leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated α‐factor pro‐leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro‐leader, but not an α‐factor prepro‐leader lacking N‐glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>9889087</pmid><doi>10.1111/j.1470-8744.1999.tb01151.x</doi><tpages>8</tpages></addata></record> |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Amino Acid Sequence Biological and medical sciences Biotechnology Chromatography, High Pressure Liquid - methods Fundamental and applied biological sciences. Psychology Genetic engineering Genetic technics Humans Insulin - genetics Insulin - isolation & purification Insulin - metabolism Insulin Secretion Mass Spectrometry Methods. Procedures. Technologies Modification of gene expression level Molecular Sequence Data Molecular Weight Peptide Mapping Pichia - genetics Pichia - metabolism Proinsulin - genetics Proinsulin - isolation & purification Proinsulin - metabolism Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - metabolism |
| title | Secretory expression and characterization of insulin in Pichia pastoris |
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