The conformation of mature human α-amylase conditions its secretion from yeast
The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary α-amylase (Amy) under control of the yeast PH05 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretio...
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| Veröffentlicht in: | Gene 1989-11, Vol.83 (2), p.355-365 |
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| creator | Takaaki, Sato Haruki, Uemura Yoshitaka, Izumoto Junji, Nakao Yusuke, Nakamura Kenichi, Matsubara |
| description | The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary α-amylase (Amy) under control of the yeast
PH05 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted
Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted. |
| doi_str_mv | 10.1016/0378-1119(89)90122-4 |
| format | Article |
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PH05 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted
Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.</description><identifier>ISSN: 0378-1119</identifier><identifier>EISSN: 1879-0038</identifier><identifier>DOI: 10.1016/0378-1119(89)90122-4</identifier><identifier>PMID: 2684791</identifier><identifier>CODEN: GENED6</identifier><language>eng</language><publisher>Lausanne: Elsevier B.V</publisher><subject>alpha-Amylases - biosynthesis ; alpha-Amylases - genetics ; alpha-Amylases - metabolism ; Amino Acid Sequence ; Base Sequence ; Biological and medical sciences ; Biotechnology ; Chromosome Deletion ; DNA - genetics ; Fundamental and applied biological sciences. Psychology ; Genes ; Genetic engineering ; Genetic technics ; Genotype ; glycosylation ; human salivary enzymes ; Humans ; Methods. Procedures. Technologies ; Miscellaneous ; Molecular Sequence Data ; Mutation ; N-terminal pyroglutamate ; Oligonucleotide Probes ; Plasmids ; Promoter Regions, Genetic ; Protein Conformation ; Recombinant DNA ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - metabolism ; Restriction Mapping ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - genetics ; Salivary Glands - metabolism ; secretory pathway ; signal peptide sequence ; Synthetic digonucleotides and genes. Sequencing</subject><ispartof>Gene, 1989-11, Vol.83 (2), p.355-365</ispartof><rights>1989</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-a00e95d77e32a4be04f8d902448edf5f102545336ab6505026e9460aea66919d3</citedby><cites>FETCH-LOGICAL-c386t-a00e95d77e32a4be04f8d902448edf5f102545336ab6505026e9460aea66919d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0378-1119(89)90122-4$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6620346$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2684791$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Takaaki, Sato</creatorcontrib><creatorcontrib>Haruki, Uemura</creatorcontrib><creatorcontrib>Yoshitaka, Izumoto</creatorcontrib><creatorcontrib>Junji, Nakao</creatorcontrib><creatorcontrib>Yusuke, Nakamura</creatorcontrib><creatorcontrib>Kenichi, Matsubara</creatorcontrib><title>The conformation of mature human α-amylase conditions its secretion from yeast</title><title>Gene</title><addtitle>Gene</addtitle><description>The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary α-amylase (Amy) under control of the yeast
PH05 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted
Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.</description><subject>alpha-Amylases - biosynthesis</subject><subject>alpha-Amylases - genetics</subject><subject>alpha-Amylases - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chromosome Deletion</subject><subject>DNA - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>Genotype</subject><subject>glycosylation</subject><subject>human salivary enzymes</subject><subject>Humans</subject><subject>Methods. Procedures. Technologies</subject><subject>Miscellaneous</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>N-terminal pyroglutamate</subject><subject>Oligonucleotide Probes</subject><subject>Plasmids</subject><subject>Promoter Regions, Genetic</subject><subject>Protein Conformation</subject><subject>Recombinant DNA</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - metabolism</subject><subject>Restriction Mapping</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Salivary Glands - metabolism</subject><subject>secretory pathway</subject><subject>signal peptide sequence</subject><subject>Synthetic digonucleotides and genes. Sequencing</subject><issn>0378-1119</issn><issn>1879-0038</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kMtq3DAUhkVJmUwmfYMGvAglXbg5uliWNoEwpBcIZDNdC418RFRsK5HswDxWX6TPVHtmmGXORgfO9_-Ij5DPFL5RoPIWeK1KSqm-UfqrBspYKT6QJVW1LgG4OiPLE3JOLnL-A9NUFVuQBZNK1JouydPmGQsXex9TZ4cQ-yL6YtrGhMXz2Nm--Pe3tN2utXnPNWGGchGGXGR0CfcZn2JX7NDm4ZJ89LbN-On4rsjv7w-b9c_y8enHr_X9Y-m4kkNpAVBXTV0jZ1ZsEYRXjQYmhMLGV54Cq0TFubRbWUEFTKIWEixaKTXVDV-RL4felxRfR8yD6UJ22La2xzhmU2vOazpVrIg4gC7FnBN685JCZ9POUDCzRzNLMrMko7TZezRiil0d-8dth80pdBQ33a-Pd5udbX2yvQv5hEnJgAs5YXcHDCcXbwGTyS5g77AJCd1gmhje_8d_VKCOmA</recordid><startdate>19891130</startdate><enddate>19891130</enddate><creator>Takaaki, Sato</creator><creator>Haruki, Uemura</creator><creator>Yoshitaka, Izumoto</creator><creator>Junji, Nakao</creator><creator>Yusuke, Nakamura</creator><creator>Kenichi, Matsubara</creator><general>Elsevier B.V</general><general>Elsevier</general><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>7X8</scope></search><sort><creationdate>19891130</creationdate><title>The conformation of mature human α-amylase conditions its secretion from yeast</title><author>Takaaki, Sato ; Haruki, Uemura ; Yoshitaka, Izumoto ; Junji, Nakao ; Yusuke, Nakamura ; Kenichi, Matsubara</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-a00e95d77e32a4be04f8d902448edf5f102545336ab6505026e9460aea66919d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>alpha-Amylases - biosynthesis</topic><topic>alpha-Amylases - genetics</topic><topic>alpha-Amylases - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Chromosome Deletion</topic><topic>DNA - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genes</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>Genotype</topic><topic>glycosylation</topic><topic>human salivary enzymes</topic><topic>Humans</topic><topic>Methods. Procedures. Technologies</topic><topic>Miscellaneous</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>N-terminal pyroglutamate</topic><topic>Oligonucleotide Probes</topic><topic>Plasmids</topic><topic>Promoter Regions, Genetic</topic><topic>Protein Conformation</topic><topic>Recombinant DNA</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - metabolism</topic><topic>Restriction Mapping</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Salivary Glands - metabolism</topic><topic>secretory pathway</topic><topic>signal peptide sequence</topic><topic>Synthetic digonucleotides and genes. Sequencing</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Takaaki, Sato</creatorcontrib><creatorcontrib>Haruki, Uemura</creatorcontrib><creatorcontrib>Yoshitaka, Izumoto</creatorcontrib><creatorcontrib>Junji, Nakao</creatorcontrib><creatorcontrib>Yusuke, Nakamura</creatorcontrib><creatorcontrib>Kenichi, Matsubara</creatorcontrib><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>MEDLINE - Academic</collection><jtitle>Gene</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Takaaki, Sato</au><au>Haruki, Uemura</au><au>Yoshitaka, Izumoto</au><au>Junji, Nakao</au><au>Yusuke, Nakamura</au><au>Kenichi, Matsubara</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The conformation of mature human α-amylase conditions its secretion from yeast</atitle><jtitle>Gene</jtitle><addtitle>Gene</addtitle><date>1989-11-30</date><risdate>1989</risdate><volume>83</volume><issue>2</issue><spage>355</spage><epage>365</epage><pages>355-365</pages><issn>0378-1119</issn><eissn>1879-0038</eissn><coden>GENED6</coden><abstract>The yeast Saccharomyces cerevisiae expresses the cloned cDNA (Amy) encoding human salivary α-amylase (Amy) under control of the yeast
PH05 promoter, and secretes the active enzyme into the culture medium. Two approaches were utilized to define the moiety of Amy, which is required for proper secretion and glycosylation. In one approach, chimeras were constructed with a variety of secretion signal sequences (yeast mating factor precursor sequence, yeast acid phosphatase signal sequence and human gastrin signal sequence) fused to the secretion signal-deleted
Amy cDNA. The other approach involved analysis of a set of deletion series and a set of point mutations in the Amy-encoding region. The results showed that heterologous signal sequences were sufficient for proper secretion in yeast, irrespective of the insertion of some extra amino acids. In most cases, enzymes with deletions and Cys-465 substitution were not secreted, even though they had complete secretion signal sequences. Instead, they accumulated in the cell in a glycosylated form. Thus, proper secretion seems to require an appropriate conformation in the polypeptide moiety to be secreted.</abstract><cop>Lausanne</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>2684791</pmid><doi>10.1016/0378-1119(89)90122-4</doi><tpages>11</tpages></addata></record> |
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| subjects | alpha-Amylases - biosynthesis alpha-Amylases - genetics alpha-Amylases - metabolism Amino Acid Sequence Base Sequence Biological and medical sciences Biotechnology Chromosome Deletion DNA - genetics Fundamental and applied biological sciences. Psychology Genes Genetic engineering Genetic technics Genotype glycosylation human salivary enzymes Humans Methods. Procedures. Technologies Miscellaneous Molecular Sequence Data Mutation N-terminal pyroglutamate Oligonucleotide Probes Plasmids Promoter Regions, Genetic Protein Conformation Recombinant DNA Recombinant Proteins - biosynthesis Recombinant Proteins - metabolism Restriction Mapping Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Salivary Glands - metabolism secretory pathway signal peptide sequence Synthetic digonucleotides and genes. Sequencing |
| title | The conformation of mature human α-amylase conditions its secretion from yeast |
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