Purification and characterization of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae
Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted...
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| Veröffentlicht in: | European journal of biochemistry 1991-03, Vol.196 (2), p.431-438 |
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| container_title | European journal of biochemistry |
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| creator | Steven, J. (Delta Biotechnology Ltd., Nottingham, England) Cottingham, I.R Berry, S.J Chinery, S.A Goodey, A.R Courtney, M Ballance, D.J |
| description | Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 X 10(6) M-1 s-1, and for two-chain tPA, 0.7 X 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases |
| doi_str_mv | 10.1111/j.1432-1033.1991.tb15834.x |
| format | Article |
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(Delta Biotechnology Ltd., Nottingham, England) ; Cottingham, I.R ; Berry, S.J ; Chinery, S.A ; Goodey, A.R ; Courtney, M ; Ballance, D.J</creator><creatorcontrib>Steven, J. (Delta Biotechnology Ltd., Nottingham, England) ; Cottingham, I.R ; Berry, S.J ; Chinery, S.A ; Goodey, A.R ; Courtney, M ; Ballance, D.J</creatorcontrib><description>Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 X 10(6) M-1 s-1, and for two-chain tPA, 0.7 X 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases</description><identifier>ISSN: 0014-2956</identifier><identifier>EISSN: 1432-1033</identifier><identifier>DOI: 10.1111/j.1432-1033.1991.tb15834.x</identifier><identifier>PMID: 1901039</identifier><identifier>CODEN: EJBCAI</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>ACTIVADOR PLASMINOGENO ; ACTIVATEUR DE PLASMINOGENE ; Analytical, structural and metabolic biochemistry ; Base Sequence ; Biological and medical sciences ; Chromatography, Ion Exchange ; DNA - genetics ; Electrophoresis, Polyacrylamide Gel ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Humans ; Hydrolases ; INHIBIDORES DE ENZIMAS ; INHIBITEUR D'ENZYME ; Kinetics ; Molecular Sequence Data ; Plasminogen Activators - antagonists & inhibitors ; Plasminogen Inactivators - isolation & purification ; Plasminogen Inactivators - metabolism ; PURIFICACION ; PURIFICATION ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; SACCHAROMYCES CEREVISIAE ; Saccharomyces cerevisiae - genetics ; Tissue Plasminogen Activator - metabolism ; Urokinase-Type Plasminogen Activator - metabolism</subject><ispartof>European journal of biochemistry, 1991-03, Vol.196 (2), p.431-438</ispartof><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4711-3f8d104d19cd8389a57c75d96a82d4a9ff0b72a4ac29116cae48dec56f3405633</citedby><cites>FETCH-LOGICAL-c4711-3f8d104d19cd8389a57c75d96a82d4a9ff0b72a4ac29116cae48dec56f3405633</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19638955$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1901039$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Steven, J. (Delta Biotechnology Ltd., Nottingham, England)</creatorcontrib><creatorcontrib>Cottingham, I.R</creatorcontrib><creatorcontrib>Berry, S.J</creatorcontrib><creatorcontrib>Chinery, S.A</creatorcontrib><creatorcontrib>Goodey, A.R</creatorcontrib><creatorcontrib>Courtney, M</creatorcontrib><creatorcontrib>Ballance, D.J</creatorcontrib><title>Purification and characterization of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae</title><title>European journal of biochemistry</title><addtitle>Eur J Biochem</addtitle><description>Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 X 10(6) M-1 s-1, and for two-chain tPA, 0.7 X 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases</description><subject>ACTIVADOR PLASMINOGENO</subject><subject>ACTIVATEUR DE PLASMINOGENE</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Chromatography, Ion Exchange</subject><subject>DNA - genetics</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>Humans</subject><subject>Hydrolases</subject><subject>INHIBIDORES DE ENZIMAS</subject><subject>INHIBITEUR D'ENZYME</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Plasminogen Activators - antagonists & inhibitors</subject><subject>Plasminogen Inactivators - isolation & purification</subject><subject>Plasminogen Inactivators - metabolism</subject><subject>PURIFICACION</subject><subject>PURIFICATION</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>SACCHAROMYCES CEREVISIAE</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Tissue Plasminogen Activator - metabolism</subject><subject>Urokinase-Type Plasminogen Activator - metabolism</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkFtv1DAQhS0EKtvCH0BCipDoW4IndhKbFwRVWypVAmnpszXrS-sll8VOSpdfj6OsCq_1i60535wZH0LeAS0gnQ_bAjgrc6CMFSAlFOMGKsF48fCMrB6l52RFKfC8lFX9khzHuKWU1rJujsgRSJoQuSI_v0_BO69x9EOfYW8yfYcB9WiD_7MUB5ftWoyd74dbmxg9-nsch5D5_s5v_Pwqs10YzKStScVsjXo2Gbq9tjHTNth7Hz3aV-SFwzba14f7hNxcnP84-5pff7u8Ovt8nWveAOTMCQOUG5DaCCYkVo1uKiNrFKXhKJ2jm6ZEjrqUALVGy4Wxuqod47SqGTshp4tvWurXZOOoOh-1bVvs7TBFJSgXQnKawI8LqMMQY7BO7YLvMOwVUDUnrbZqjlPNcao5aXVIWj2k5reHKdOms-Zf6xJt0t8fdIwaWxew1z7-h9Xpb1WVuE8L99u3dv-EDdTF-Zc1Z5Ac3iwODgeFtyFNuVlLYJSJhv0FLeSlRA</recordid><startdate>19910314</startdate><enddate>19910314</enddate><creator>Steven, J. (Delta Biotechnology Ltd., Nottingham, England)</creator><creator>Cottingham, I.R</creator><creator>Berry, S.J</creator><creator>Chinery, S.A</creator><creator>Goodey, A.R</creator><creator>Courtney, M</creator><creator>Ballance, D.J</creator><general>Blackwell Publishing Ltd</general><general>Blackwell</general><scope>FBQ</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>7X8</scope></search><sort><creationdate>19910314</creationdate><title>Purification and characterization of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae</title><author>Steven, J. (Delta Biotechnology Ltd., Nottingham, England) ; Cottingham, I.R ; Berry, S.J ; Chinery, S.A ; Goodey, A.R ; Courtney, M ; Ballance, D.J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4711-3f8d104d19cd8389a57c75d96a82d4a9ff0b72a4ac29116cae48dec56f3405633</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>ACTIVADOR PLASMINOGENO</topic><topic>ACTIVATEUR DE PLASMINOGENE</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Chromatography, Ion Exchange</topic><topic>DNA - genetics</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression</topic><topic>Humans</topic><topic>Hydrolases</topic><topic>INHIBIDORES DE ENZIMAS</topic><topic>INHIBITEUR D'ENZYME</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Plasminogen Activators - antagonists & inhibitors</topic><topic>Plasminogen Inactivators - isolation & purification</topic><topic>Plasminogen Inactivators - metabolism</topic><topic>PURIFICACION</topic><topic>PURIFICATION</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>SACCHAROMYCES CEREVISIAE</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Tissue Plasminogen Activator - metabolism</topic><topic>Urokinase-Type Plasminogen Activator - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Steven, J. (Delta Biotechnology Ltd., Nottingham, England)</creatorcontrib><creatorcontrib>Cottingham, I.R</creatorcontrib><creatorcontrib>Berry, S.J</creatorcontrib><creatorcontrib>Chinery, S.A</creatorcontrib><creatorcontrib>Goodey, A.R</creatorcontrib><creatorcontrib>Courtney, M</creatorcontrib><creatorcontrib>Ballance, D.J</creatorcontrib><collection>AGRIS</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>MEDLINE - Academic</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Steven, J. (Delta Biotechnology Ltd., Nottingham, England)</au><au>Cottingham, I.R</au><au>Berry, S.J</au><au>Chinery, S.A</au><au>Goodey, A.R</au><au>Courtney, M</au><au>Ballance, D.J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>1991-03-14</date><risdate>1991</risdate><volume>196</volume><issue>2</issue><spage>431</spage><epage>438</epage><pages>431-438</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><coden>EJBCAI</coden><abstract>Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 X 10(6) M-1 s-1, and for two-chain tPA, 0.7 X 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>1901039</pmid><doi>10.1111/j.1432-1033.1991.tb15834.x</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | European journal of biochemistry, 1991-03, Vol.196 (2), p.431-438 |
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| language | eng |
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| subjects | ACTIVADOR PLASMINOGENO ACTIVATEUR DE PLASMINOGENE Analytical, structural and metabolic biochemistry Base Sequence Biological and medical sciences Chromatography, Ion Exchange DNA - genetics Electrophoresis, Polyacrylamide Gel Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Gene Expression Humans Hydrolases INHIBIDORES DE ENZIMAS INHIBITEUR D'ENZYME Kinetics Molecular Sequence Data Plasminogen Activators - antagonists & inhibitors Plasminogen Inactivators - isolation & purification Plasminogen Inactivators - metabolism PURIFICACION PURIFICATION Recombinant Proteins - biosynthesis Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism SACCHAROMYCES CEREVISIAE Saccharomyces cerevisiae - genetics Tissue Plasminogen Activator - metabolism Urokinase-Type Plasminogen Activator - metabolism |
| title | Purification and characterization of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae |
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