Development of an optimized refolding process for recombinant Ala–Glu–IGF-1
Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant...
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| Veröffentlicht in: | Protein engineering 1992-12, Vol.5 (8), p.797-806 |
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| container_title | Protein engineering |
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| creator | Hejnaes, Kim Ry Bayne, Stephen Nørskov, Leif Holmegaard, Hans Sørensen, H.H. Thomsen, Johannes Schäffer, Lauge Wollmer, Axel Skriver, Lars |
| description | Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w)was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements: (i) Cys6–Cys47, Cys18–Cys61, Cys48–Cys52; or (if) Cys6–Cys52, Cysl8–Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two. |
| doi_str_mv | 10.1093/protein/5.8.797 |
| format | Article |
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The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w)was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements: (i) Cys6–Cys47, Cys18–Cys61, Cys48–Cys52; or (if) Cys6–Cys52, Cysl8–Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.</description><identifier>ISSN: 1741-0126</identifier><identifier>ISSN: 0269-2139</identifier><identifier>EISSN: 1741-0134</identifier><identifier>EISSN: 1460-213X</identifier><identifier>DOI: 10.1093/protein/5.8.797</identifier><identifier>PMID: 1287661</identifier><identifier>CODEN: PRENE9</identifier><language>eng</language><publisher>Oxford: Oxford University Press</publisher><subject>Amino Acid Sequence ; Aminopeptidases - pharmacology ; Binding, Competitive ; Biological and medical sciences ; Circular Dichroism ; Conformational dynamics in molecular biology ; Cysteine ; Endopeptidases - genetics ; energy minimization ; Escherichia coli ; folding ; Fundamental and applied biological sciences. Psychology ; Humans ; Insulin-Like Growth Factor I - analogs & derivatives ; Insulin-Like Growth Factor I - drug effects ; Insulin-Like Growth Factor I - genetics ; Insulin-Like Growth Factor I - metabolism ; Models, Molecular ; Molecular biophysics ; molecular modelling ; Molecular Sequence Data ; Molecular Weight ; Peptide Mapping ; Protein Denaturation ; Protein Engineering ; Protein Folding ; Receptor, IGF Type 1 - metabolism ; recombinant human IGF-1 ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Sequence Homology, Amino Acid</subject><ispartof>Protein engineering, 1992-12, Vol.5 (8), p.797-806</ispartof><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c390t-418b51d3b200210ff5f60460a2c11f2d4aaa2b1ff5e82acc6a516ec020a0afda3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4587224$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1287661$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hejnaes, Kim Ry</creatorcontrib><creatorcontrib>Bayne, Stephen</creatorcontrib><creatorcontrib>Nørskov, Leif</creatorcontrib><creatorcontrib>Holmegaard, Hans</creatorcontrib><creatorcontrib>Sørensen, H.H.</creatorcontrib><creatorcontrib>Thomsen, Johannes</creatorcontrib><creatorcontrib>Schäffer, Lauge</creatorcontrib><creatorcontrib>Wollmer, Axel</creatorcontrib><creatorcontrib>Skriver, Lars</creatorcontrib><title>Development of an optimized refolding process for recombinant Ala–Glu–IGF-1</title><title>Protein engineering</title><addtitle>Protein Eng</addtitle><description>Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w)was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements: (i) Cys6–Cys47, Cys18–Cys61, Cys48–Cys52; or (if) Cys6–Cys52, Cysl8–Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.</description><subject>Amino Acid Sequence</subject><subject>Aminopeptidases - pharmacology</subject><subject>Binding, Competitive</subject><subject>Biological and medical sciences</subject><subject>Circular Dichroism</subject><subject>Conformational dynamics in molecular biology</subject><subject>Cysteine</subject><subject>Endopeptidases - genetics</subject><subject>energy minimization</subject><subject>Escherichia coli</subject><subject>folding</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Insulin-Like Growth Factor I - analogs & derivatives</subject><subject>Insulin-Like Growth Factor I - drug effects</subject><subject>Insulin-Like Growth Factor I - genetics</subject><subject>Insulin-Like Growth Factor I - metabolism</subject><subject>Models, Molecular</subject><subject>Molecular biophysics</subject><subject>molecular modelling</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Peptide Mapping</subject><subject>Protein Denaturation</subject><subject>Protein Engineering</subject><subject>Protein Folding</subject><subject>Receptor, IGF Type 1 - metabolism</subject><subject>recombinant human IGF-1</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Homology, Amino Acid</subject><issn>1741-0126</issn><issn>0269-2139</issn><issn>1741-0134</issn><issn>1460-213X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkMtu1DAUhi1E1ZbCmhVSFqi7zPj4mllWvaSVpiqIIiE21oljI5cknsaZClj1HfqGPAmuZjQsu_Gxzv-d20_Ie6AzoAs-X41xcmGYy1k10wv9ihyCFlBS4OL17s_UAXmT0h2lTGmAfbIPrNJKwSG5OXMProur3g1TEX2BQxFXU-jDH9cWo_Oxa8Pwo8hjrEup8HHMWRv7JgyYK046_Pv4VHfr_F7VFyW8JXseu-TebeMR-Xpxfnt6WS5v6qvTk2Vp-YJOpYCqkdDyhuWdgHovvaJCUWQWwLNWICJrIOddxdBahRKUs5RRpOhb5EfkeNM3b3a_dmkyfUjWdR0OLq6T0VxUgkr1IghKLCiTMoPzDWjHmFI-3azG0OP42wA1z16brddGmspkr3PFh23rddO79j-_MTfrH7c6JoudH3GwIe0wISvNmMhYucFCmtyvnYzjT6M019Jcfvtuvlzz22Vdfzaf-D9EFJno</recordid><startdate>19921201</startdate><enddate>19921201</enddate><creator>Hejnaes, Kim Ry</creator><creator>Bayne, Stephen</creator><creator>Nørskov, Leif</creator><creator>Holmegaard, Hans</creator><creator>Sørensen, H.H.</creator><creator>Thomsen, Johannes</creator><creator>Schäffer, Lauge</creator><creator>Wollmer, Axel</creator><creator>Skriver, Lars</creator><general>Oxford University 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>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>19921201</creationdate><title>Development of an optimized refolding process for recombinant Ala–Glu–IGF-1</title><author>Hejnaes, Kim Ry ; Bayne, Stephen ; Nørskov, Leif ; Holmegaard, Hans ; Sørensen, H.H. ; Thomsen, Johannes ; Schäffer, Lauge ; Wollmer, Axel ; Skriver, Lars</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c390t-418b51d3b200210ff5f60460a2c11f2d4aaa2b1ff5e82acc6a516ec020a0afda3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Amino Acid Sequence</topic><topic>Aminopeptidases - pharmacology</topic><topic>Binding, Competitive</topic><topic>Biological and medical sciences</topic><topic>Circular Dichroism</topic><topic>Conformational dynamics in molecular biology</topic><topic>Cysteine</topic><topic>Endopeptidases - genetics</topic><topic>energy minimization</topic><topic>Escherichia coli</topic><topic>folding</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Insulin-Like Growth Factor I - analogs & derivatives</topic><topic>Insulin-Like Growth Factor I - drug effects</topic><topic>Insulin-Like Growth Factor I - genetics</topic><topic>Insulin-Like Growth Factor I - metabolism</topic><topic>Models, Molecular</topic><topic>Molecular biophysics</topic><topic>molecular modelling</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Peptide Mapping</topic><topic>Protein Denaturation</topic><topic>Protein Engineering</topic><topic>Protein Folding</topic><topic>Receptor, IGF Type 1 - metabolism</topic><topic>recombinant human IGF-1</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Homology, Amino Acid</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hejnaes, Kim Ry</creatorcontrib><creatorcontrib>Bayne, Stephen</creatorcontrib><creatorcontrib>Nørskov, Leif</creatorcontrib><creatorcontrib>Holmegaard, Hans</creatorcontrib><creatorcontrib>Sørensen, H.H.</creatorcontrib><creatorcontrib>Thomsen, Johannes</creatorcontrib><creatorcontrib>Schäffer, Lauge</creatorcontrib><creatorcontrib>Wollmer, Axel</creatorcontrib><creatorcontrib>Skriver, Lars</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Protein engineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hejnaes, Kim Ry</au><au>Bayne, Stephen</au><au>Nørskov, Leif</au><au>Holmegaard, Hans</au><au>Sørensen, H.H.</au><au>Thomsen, Johannes</au><au>Schäffer, Lauge</au><au>Wollmer, Axel</au><au>Skriver, Lars</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of an optimized refolding process for recombinant Ala–Glu–IGF-1</atitle><jtitle>Protein engineering</jtitle><addtitle>Protein Eng</addtitle><date>1992-12-01</date><risdate>1992</risdate><volume>5</volume><issue>8</issue><spage>797</spage><epage>806</epage><pages>797-806</pages><issn>1741-0126</issn><issn>0269-2139</issn><eissn>1741-0134</eissn><eissn>1460-213X</eissn><coden>PRENE9</coden><abstract>Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding. The renaturation process was shown to be a function of the redox potential of the solution. Folding by different methods had no significant effect on the renaturation. A maximal yield of 60% (w/w)was obtained. The folded AE-IGF-1 was enzymatically converted to IGF-1. The major by-product (20% w/w) was identified as scrambled IGF-1. Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements: (i) Cys6–Cys47, Cys18–Cys61, Cys48–Cys52; or (if) Cys6–Cys52, Cysl8–Cys61, Cys47 and Cys48 being in their reduced forms. Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two.</abstract><cop>Oxford</cop><pub>Oxford University Press</pub><pmid>1287661</pmid><doi>10.1093/protein/5.8.797</doi><tpages>10</tpages></addata></record> |
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| source | Oxford University Press Journals Digital Archive legacy; MEDLINE; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Aminopeptidases - pharmacology Binding, Competitive Biological and medical sciences Circular Dichroism Conformational dynamics in molecular biology Cysteine Endopeptidases - genetics energy minimization Escherichia coli folding Fundamental and applied biological sciences. Psychology Humans Insulin-Like Growth Factor I - analogs & derivatives Insulin-Like Growth Factor I - drug effects Insulin-Like Growth Factor I - genetics Insulin-Like Growth Factor I - metabolism Models, Molecular Molecular biophysics molecular modelling Molecular Sequence Data Molecular Weight Peptide Mapping Protein Denaturation Protein Engineering Protein Folding Receptor, IGF Type 1 - metabolism recombinant human IGF-1 Recombinant Proteins - genetics Recombinant Proteins - metabolism Sequence Homology, Amino Acid |
| title | Development of an optimized refolding process for recombinant Ala–Glu–IGF-1 |
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