Expression and Site-Specific Mutagenesis of the Poliovirus 3C Protease in Escherichia coli
We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expres...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1986-08, Vol.83 (15), p.5392-5396 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Ivanoff, Lucinda A. Towatari, Takae Ray, Jasodhara Korant, Bruce D. Petteway, Stephen R. |
| description | We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity. |
| doi_str_mv | 10.1073/pnas.83.15.5392 |
| format | Article |
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The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.83.15.5392</identifier><identifier>PMID: 3016701</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Active sites ; Amino acids ; Biological and medical sciences ; Biotechnology ; Cloning, Molecular ; Codons ; DNA ; DNA - genetics ; DNA, Viral - genetics ; Enzyme Precursors - genetics ; Enzymes ; Escherichia coli ; Fundamental and applied biological sciences. Psychology ; Genetic engineering ; Genetic mutation ; Genetic technics ; Genetic Vectors ; HeLa cells ; Methods. Procedures. Technologies ; Molecular cloning ; Mutation ; Peptide Hydrolases - genetics ; Poliovirus ; Poliovirus - enzymology ; Poliovirus - genetics ; Protein precursors ; Protein Processing, Post-Translational ; Proteins ; Structure-Activity Relationship ; Viral Proteins - genetics</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1986-08, Vol.83 (15), p.5392-5396</ispartof><rights>1986 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c521t-ab2bbe8c66906a6d3cdd1ada45b76a1d5cb2ba46fa43197de6236aed1660e1dc3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/83/15.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/27644$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/27644$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27924,27925,53791,53793,58017,58250</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8794913$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3016701$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ivanoff, Lucinda A.</creatorcontrib><creatorcontrib>Towatari, Takae</creatorcontrib><creatorcontrib>Ray, Jasodhara</creatorcontrib><creatorcontrib>Korant, Bruce D.</creatorcontrib><creatorcontrib>Petteway, Stephen R.</creatorcontrib><title>Expression and Site-Specific Mutagenesis of the Poliovirus 3C Protease in Escherichia coli</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.</description><subject>Active sites</subject><subject>Amino acids</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cloning, Molecular</subject><subject>Codons</subject><subject>DNA</subject><subject>DNA - genetics</subject><subject>DNA, Viral - genetics</subject><subject>Enzyme Precursors - genetics</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genetic engineering</subject><subject>Genetic mutation</subject><subject>Genetic technics</subject><subject>Genetic Vectors</subject><subject>HeLa cells</subject><subject>Methods. Procedures. Technologies</subject><subject>Molecular cloning</subject><subject>Mutation</subject><subject>Peptide Hydrolases - genetics</subject><subject>Poliovirus</subject><subject>Poliovirus - enzymology</subject><subject>Poliovirus - genetics</subject><subject>Protein precursors</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteins</subject><subject>Structure-Activity Relationship</subject><subject>Viral Proteins - genetics</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp90c2L1DAYBvAgyjqungVByUH01Nl8tGl78CDD-AErLqxevIS3ydudLJ2mm6TL-t_bMqXoxVMOz-_JG_IS8pKzLWelvBh6iNtKbnmxLWQtHpENZzXPVF6zx2TDmCizKhf5U_IsxlvGWF1U7IycScZVyfiG_No_DAFjdL6n0Ft67RJm1wMa1zpDv40JbrDH6CL1LU0HpFe-c_7ehTFSuaNXwSeEiNT1dB_NAYMzBwfUTOo5edJCF_HFcp6Tn5_2P3Zfssvvn7_uPl5mphA8ZdCIpsHKKFUzBcpKYy0HC3nRlAq4LcwEIFct5JLXpUUlpAK0XCmG3Bp5Tj6c7h3G5ojWYJ8CdHoI7gjht_bg9L9J7w76xt9rWSlRi6n_bukHfzdiTProosGugx79GDUvGBeFkBO8OEETfIwB23UGZ3rehp63oSs5VfS8janx-u-nrX75_il_u-QQDXRtgN64uLKqrPOaz4PfL2y-f03XOboduy7hQ5rkm__KCbw6gduYfFiFKFWeyz_RVrYf</recordid><startdate>19860801</startdate><enddate>19860801</enddate><creator>Ivanoff, Lucinda A.</creator><creator>Towatari, Takae</creator><creator>Ray, Jasodhara</creator><creator>Korant, Bruce D.</creator><creator>Petteway, Stephen R.</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</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>7U9</scope><scope>H94</scope><scope>5PM</scope></search><sort><creationdate>19860801</creationdate><title>Expression and Site-Specific Mutagenesis of the Poliovirus 3C Protease in Escherichia coli</title><author>Ivanoff, Lucinda A. ; Towatari, Takae ; Ray, Jasodhara ; Korant, Bruce D. ; Petteway, Stephen R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c521t-ab2bbe8c66906a6d3cdd1ada45b76a1d5cb2ba46fa43197de6236aed1660e1dc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>Active sites</topic><topic>Amino acids</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Cloning, Molecular</topic><topic>Codons</topic><topic>DNA</topic><topic>DNA - genetics</topic><topic>DNA, Viral - genetics</topic><topic>Enzyme Precursors - genetics</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genetic engineering</topic><topic>Genetic mutation</topic><topic>Genetic technics</topic><topic>Genetic Vectors</topic><topic>HeLa cells</topic><topic>Methods. Procedures. Technologies</topic><topic>Molecular cloning</topic><topic>Mutation</topic><topic>Peptide Hydrolases - genetics</topic><topic>Poliovirus</topic><topic>Poliovirus - enzymology</topic><topic>Poliovirus - genetics</topic><topic>Protein precursors</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteins</topic><topic>Structure-Activity Relationship</topic><topic>Viral Proteins - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ivanoff, Lucinda A.</creatorcontrib><creatorcontrib>Towatari, Takae</creatorcontrib><creatorcontrib>Ray, Jasodhara</creatorcontrib><creatorcontrib>Korant, Bruce D.</creatorcontrib><creatorcontrib>Petteway, Stephen R.</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>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ivanoff, Lucinda A.</au><au>Towatari, Takae</au><au>Ray, Jasodhara</au><au>Korant, Bruce D.</au><au>Petteway, Stephen R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression and Site-Specific Mutagenesis of the Poliovirus 3C Protease in Escherichia coli</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1986-08-01</date><risdate>1986</risdate><volume>83</volume><issue>15</issue><spage>5392</spage><epage>5396</epage><pages>5392-5396</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>3016701</pmid><doi>10.1073/pnas.83.15.5392</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Active sites Amino acids Biological and medical sciences Biotechnology Cloning, Molecular Codons DNA DNA - genetics DNA, Viral - genetics Enzyme Precursors - genetics Enzymes Escherichia coli Fundamental and applied biological sciences. Psychology Genetic engineering Genetic mutation Genetic technics Genetic Vectors HeLa cells Methods. Procedures. Technologies Molecular cloning Mutation Peptide Hydrolases - genetics Poliovirus Poliovirus - enzymology Poliovirus - genetics Protein precursors Protein Processing, Post-Translational Proteins Structure-Activity Relationship Viral Proteins - genetics |
| title | Expression and Site-Specific Mutagenesis of the Poliovirus 3C Protease in Escherichia coli |
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