Recombinant Human Pancreatic Ribonuclease Produced in E. coli : Importance of the Amino-Terminal Sequence
Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-...
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| Veröffentlicht in: | Biochemical and biophysical research communications 1995-11, Vol.216 (1), p.406-413 |
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| creator | Futami, J. Seno, M. Kosaka, M. Tada, H. Seno, S. Yamada, H. |
| description | Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein. |
| doi_str_mv | 10.1006/bbrc.1995.2638 |
| format | Article |
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The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1006/bbrc.1995.2638</identifier><identifier>PMID: 7488119</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Animals ; Base Sequence ; Cattle ; Chromatography, Ion Exchange ; Circular Dichroism ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Humans ; Kinetics ; Molecular Sequence Data ; Molecular Weight ; Mutagenesis ; Mutagenesis, Insertional ; Oligodeoxyribonucleotides ; Plasmids ; Promoter Regions, Genetic ; Protein Conformation ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - isolation & purification ; Ribonuclease, Pancreatic - biosynthesis ; Ribonuclease, Pancreatic - chemistry ; Ribonuclease, Pancreatic - isolation & purification ; Sequence Deletion ; Substrate Specificity</subject><ispartof>Biochemical and biophysical research communications, 1995-11, Vol.216 (1), p.406-413</ispartof><rights>1995 Academic Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c349t-8e60d0a37f3c3635c73ebfa5e08d0f6acf87f9dd948e51d340c7cd84238b209a3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0006291X85726381$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7488119$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Futami, J.</creatorcontrib><creatorcontrib>Seno, M.</creatorcontrib><creatorcontrib>Kosaka, M.</creatorcontrib><creatorcontrib>Tada, H.</creatorcontrib><creatorcontrib>Seno, S.</creatorcontrib><creatorcontrib>Yamada, H.</creatorcontrib><title>Recombinant Human Pancreatic Ribonuclease Produced in E. coli : Importance of the Amino-Terminal Sequence</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Cattle</subject><subject>Chromatography, Ion Exchange</subject><subject>Circular Dichroism</subject><subject>Cloning, Molecular</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Escherichia coli</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Insertional</subject><subject>Oligodeoxyribonucleotides</subject><subject>Plasmids</subject><subject>Promoter Regions, Genetic</subject><subject>Protein Conformation</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Ribonuclease, Pancreatic - biosynthesis</subject><subject>Ribonuclease, Pancreatic - chemistry</subject><subject>Ribonuclease, Pancreatic - isolation & purification</subject><subject>Sequence Deletion</subject><subject>Substrate Specificity</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kF1LwzAUhoMoc05vvRPyB1qTph-Jd2NMNxAcc4J3JT05wcjazLQV_Pe2bHjn1XvxfnDOQ8gtZzFnLL-vqgAxVyqLk1zIMzLlTLEo4Sw9J1M2JKJE8fdLctW2n4xxnuZqQiZFKiXnakrcFsHXlWt009FVX-uGbnQDAXXngG5d5Zse9qhbpJvgTQ9oqGvoMqbg944-0HV98KEbKki9pd0H0nntGh_tMAyq9_QVv3oc7GtyYfW-xZuTzsjb43K3WEXPL0_rxfw5ApGqLpKYM8O0KKwAkYsMCoGV1RkyaZjNNVhZWGWMSiVm3IiUQQFGpomQVcKUFjMSH3ch-LYNaMtDcLUOPyVn5YisHJGVI7JyRDYU7o6FQ1_VaP7iJ0aDL48-Dld_OwxlC278yLiA0JXGu_-mfwHqCnuR</recordid><startdate>19951102</startdate><enddate>19951102</enddate><creator>Futami, J.</creator><creator>Seno, M.</creator><creator>Kosaka, M.</creator><creator>Tada, H.</creator><creator>Seno, S.</creator><creator>Yamada, H.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>19951102</creationdate><title>Recombinant Human Pancreatic Ribonuclease Produced in E. coli : Importance of the Amino-Terminal Sequence</title><author>Futami, J. ; Seno, M. ; Kosaka, M. ; Tada, H. ; Seno, S. ; Yamada, H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c349t-8e60d0a37f3c3635c73ebfa5e08d0f6acf87f9dd948e51d340c7cd84238b209a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Cattle</topic><topic>Chromatography, Ion Exchange</topic><topic>Circular Dichroism</topic><topic>Cloning, Molecular</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Escherichia coli</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>Mutagenesis</topic><topic>Mutagenesis, Insertional</topic><topic>Oligodeoxyribonucleotides</topic><topic>Plasmids</topic><topic>Promoter Regions, Genetic</topic><topic>Protein Conformation</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Ribonuclease, Pancreatic - biosynthesis</topic><topic>Ribonuclease, Pancreatic - chemistry</topic><topic>Ribonuclease, Pancreatic - isolation & purification</topic><topic>Sequence Deletion</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Futami, J.</creatorcontrib><creatorcontrib>Seno, M.</creatorcontrib><creatorcontrib>Kosaka, M.</creatorcontrib><creatorcontrib>Tada, H.</creatorcontrib><creatorcontrib>Seno, S.</creatorcontrib><creatorcontrib>Yamada, H.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Futami, J.</au><au>Seno, M.</au><au>Kosaka, M.</au><au>Tada, H.</au><au>Seno, S.</au><au>Yamada, H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Recombinant Human Pancreatic Ribonuclease Produced in E. coli : Importance of the Amino-Terminal Sequence</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>1995-11-02</date><risdate>1995</risdate><volume>216</volume><issue>1</issue><spage>406</spage><epage>413</epage><pages>406-413</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><abstract>Human pancreatic ribonuclease I (hRNase 1) in the mature form has been produced in E.coli using T7 expression system. The recombinant hRNase 1 protein was solubilized from the inclusion bodies, refolded in glutathione redox system, and purified through chromatographic procedures by utilizing cation-exchange and reversed-phase columns. The ribonucleolytic activity of recombinant hRNase 1 was examined on yeast RNA and cytidylyl-3′,5′-adenosine revealing the distinctive ribonucleolytic activity. The activity was perfectly inhibited by human placental RNase inhibitor. Truncation of 7 amino acid residues in the amino-terminal sequence resulted in much reduction in ribonucleolytic activity and in affinity to human placental RNase inhibitor with the disintegration of secondary structures of the protein observed by circular dichroism spectra. The present study has revealed the important contribution of the amino-terminal sequence of hRNase I to the characteristics of the protein.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>7488119</pmid><doi>10.1006/bbrc.1995.2638</doi><tpages>8</tpages></addata></record> |
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| ispartof | Biochemical and biophysical research communications, 1995-11, Vol.216 (1), p.406-413 |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Amino Acid Sequence Animals Base Sequence Cattle Chromatography, Ion Exchange Circular Dichroism Cloning, Molecular Electrophoresis, Polyacrylamide Gel Escherichia coli Humans Kinetics Molecular Sequence Data Molecular Weight Mutagenesis Mutagenesis, Insertional Oligodeoxyribonucleotides Plasmids Promoter Regions, Genetic Protein Conformation Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Ribonuclease, Pancreatic - biosynthesis Ribonuclease, Pancreatic - chemistry Ribonuclease, Pancreatic - isolation & purification Sequence Deletion Substrate Specificity |
| title | Recombinant Human Pancreatic Ribonuclease Produced in E. coli : Importance of the Amino-Terminal Sequence |
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