High-level synthesis of the 12-kDa human FK506-binding protein in Escherichia coli using translational coupling
The gene encoding the 12-kDa cytosolic form of human FK506-binding protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the trc promoter vector, pKK233-2, yield...
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| Veröffentlicht in: | Gene 1993-06, Vol.128 (2), p.219-225 |
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| creator | Pilot-Matias, Tami J. Pratt, Steven D. Lane, Benjamin C. |
| description | The gene encoding the 12-kDa cytosolic form of human FK506-binding protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in
Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the
trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified
lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational coupling to an upstream segment of
kdsB under the control of this modified
lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was shown to have the authentic N terminus. The level of active re-hFKPB-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase. |
| doi_str_mv | 10.1016/0378-1119(93)90566-L |
| format | Article |
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Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the
trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified
lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational coupling to an upstream segment of
kdsB under the control of this modified
lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was shown to have the authentic N terminus. The level of active re-hFKPB-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.</description><identifier>ISSN: 0378-1119</identifier><identifier>EISSN: 1879-0038</identifier><identifier>DOI: 10.1016/0378-1119(93)90566-L</identifier><identifier>PMID: 7685727</identifier><identifier>CODEN: GENED6</identifier><language>eng</language><publisher>Lausanne: Elsevier B.V</publisher><subject>Alkaline Phosphatase - metabolism ; Ascomycin ; Base Sequence ; Binding, Competitive ; Biological and medical sciences ; Carrier Proteins - biosynthesis ; Carrier Proteins - genetics ; CMP-3-deoxy-D- manno-octulosonate cytidylyltransferase ; CTP ; dicistronic ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli - genetics ; Fundamental and applied biological sciences. Psychology ; Gene expression ; Gene Expression Regulation, Bacterial ; Genes ; Humans ; immunosuppressant ; Lac Operon ; Molecular and cellular biology ; Molecular genetics ; Molecular Sequence Data ; Nucleotidyltransferases - metabolism ; PCR ; Peptide Chain Initiation, Translational ; Plasmids ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Protein Biosynthesis ; rapamycin ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - genetics ; Sequence Analysis, DNA ; Tacrolimus - analogs & derivatives ; Tacrolimus Binding Proteins</subject><ispartof>Gene, 1993-06, Vol.128 (2), p.219-225</ispartof><rights>1993</rights><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c417t-4a4849cbb9d7f72dbf0a25a2bada27d1fe0a8a22c0bdc1231b21d331726f59733</citedby><cites>FETCH-LOGICAL-c417t-4a4849cbb9d7f72dbf0a25a2bada27d1fe0a8a22c0bdc1231b21d331726f59733</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/037811199390566L$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65534</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4826472$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7685727$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pilot-Matias, Tami J.</creatorcontrib><creatorcontrib>Pratt, Steven D.</creatorcontrib><creatorcontrib>Lane, Benjamin C.</creatorcontrib><title>High-level synthesis of the 12-kDa human FK506-binding protein in Escherichia coli using translational coupling</title><title>Gene</title><addtitle>Gene</addtitle><description>The gene encoding the 12-kDa cytosolic form of human FK506-binding protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in
Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the
trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified
lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational coupling to an upstream segment of
kdsB under the control of this modified
lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was shown to have the authentic N terminus. The level of active re-hFKPB-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.</description><subject>Alkaline Phosphatase - metabolism</subject><subject>Ascomycin</subject><subject>Base Sequence</subject><subject>Binding, Competitive</subject><subject>Biological and medical sciences</subject><subject>Carrier Proteins - biosynthesis</subject><subject>Carrier Proteins - genetics</subject><subject>CMP-3-deoxy-D- manno-octulosonate cytidylyltransferase</subject><subject>CTP</subject><subject>dicistronic</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene expression</subject><subject>Gene Expression Regulation, Bacterial</subject><subject>Genes</subject><subject>Humans</subject><subject>immunosuppressant</subject><subject>Lac Operon</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Molecular Sequence Data</subject><subject>Nucleotidyltransferases - metabolism</subject><subject>PCR</subject><subject>Peptide Chain Initiation, Translational</subject><subject>Plasmids</subject><subject>Polymerase Chain Reaction</subject><subject>Promoter Regions, Genetic</subject><subject>Protein Biosynthesis</subject><subject>rapamycin</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Sequence Analysis, DNA</subject><subject>Tacrolimus - analogs & derivatives</subject><subject>Tacrolimus Binding Proteins</subject><issn>0378-1119</issn><issn>1879-0038</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkV1vFCEUhonR1LX6DzThwjT2AuVjgJkbE9MPa9zEm_aanAGmg7LMCjNN-u9lu5u9rIQEwvucl5P3IPSe0c-MMvWFCt0Sxlj3qRPnHZVKkfULtGKt7gilon2JVkfkNXpTym9al5T8BJ1o1UrN9QpNN-F-JNE_-IjLY5pHX0LB04DrDTNO_lwCHpcNJHz9U1JF-pBcSPd4m6fZh4Trvip29DnYMQC2Uwx4KTtizpBKhDlMCWIVlm2sz2_RqwFi8e8O5ym6u766vbgh61_ff1x8WxPbMD2TBpq26Wzfd04Pmrt-oMAl8B4ccO3Y4Cm0wLmlvbOMC9Zz5oRgmqtBdlqIU3S2962N_l18mc0mFOtjhOSnpRgtddcIyf4LMqWqn-YVbPagzVMp2Q9mm8MG8qNh1OwGYnZpm13aphPmaSBmXcs-HPyXfuPdsegwgap_POhQLMShpmZDOWJNy1Xz9PvXPeZraA_BZ1Ns8Ml6F7K3s3FTeL6Pf3uKpyA</recordid><startdate>19930630</startdate><enddate>19930630</enddate><creator>Pilot-Matias, Tami J.</creator><creator>Pratt, Steven D.</creator><creator>Lane, Benjamin C.</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>7TM</scope><scope>7X8</scope></search><sort><creationdate>19930630</creationdate><title>High-level synthesis of the 12-kDa human FK506-binding protein in Escherichia coli using translational coupling</title><author>Pilot-Matias, Tami J. ; Pratt, Steven D. ; Lane, Benjamin C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c417t-4a4849cbb9d7f72dbf0a25a2bada27d1fe0a8a22c0bdc1231b21d331726f59733</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Alkaline Phosphatase - metabolism</topic><topic>Ascomycin</topic><topic>Base Sequence</topic><topic>Binding, Competitive</topic><topic>Biological and medical sciences</topic><topic>Carrier Proteins - biosynthesis</topic><topic>Carrier Proteins - genetics</topic><topic>CMP-3-deoxy-D- manno-octulosonate cytidylyltransferase</topic><topic>CTP</topic><topic>dicistronic</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Escherichia coli - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene expression</topic><topic>Gene Expression Regulation, Bacterial</topic><topic>Genes</topic><topic>Humans</topic><topic>immunosuppressant</topic><topic>Lac Operon</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Sequence Data</topic><topic>Nucleotidyltransferases - metabolism</topic><topic>PCR</topic><topic>Peptide Chain Initiation, Translational</topic><topic>Plasmids</topic><topic>Polymerase Chain Reaction</topic><topic>Promoter Regions, Genetic</topic><topic>Protein Biosynthesis</topic><topic>rapamycin</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Sequence Analysis, DNA</topic><topic>Tacrolimus - analogs & derivatives</topic><topic>Tacrolimus Binding Proteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pilot-Matias, Tami J.</creatorcontrib><creatorcontrib>Pratt, Steven D.</creatorcontrib><creatorcontrib>Lane, Benjamin C.</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>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Gene</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pilot-Matias, Tami J.</au><au>Pratt, Steven D.</au><au>Lane, Benjamin C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-level synthesis of the 12-kDa human FK506-binding protein in Escherichia coli using translational coupling</atitle><jtitle>Gene</jtitle><addtitle>Gene</addtitle><date>1993-06-30</date><risdate>1993</risdate><volume>128</volume><issue>2</issue><spage>219</spage><epage>225</epage><pages>219-225</pages><issn>0378-1119</issn><eissn>1879-0038</eissn><coden>GENED6</coden><abstract>The gene encoding the 12-kDa cytosolic form of human FK506-binding protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in
Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the
trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified
lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational coupling to an upstream segment of
kdsB under the control of this modified
lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was shown to have the authentic N terminus. The level of active re-hFKPB-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.</abstract><cop>Lausanne</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>7685727</pmid><doi>10.1016/0378-1119(93)90566-L</doi><tpages>7</tpages></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Alkaline Phosphatase - metabolism Ascomycin Base Sequence Binding, Competitive Biological and medical sciences Carrier Proteins - biosynthesis Carrier Proteins - genetics CMP-3-deoxy-D- manno-octulosonate cytidylyltransferase CTP dicistronic Electrophoresis, Polyacrylamide Gel Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Gene expression Gene Expression Regulation, Bacterial Genes Humans immunosuppressant Lac Operon Molecular and cellular biology Molecular genetics Molecular Sequence Data Nucleotidyltransferases - metabolism PCR Peptide Chain Initiation, Translational Plasmids Polymerase Chain Reaction Promoter Regions, Genetic Protein Biosynthesis rapamycin Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics Sequence Analysis, DNA Tacrolimus - analogs & derivatives Tacrolimus Binding Proteins |
| title | High-level synthesis of the 12-kDa human FK506-binding protein in Escherichia coli using translational coupling |
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