A Modular Set of Prokaryotic and Eukaryotic Expression Vectors
A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine–GST) to yield extremely pure protein even with very low expression rates, (ii) highly eff...
Gespeichert in:
| Veröffentlicht in: | Analytical biochemistry 2000-01, Vol.277 (1), p.109-120 |
|---|---|
| 1. Verfasser: | |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 120 |
|---|---|
| container_issue | 1 |
| container_start_page | 109 |
| container_title | Analytical biochemistry |
| container_volume | 277 |
| creator | Melcher, Karsten |
| description | A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine–GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed. |
| doi_str_mv | 10.1006/abio.1999.4383 |
| format | Article |
| fullrecord | <record><control><sourceid>pubmed_cross</sourceid><recordid>TN_cdi_crossref_primary_10_1006_abio_1999_4383</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S000326979994383X</els_id><sourcerecordid>10610695</sourcerecordid><originalsourceid>FETCH-LOGICAL-c340t-8c4586eb3eadd1ce0826809fe0763fd63e26a1d9ef16eec0c964205b0501c2f93</originalsourceid><addsrcrecordid>eNp1kN1LwzAUxYMobk5ffZT8A603TZs1L8IY8wMmCn68hjS5gejWjKQT_e9tqYgvwoXLhXMO9_wIOWeQMwBxqRsfcialzEte8wMyZSBFBhzkIZkCAM8KIecTcpLSGwBjZSWOyYSB6EdWU3K1oPfB7jc60ifsaHD0MYZ3Hb9C5w3VraWr_e-5-txFTMmHlr6i6UJMp-TI6U3Cs589Iy_Xq-flbbZ-uLlbLtaZ4SV0WW3KqhbYcNTWMoNQF6IG6RDmgjsrOBZCMyvRMYFowEhRFlA1UAEzhZN8RvIx18SQUkSndtFv-78UAzWAUAMINYBQA4jecDEadvtmi_aPfGzeC-pRgP3bHx6jSsZja9D62HdTNvj_sr8BYyJsfw</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>A Modular Set of Prokaryotic and Eukaryotic Expression Vectors</title><source>MEDLINE</source><source>ScienceDirect Journals (5 years ago - present)</source><creator>Melcher, Karsten</creator><creatorcontrib>Melcher, Karsten</creatorcontrib><description>A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine–GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1006/abio.1999.4383</identifier><identifier>PMID: 10610695</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>ADH1 promoter ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular - methods ; DNA Primers ; DNA-Binding Proteins ; Electrophoresis, Polyacrylamide Gel - methods ; Escherichia coli - genetics ; Fungal Proteins - biosynthesis ; Fungal Proteins - chemistry ; Fungal Proteins - genetics ; GAL genes ; GAL1 promoter ; Genetic Vectors ; Glutathione Transferase - biosynthesis ; Glutathione Transferase - chemistry ; Glutathione Transferase - genetics ; GST ; hexahistidine ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Open Reading Frames ; Plasmids ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - isolation & purification ; Restriction Mapping ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae Proteins ; T7 promoter ; TEV protease ; Transcription Factors - biosynthesis ; Transcription Factors - chemistry ; Transcription Factors - genetics</subject><ispartof>Analytical biochemistry, 2000-01, Vol.277 (1), p.109-120</ispartof><rights>2000 Academic Press</rights><rights>Copyright 2000 Academic Press.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c340t-8c4586eb3eadd1ce0826809fe0763fd63e26a1d9ef16eec0c964205b0501c2f93</citedby><cites>FETCH-LOGICAL-c340t-8c4586eb3eadd1ce0826809fe0763fd63e26a1d9ef16eec0c964205b0501c2f93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/abio.1999.4383$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3549,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10610695$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Melcher, Karsten</creatorcontrib><title>A Modular Set of Prokaryotic and Eukaryotic Expression Vectors</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine–GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.</description><subject>ADH1 promoter</subject><subject>Amino Acid Sequence</subject><subject>Base Sequence</subject><subject>Cloning, Molecular - methods</subject><subject>DNA Primers</subject><subject>DNA-Binding Proteins</subject><subject>Electrophoresis, Polyacrylamide Gel - methods</subject><subject>Escherichia coli - genetics</subject><subject>Fungal Proteins - biosynthesis</subject><subject>Fungal Proteins - chemistry</subject><subject>Fungal Proteins - genetics</subject><subject>GAL genes</subject><subject>GAL1 promoter</subject><subject>Genetic Vectors</subject><subject>Glutathione Transferase - biosynthesis</subject><subject>Glutathione Transferase - chemistry</subject><subject>Glutathione Transferase - genetics</subject><subject>GST</subject><subject>hexahistidine</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Open Reading Frames</subject><subject>Plasmids</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Restriction Mapping</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae Proteins</subject><subject>T7 promoter</subject><subject>TEV protease</subject><subject>Transcription Factors - biosynthesis</subject><subject>Transcription Factors - chemistry</subject><subject>Transcription Factors - genetics</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kN1LwzAUxYMobk5ffZT8A603TZs1L8IY8wMmCn68hjS5gejWjKQT_e9tqYgvwoXLhXMO9_wIOWeQMwBxqRsfcialzEte8wMyZSBFBhzkIZkCAM8KIecTcpLSGwBjZSWOyYSB6EdWU3K1oPfB7jc60ifsaHD0MYZ3Hb9C5w3VraWr_e-5-txFTMmHlr6i6UJMp-TI6U3Cs589Iy_Xq-flbbZ-uLlbLtaZ4SV0WW3KqhbYcNTWMoNQF6IG6RDmgjsrOBZCMyvRMYFowEhRFlA1UAEzhZN8RvIx18SQUkSndtFv-78UAzWAUAMINYBQA4jecDEadvtmi_aPfGzeC-pRgP3bHx6jSsZja9D62HdTNvj_sr8BYyJsfw</recordid><startdate>20000101</startdate><enddate>20000101</enddate><creator>Melcher, Karsten</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>20000101</creationdate><title>A Modular Set of Prokaryotic and Eukaryotic Expression Vectors</title><author>Melcher, Karsten</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c340t-8c4586eb3eadd1ce0826809fe0763fd63e26a1d9ef16eec0c964205b0501c2f93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>ADH1 promoter</topic><topic>Amino Acid Sequence</topic><topic>Base Sequence</topic><topic>Cloning, Molecular - methods</topic><topic>DNA Primers</topic><topic>DNA-Binding Proteins</topic><topic>Electrophoresis, Polyacrylamide Gel - methods</topic><topic>Escherichia coli - genetics</topic><topic>Fungal Proteins - biosynthesis</topic><topic>Fungal Proteins - chemistry</topic><topic>Fungal Proteins - genetics</topic><topic>GAL genes</topic><topic>GAL1 promoter</topic><topic>Genetic Vectors</topic><topic>Glutathione Transferase - biosynthesis</topic><topic>Glutathione Transferase - chemistry</topic><topic>Glutathione Transferase - genetics</topic><topic>GST</topic><topic>hexahistidine</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Open Reading Frames</topic><topic>Plasmids</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Restriction Mapping</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae Proteins</topic><topic>T7 promoter</topic><topic>TEV protease</topic><topic>Transcription Factors - biosynthesis</topic><topic>Transcription Factors - chemistry</topic><topic>Transcription Factors - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Melcher, Karsten</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Melcher, Karsten</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Modular Set of Prokaryotic and Eukaryotic Expression Vectors</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2000-01-01</date><risdate>2000</risdate><volume>277</volume><issue>1</issue><spage>109</spage><epage>120</epage><pages>109-120</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine–GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>10610695</pmid><doi>10.1006/abio.1999.4383</doi><tpages>12</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Analytical biochemistry, 2000-01, Vol.277 (1), p.109-120 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_crossref_primary_10_1006_abio_1999_4383 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | ADH1 promoter Amino Acid Sequence Base Sequence Cloning, Molecular - methods DNA Primers DNA-Binding Proteins Electrophoresis, Polyacrylamide Gel - methods Escherichia coli - genetics Fungal Proteins - biosynthesis Fungal Proteins - chemistry Fungal Proteins - genetics GAL genes GAL1 promoter Genetic Vectors Glutathione Transferase - biosynthesis Glutathione Transferase - chemistry Glutathione Transferase - genetics GST hexahistidine Molecular Sequence Data Mutagenesis, Site-Directed Open Reading Frames Plasmids Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - isolation & purification Restriction Mapping Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins T7 promoter TEV protease Transcription Factors - biosynthesis Transcription Factors - chemistry Transcription Factors - genetics |
| title | A Modular Set of Prokaryotic and Eukaryotic Expression Vectors |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-08T06%3A55%3A48IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-pubmed_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20Modular%20Set%20of%20Prokaryotic%20and%20Eukaryotic%20Expression%20Vectors&rft.jtitle=Analytical%20biochemistry&rft.au=Melcher,%20Karsten&rft.date=2000-01-01&rft.volume=277&rft.issue=1&rft.spage=109&rft.epage=120&rft.pages=109-120&rft.issn=0003-2697&rft.eissn=1096-0309&rft_id=info:doi/10.1006/abio.1999.4383&rft_dat=%3Cpubmed_cross%3E10610695%3C/pubmed_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_id=info:pmid/10610695&rft_els_id=S000326979994383X&rfr_iscdi=true |