Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium
A multiple vector system for the intracellular high‐level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N‐ and C‐terminal fusion of a protein of interest to a Strep II and a His6‐tag is possible. Corresponding genes are expressed under the control of a...
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| Veröffentlicht in: | Biotechnology and bioengineering 2007-02, Vol.96 (3), p.525-537 |
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| creator | Biedendieck, Rebekka Yang, Yang Deckwer, Wolf-Dieter Malten, Marco Jahn, Dieter |
| description | A multiple vector system for the intracellular high‐level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N‐ and C‐terminal fusion of a protein of interest to a Strep II and a His6‐tag is possible. Corresponding genes are expressed under the control of a xylose‐inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N‐ or C‐terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one‐step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc. |
| doi_str_mv | 10.1002/bit.21145 |
| format | Article |
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The N‐ and C‐terminal fusion of a protein of interest to a Strep II and a His6‐tag is possible. Corresponding genes are expressed under the control of a xylose‐inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N‐ or C‐terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one‐step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.21145</identifier><identifier>PMID: 16964623</identifier><identifier>CODEN: BIBIAU</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>affinity tags ; Bacillus megaterium ; Bacillus megaterium - chemistry ; Bacillus megaterium - genetics ; Biological and medical sciences ; Biotechnology ; Chromatography, Affinity - methods ; Cloning, Molecular ; Cytoplasm - chemistry ; Cytoplasm - genetics ; Endopeptidases - chemistry ; Endopeptidases - genetics ; Factor Xa - chemistry ; Factor Xa - genetics ; fed batch cultivation ; Fundamental and applied biological sciences. Psychology ; Genetic Vectors ; green fluorescent protein ; Green Fluorescent Proteins - chemistry ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - isolation & purification ; Histidine - chemistry ; Histidine - genetics ; Plasmids - genetics ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; recombinant protein production ; Tobacco etch virus</subject><ispartof>Biotechnology and bioengineering, 2007-02, Vol.96 (3), p.525-537</ispartof><rights>Copyright © 2006 Wiley Periodicals, Inc.</rights><rights>2007 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4535-5c2997661b69bfd145b22f5546d200ec163e6420edc1e7bc23ae562d2338a5ac3</citedby><cites>FETCH-LOGICAL-c4535-5c2997661b69bfd145b22f5546d200ec163e6420edc1e7bc23ae562d2338a5ac3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fbit.21145$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbit.21145$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27915,27916,45565,45566</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=18466987$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16964623$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Biedendieck, Rebekka</creatorcontrib><creatorcontrib>Yang, Yang</creatorcontrib><creatorcontrib>Deckwer, Wolf-Dieter</creatorcontrib><creatorcontrib>Malten, Marco</creatorcontrib><creatorcontrib>Jahn, Dieter</creatorcontrib><title>Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol. Bioeng</addtitle><description>A multiple vector system for the intracellular high‐level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N‐ and C‐terminal fusion of a protein of interest to a Strep II and a His6‐tag is possible. Corresponding genes are expressed under the control of a xylose‐inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N‐ or C‐terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one‐step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc.</description><subject>affinity tags</subject><subject>Bacillus megaterium</subject><subject>Bacillus megaterium - chemistry</subject><subject>Bacillus megaterium - genetics</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chromatography, Affinity - methods</subject><subject>Cloning, Molecular</subject><subject>Cytoplasm - chemistry</subject><subject>Cytoplasm - genetics</subject><subject>Endopeptidases - chemistry</subject><subject>Endopeptidases - genetics</subject><subject>Factor Xa - chemistry</subject><subject>Factor Xa - genetics</subject><subject>fed batch cultivation</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genetic Vectors</subject><subject>green fluorescent protein</subject><subject>Green Fluorescent Proteins - chemistry</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - isolation & purification</subject><subject>Histidine - chemistry</subject><subject>Histidine - genetics</subject><subject>Plasmids - genetics</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>recombinant protein production</subject><subject>Tobacco etch virus</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1vEzEQhi0EoqFw4A8gX0DisK0_1vb6SCpaKpWAUBBHa9ZrB8N-BNsrmn-P0wR6Qj1Ztp55Z8YPQi8pOaOEsPM25DNGaS0eoQUlWlWEafIYLQghsuJCsxP0LKUf5aoaKZ-iEyq1rCXjC5Q-95CG0OG0S9kN2E8R5-8OhzFHsK7v5x4i3sapm20O04hh7PB2jsEHC3cPk8fgfRhD3lUZNhvX7fHswphKCl6CDSUl4cFtILsY5uE5euKhT-7F8TxFXy_fry8-VDefrq4v3t1UthZcVMIyrZWUtJW69V1Zr2XMC1HLjhHiLJXcyZoR11nqVGsZByck6xjnDQiw_BS9OeSWeX7NLmUzhLTfCUY3zcnIhjeNqpsHQV4-TjPFHwSpbphUmhbw7QG0cUopOm-2MQwQd4YSs3dmijNz56ywr46hczu47p48SirA6yMAyULvI4w2pHuuqaXUjSrc-YH7HXq3-39Hs7xe_21dHSpCkX_7rwLiTyMVV8J8W12Z9ZflR7WSS7PifwC9lb3u</recordid><startdate>20070215</startdate><enddate>20070215</enddate><creator>Biedendieck, Rebekka</creator><creator>Yang, Yang</creator><creator>Deckwer, Wolf-Dieter</creator><creator>Malten, Marco</creator><creator>Jahn, Dieter</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><general>Wiley</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>7QO</scope><scope>7T7</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope><scope>7U5</scope><scope>F28</scope><scope>L7M</scope><scope>7X8</scope></search><sort><creationdate>20070215</creationdate><title>Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium</title><author>Biedendieck, Rebekka ; Yang, Yang ; Deckwer, Wolf-Dieter ; Malten, Marco ; Jahn, Dieter</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4535-5c2997661b69bfd145b22f5546d200ec163e6420edc1e7bc23ae562d2338a5ac3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>affinity tags</topic><topic>Bacillus megaterium</topic><topic>Bacillus megaterium - chemistry</topic><topic>Bacillus megaterium - genetics</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Chromatography, Affinity - methods</topic><topic>Cloning, Molecular</topic><topic>Cytoplasm - chemistry</topic><topic>Cytoplasm - genetics</topic><topic>Endopeptidases - chemistry</topic><topic>Endopeptidases - genetics</topic><topic>Factor Xa - chemistry</topic><topic>Factor Xa - genetics</topic><topic>fed batch cultivation</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genetic Vectors</topic><topic>green fluorescent protein</topic><topic>Green Fluorescent Proteins - chemistry</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - isolation & purification</topic><topic>Histidine - chemistry</topic><topic>Histidine - genetics</topic><topic>Plasmids - genetics</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>recombinant protein production</topic><topic>Tobacco etch virus</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Biedendieck, Rebekka</creatorcontrib><creatorcontrib>Yang, Yang</creatorcontrib><creatorcontrib>Deckwer, Wolf-Dieter</creatorcontrib><creatorcontrib>Malten, Marco</creatorcontrib><creatorcontrib>Jahn, Dieter</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>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology and bioengineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Biedendieck, Rebekka</au><au>Yang, Yang</au><au>Deckwer, Wolf-Dieter</au><au>Malten, Marco</au><au>Jahn, Dieter</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium</atitle><jtitle>Biotechnology and bioengineering</jtitle><addtitle>Biotechnol. Bioeng</addtitle><date>2007-02-15</date><risdate>2007</risdate><volume>96</volume><issue>3</issue><spage>525</spage><epage>537</epage><pages>525-537</pages><issn>0006-3592</issn><eissn>1097-0290</eissn><coden>BIBIAU</coden><abstract>A multiple vector system for the intracellular high‐level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N‐ and C‐terminal fusion of a protein of interest to a Strep II and a His6‐tag is possible. Corresponding genes are expressed under the control of a xylose‐inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N‐ or C‐terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one‐step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>16964623</pmid><doi>10.1002/bit.21145</doi><tpages>13</tpages></addata></record> |
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| subjects | affinity tags Bacillus megaterium Bacillus megaterium - chemistry Bacillus megaterium - genetics Biological and medical sciences Biotechnology Chromatography, Affinity - methods Cloning, Molecular Cytoplasm - chemistry Cytoplasm - genetics Endopeptidases - chemistry Endopeptidases - genetics Factor Xa - chemistry Factor Xa - genetics fed batch cultivation Fundamental and applied biological sciences. Psychology Genetic Vectors green fluorescent protein Green Fluorescent Proteins - chemistry Green Fluorescent Proteins - genetics Green Fluorescent Proteins - isolation & purification Histidine - chemistry Histidine - genetics Plasmids - genetics Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification recombinant protein production Tobacco etch virus |
| title | Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium |
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