Construction of a host-independent T7 expression system with small RNA regulation
•We integrate T7 RNA Polymerase gene and T7 Promoter into a single plasmid.•We use small antisense RNA to regulate T7 RNA Polymerase expression on plasmid.•We construct a novel T7 expression system independent on DE3 lysogenic hosts.•We prove the expression ability of this system in 4 non-DE3 E. col...
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Veröffentlicht in: | Journal of biotechnology 2014-11, Vol.189, p.72-75 |
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creator | Wang, Gang Li, Qiang Xu, Dikai Cui, Mingxin Sun, Xiao Xu, Yanyan Wang, Wenya |
description | •We integrate T7 RNA Polymerase gene and T7 Promoter into a single plasmid.•We use small antisense RNA to regulate T7 RNA Polymerase expression on plasmid.•We construct a novel T7 expression system independent on DE3 lysogenic hosts.•We prove the expression ability of this system in 4 non-DE3 E. coli strains.•We enhance the protein expression in an industrial wild-type strain by this system.
It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572. |
doi_str_mv | 10.1016/j.jbiotec.2014.08.039 |
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It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2014.08.039</identifier><identifier>PMID: 25193711</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Antisense RNA ; Bacteriophage T7 - genetics ; Biotechnology ; Construction ; DNA-Directed RNA Polymerases - genetics ; Escherichia coli ; Gene expression ; Host-independent expression system ; Polymerase ; Proteins ; Ribonucleic acids ; RNA, Antisense - genetics ; Sinorhizobium ; Strain ; T7 Promoter ; T7 RNA Polymerase ; Viral Proteins - genetics</subject><ispartof>Journal of biotechnology, 2014-11, Vol.189, p.72-75</ispartof><rights>2014 Elsevier B.V.</rights><rights>Copyright © 2014 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c431t-6622acef5f216f4f04e6d010f84df33317a4469c067bc0f8796918f6f60e40573</citedby><cites>FETCH-LOGICAL-c431t-6622acef5f216f4f04e6d010f84df33317a4469c067bc0f8796918f6f60e40573</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jbiotec.2014.08.039$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27926,27927,45997</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25193711$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Gang</creatorcontrib><creatorcontrib>Li, Qiang</creatorcontrib><creatorcontrib>Xu, Dikai</creatorcontrib><creatorcontrib>Cui, Mingxin</creatorcontrib><creatorcontrib>Sun, Xiao</creatorcontrib><creatorcontrib>Xu, Yanyan</creatorcontrib><creatorcontrib>Wang, Wenya</creatorcontrib><title>Construction of a host-independent T7 expression system with small RNA regulation</title><title>Journal of biotechnology</title><addtitle>J Biotechnol</addtitle><description>•We integrate T7 RNA Polymerase gene and T7 Promoter into a single plasmid.•We use small antisense RNA to regulate T7 RNA Polymerase expression on plasmid.•We construct a novel T7 expression system independent on DE3 lysogenic hosts.•We prove the expression ability of this system in 4 non-DE3 E. coli strains.•We enhance the protein expression in an industrial wild-type strain by this system.
It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572.</description><subject>Antisense RNA</subject><subject>Bacteriophage T7 - genetics</subject><subject>Biotechnology</subject><subject>Construction</subject><subject>DNA-Directed RNA Polymerases - genetics</subject><subject>Escherichia coli</subject><subject>Gene expression</subject><subject>Host-independent expression system</subject><subject>Polymerase</subject><subject>Proteins</subject><subject>Ribonucleic acids</subject><subject>RNA, Antisense - genetics</subject><subject>Sinorhizobium</subject><subject>Strain</subject><subject>T7 Promoter</subject><subject>T7 RNA Polymerase</subject><subject>Viral Proteins - genetics</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkUtv1DAURi1ERYfCTwB5ySbhOnb8WKFqVB5SRVVU1lbGuaYe5THYDtB_X0czsKUL25J17vfJPoS8YVAzYPL9vt7vwpzR1Q0wUYOugZtnZMO04pXQkj8nm8LpislWnpOXKe0BQJiWvSDnTcsMV4xtyO12nlKOi8thnujsaUfv55SrMPV4wLJNmd4pin8OEVNamfSQMo70d8j3NI3dMNBvXy9pxB_L0K0hr8iZ74aEr0_nBfn-8epu-7m6vvn0ZXt5XTnBWa6kbJrOoW99w6QXHgTKHhh4LXrPOWeqE0IaB1LtXLlVRhqmvfQSUECr-AV5d8w9xPnnginbMSSHw9BNOC_JlnczbhQY8wRUqFKtQT8BbYwpq1lT2yPq4pxSRG8PMYxdfLAM7OrI7u3JkV0dWdC2OCpzb08Vy27E_t_UXykF-HAEsHzfr4DRJhdwctiHiC7bfg7_qXgE4tijsQ</recordid><startdate>20141110</startdate><enddate>20141110</enddate><creator>Wang, Gang</creator><creator>Li, Qiang</creator><creator>Xu, Dikai</creator><creator>Cui, Mingxin</creator><creator>Sun, Xiao</creator><creator>Xu, Yanyan</creator><creator>Wang, Wenya</creator><general>Elsevier B.V</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><scope>7X8</scope><scope>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7U5</scope><scope>L7M</scope></search><sort><creationdate>20141110</creationdate><title>Construction of a host-independent T7 expression system with small RNA regulation</title><author>Wang, Gang ; Li, Qiang ; Xu, Dikai ; Cui, Mingxin ; Sun, Xiao ; Xu, Yanyan ; Wang, Wenya</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c431t-6622acef5f216f4f04e6d010f84df33317a4469c067bc0f8796918f6f60e40573</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Antisense RNA</topic><topic>Bacteriophage T7 - genetics</topic><topic>Biotechnology</topic><topic>Construction</topic><topic>DNA-Directed RNA Polymerases - genetics</topic><topic>Escherichia coli</topic><topic>Gene expression</topic><topic>Host-independent expression system</topic><topic>Polymerase</topic><topic>Proteins</topic><topic>Ribonucleic acids</topic><topic>RNA, Antisense - genetics</topic><topic>Sinorhizobium</topic><topic>Strain</topic><topic>T7 Promoter</topic><topic>T7 RNA Polymerase</topic><topic>Viral Proteins - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Gang</creatorcontrib><creatorcontrib>Li, Qiang</creatorcontrib><creatorcontrib>Xu, Dikai</creatorcontrib><creatorcontrib>Cui, Mingxin</creatorcontrib><creatorcontrib>Sun, Xiao</creatorcontrib><creatorcontrib>Xu, Yanyan</creatorcontrib><creatorcontrib>Wang, Wenya</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Gang</au><au>Li, Qiang</au><au>Xu, Dikai</au><au>Cui, Mingxin</au><au>Sun, Xiao</au><au>Xu, Yanyan</au><au>Wang, Wenya</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Construction of a host-independent T7 expression system with small RNA regulation</atitle><jtitle>Journal of biotechnology</jtitle><addtitle>J Biotechnol</addtitle><date>2014-11-10</date><risdate>2014</risdate><volume>189</volume><spage>72</spage><epage>75</epage><pages>72-75</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><abstract>•We integrate T7 RNA Polymerase gene and T7 Promoter into a single plasmid.•We use small antisense RNA to regulate T7 RNA Polymerase expression on plasmid.•We construct a novel T7 expression system independent on DE3 lysogenic hosts.•We prove the expression ability of this system in 4 non-DE3 E. coli strains.•We enhance the protein expression in an industrial wild-type strain by this system.
It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>25193711</pmid><doi>10.1016/j.jbiotec.2014.08.039</doi><tpages>4</tpages></addata></record> |
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subjects | Antisense RNA Bacteriophage T7 - genetics Biotechnology Construction DNA-Directed RNA Polymerases - genetics Escherichia coli Gene expression Host-independent expression system Polymerase Proteins Ribonucleic acids RNA, Antisense - genetics Sinorhizobium Strain T7 Promoter T7 RNA Polymerase Viral Proteins - genetics |
title | Construction of a host-independent T7 expression system with small RNA regulation |
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