High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli
► A suite of N-terminal hexa-histidine tagged fusion protein vectors is presented. ► 9 fusion proteins can be made from one PCR product using Infusion™ cloning. ► The effect of fusions on the solubility of 20 proteins in E coli was tested in 2 media. ► The SUMO tag was the most effective at solubili...
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| Veröffentlicht in: | Methods (San Diego, Calif.) Calif.), 2011-09, Vol.55 (1), p.29-37 |
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| creator | Bird, Louise E. |
| description | ► A suite of N-terminal hexa-histidine tagged fusion protein vectors is presented. ► 9 fusion proteins can be made from one PCR product using Infusion™ cloning. ► The effect of fusions on the solubility of 20 proteins in E coli was tested in 2 media. ► The SUMO tag was the most effective at solubilising targets. ► Auto-induction media gave more high level expressers than IPTG induction.
A suite of protein fusion vectors is presented that has been designed so that nine separate fusion vectors can be constructed from one PCR product using InFusion™ cloning. These vectors in combination with a small scale Escherichia coli expression screen can be used to assess in parallel the effect of fusion tags on solubility. The vectors were tested with 20 target proteins and the results suggest that the vectors are useful both as a rescue strategy if the N-terminal hexa-histidine tagged construct does not express and also as part of a primary expression experiment. |
| doi_str_mv | 10.1016/j.ymeth.2011.08.002 |
| format | Article |
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A suite of protein fusion vectors is presented that has been designed so that nine separate fusion vectors can be constructed from one PCR product using InFusion™ cloning. These vectors in combination with a small scale Escherichia coli expression screen can be used to assess in parallel the effect of fusion tags on solubility. The vectors were tested with 20 target proteins and the results suggest that the vectors are useful both as a rescue strategy if the N-terminal hexa-histidine tagged construct does not express and also as part of a primary expression experiment.</description><identifier>ISSN: 1046-2023</identifier><identifier>EISSN: 1095-9130</identifier><identifier>DOI: 10.1016/j.ymeth.2011.08.002</identifier><identifier>PMID: 21856427</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Gene Expression ; Genetic Vectors - chemistry ; Genetic Vectors - genetics ; Genetic Vectors - metabolism ; Hexa-histidine tag ; High-Throughput Screening Assays ; Histidine - metabolism ; Humans ; InFusion cloning ; Mice ; Molecular Biology - methods ; Molecular Sequence Data ; N-terminal fusion ; Neisseria meningitidis ; Oligopeptides - metabolism ; Protein expression ; Pseudomonas aeruginosa ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Solubilising protein ; Solubility</subject><ispartof>Methods (San Diego, Calif.), 2011-09, Vol.55 (1), p.29-37</ispartof><rights>2011 Elsevier Inc.</rights><rights>Copyright © 2011 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c456t-9f652abfd4150a1fa327657367c7346c0fbd1501f44148adc2f0ebc92ce64b0c3</citedby><cites>FETCH-LOGICAL-c456t-9f652abfd4150a1fa327657367c7346c0fbd1501f44148adc2f0ebc92ce64b0c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.ymeth.2011.08.002$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27922,27923,45993</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21856427$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bird, Louise E.</creatorcontrib><title>High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli</title><title>Methods (San Diego, Calif.)</title><addtitle>Methods</addtitle><description>► A suite of N-terminal hexa-histidine tagged fusion protein vectors is presented. ► 9 fusion proteins can be made from one PCR product using Infusion™ cloning. ► The effect of fusions on the solubility of 20 proteins in E coli was tested in 2 media. ► The SUMO tag was the most effective at solubilising targets. ► Auto-induction media gave more high level expressers than IPTG induction.
A suite of protein fusion vectors is presented that has been designed so that nine separate fusion vectors can be constructed from one PCR product using InFusion™ cloning. These vectors in combination with a small scale Escherichia coli expression screen can be used to assess in parallel the effect of fusion tags on solubility. The vectors were tested with 20 target proteins and the results suggest that the vectors are useful both as a rescue strategy if the N-terminal hexa-histidine tagged construct does not express and also as part of a primary expression experiment.</description><subject>Animals</subject><subject>Base Sequence</subject><subject>Cloning, Molecular</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Gene Expression</subject><subject>Genetic Vectors - chemistry</subject><subject>Genetic Vectors - genetics</subject><subject>Genetic Vectors - metabolism</subject><subject>Hexa-histidine tag</subject><subject>High-Throughput Screening Assays</subject><subject>Histidine - metabolism</subject><subject>Humans</subject><subject>InFusion cloning</subject><subject>Mice</subject><subject>Molecular Biology - methods</subject><subject>Molecular Sequence Data</subject><subject>N-terminal fusion</subject><subject>Neisseria meningitidis</subject><subject>Oligopeptides - metabolism</subject><subject>Protein expression</subject><subject>Pseudomonas aeruginosa</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Solubilising protein</subject><subject>Solubility</subject><issn>1046-2023</issn><issn>1095-9130</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9v1DAQxSNERUvLJ0BCvnFKGP-Jkxw4oKpQpEpc2rPlTMYbr5J4sZ2Kfvtm2cIRTjPS_N4b6b2ieM-h4sD1p331NFMeKwGcV9BWAOJVccGhq8uOS3h93JUuBQh5XrxNaQ8AXDTtm-Jc8LbWSjQXxf7W70aWxxjW3XhYM8OwpBxXzD4szC4DS7OdJpbQTsTo1yFSSsdTwki0-GXHgmPzOmVfZrtjj4Q5xMT8wm4SjhQ9jt5urpO_Ks6cnRK9e5mXxcPXm_vr2_Lux7fv11_uSlS1zmXndC1s7wbFa7DcWSkaXTdSN9hIpRFcP2wX7pTiqrUDCgfUYyeQtOoB5WXx8eR7iOHnSimb2SekabILhTWZDhreyFrAf8m2a2tohew2Up5IjCGlSM4cop9tfDIczLENsze_2zDHNgy0ZmtjU3148V_7mYa_mj_xb8DnE0BbHo-eoknoaUEafNyCNEPw_3zwDHATnes</recordid><startdate>201109</startdate><enddate>201109</enddate><creator>Bird, Louise E.</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><scope>7X8</scope><scope>7QL</scope><scope>C1K</scope></search><sort><creationdate>201109</creationdate><title>High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli</title><author>Bird, Louise E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c456t-9f652abfd4150a1fa327657367c7346c0fbd1501f44148adc2f0ebc92ce64b0c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Base Sequence</topic><topic>Cloning, Molecular</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Gene Expression</topic><topic>Genetic Vectors - chemistry</topic><topic>Genetic Vectors - genetics</topic><topic>Genetic Vectors - metabolism</topic><topic>Hexa-histidine tag</topic><topic>High-Throughput Screening Assays</topic><topic>Histidine - metabolism</topic><topic>Humans</topic><topic>InFusion cloning</topic><topic>Mice</topic><topic>Molecular Biology - methods</topic><topic>Molecular Sequence Data</topic><topic>N-terminal fusion</topic><topic>Neisseria meningitidis</topic><topic>Oligopeptides - metabolism</topic><topic>Protein expression</topic><topic>Pseudomonas aeruginosa</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Solubilising protein</topic><topic>Solubility</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bird, Louise E.</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Methods (San Diego, Calif.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bird, Louise E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli</atitle><jtitle>Methods (San Diego, Calif.)</jtitle><addtitle>Methods</addtitle><date>2011-09</date><risdate>2011</risdate><volume>55</volume><issue>1</issue><spage>29</spage><epage>37</epage><pages>29-37</pages><issn>1046-2023</issn><eissn>1095-9130</eissn><abstract>► A suite of N-terminal hexa-histidine tagged fusion protein vectors is presented. ► 9 fusion proteins can be made from one PCR product using Infusion™ cloning. ► The effect of fusions on the solubility of 20 proteins in E coli was tested in 2 media. ► The SUMO tag was the most effective at solubilising targets. ► Auto-induction media gave more high level expressers than IPTG induction.
A suite of protein fusion vectors is presented that has been designed so that nine separate fusion vectors can be constructed from one PCR product using InFusion™ cloning. These vectors in combination with a small scale Escherichia coli expression screen can be used to assess in parallel the effect of fusion tags on solubility. The vectors were tested with 20 target proteins and the results suggest that the vectors are useful both as a rescue strategy if the N-terminal hexa-histidine tagged construct does not express and also as part of a primary expression experiment.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>21856427</pmid><doi>10.1016/j.ymeth.2011.08.002</doi><tpages>9</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1046-2023 |
| ispartof | Methods (San Diego, Calif.), 2011-09, Vol.55 (1), p.29-37 |
| issn | 1046-2023 1095-9130 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_907173520 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Animals Base Sequence Cloning, Molecular Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Gene Expression Genetic Vectors - chemistry Genetic Vectors - genetics Genetic Vectors - metabolism Hexa-histidine tag High-Throughput Screening Assays Histidine - metabolism Humans InFusion cloning Mice Molecular Biology - methods Molecular Sequence Data N-terminal fusion Neisseria meningitidis Oligopeptides - metabolism Protein expression Pseudomonas aeruginosa Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Solubilising protein Solubility |
| title | High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli |
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