A facile method for high level dual expression of recombinant and congener protein in a single expression system
Protein engineering is an emerging field for developing novel therapeutic proteins and commercial enzymes, along with a major impact on the global market. In recent decades, advanced methods employing protein modification through expansion of the genetic code have led to the development of proteins...
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Veröffentlicht in: | Protein expression and purification 2019-04, Vol.156, p.1-7 |
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creator | Ilamaran, M. Sriram Raghavan, S. Karthik, S. Sanjay Nalawade, Ketaki Samvedna, S. Routray, W. Kamini, N.R. Saravanan, P. Ayyadurai, N. |
description | Protein engineering is an emerging field for developing novel therapeutic proteins and commercial enzymes, along with a major impact on the global market. In recent decades, advanced methods employing protein modification through expansion of the genetic code have led to the development of proteins with new biochemical and physical properties. These techniques have produced engineered proteins with improved attribute comprising substrate relaxation, protein drug conjugation and high stability under extreme conditions of high temperatures, pH and organic solvents. Furthermore, residue specific incorporation is the simplest method for the global incorporation of non-canonical amino acid (NCAA) for protein modification; however it has the major drawbacks of high production cost and manpower requirement. In the present study, we developed a method for the incorporation of single NCAA in two different proteins by using Escherichia coli (E. coli) expression system. For that, the dual protein expressing Escherichia coli JW2581 strain was constructed by transforming pQE80L and pD881-PpiBT vectors with different promoters, selectable markers and AnnexinV, GFPHS gene. To modify the protein, the 3,4 dihydroxy phenyl alanine (DOPA) was globally incorporated into the GFPHS and Annexin V protein using dual protein expression system. The incorporation efficiency during the dual protein expression was achieved through optimized concentrations of amino acids, carbohydrate and inducers in minimal medium. This method for the incorporation of single NCAA into two different proteins using a single expression host system saves the production cost, manpower and time substantially.
•The recombinant strain to express dual protein was constructed.•Development of a novel protein expression and purification approach for dual expression of congener proteins.•Reports the effect of different plasmid vector for expression of unnatural amino acid containing proteins. |
doi_str_mv | 10.1016/j.pep.2018.12.003 |
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•The recombinant strain to express dual protein was constructed.•Development of a novel protein expression and purification approach for dual expression of congener proteins.•Reports the effect of different plasmid vector for expression of unnatural amino acid containing proteins.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/j.pep.2018.12.003</identifier><identifier>PMID: 30562573</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Annexin V ; Dual protein expression ; Escherichia coli ; Gene Expression ; Green fluorescent protein (GFP) ; Non canonical amino acid ; Protein engineering ; Protein Engineering - methods ; Protein Processing, Post-Translational ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics</subject><ispartof>Protein expression and purification, 2019-04, Vol.156, p.1-7</ispartof><rights>2018 Elsevier Inc.</rights><rights>Copyright © 2018 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c353t-1c79e7b26c2b0700b583ddade5413c38f152b8d5df313bded33656a09914901f3</citedby><cites>FETCH-LOGICAL-c353t-1c79e7b26c2b0700b583ddade5413c38f152b8d5df313bded33656a09914901f3</cites><orcidid>0000-0002-8302-6411</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1046592818306338$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30562573$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ilamaran, M.</creatorcontrib><creatorcontrib>Sriram Raghavan, S.</creatorcontrib><creatorcontrib>Karthik, S.</creatorcontrib><creatorcontrib>Sanjay Nalawade, Ketaki</creatorcontrib><creatorcontrib>Samvedna, S.</creatorcontrib><creatorcontrib>Routray, W.</creatorcontrib><creatorcontrib>Kamini, N.R.</creatorcontrib><creatorcontrib>Saravanan, P.</creatorcontrib><creatorcontrib>Ayyadurai, N.</creatorcontrib><title>A facile method for high level dual expression of recombinant and congener protein in a single expression system</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>Protein engineering is an emerging field for developing novel therapeutic proteins and commercial enzymes, along with a major impact on the global market. In recent decades, advanced methods employing protein modification through expansion of the genetic code have led to the development of proteins with new biochemical and physical properties. These techniques have produced engineered proteins with improved attribute comprising substrate relaxation, protein drug conjugation and high stability under extreme conditions of high temperatures, pH and organic solvents. Furthermore, residue specific incorporation is the simplest method for the global incorporation of non-canonical amino acid (NCAA) for protein modification; however it has the major drawbacks of high production cost and manpower requirement. In the present study, we developed a method for the incorporation of single NCAA in two different proteins by using Escherichia coli (E. coli) expression system. For that, the dual protein expressing Escherichia coli JW2581 strain was constructed by transforming pQE80L and pD881-PpiBT vectors with different promoters, selectable markers and AnnexinV, GFPHS gene. To modify the protein, the 3,4 dihydroxy phenyl alanine (DOPA) was globally incorporated into the GFPHS and Annexin V protein using dual protein expression system. The incorporation efficiency during the dual protein expression was achieved through optimized concentrations of amino acids, carbohydrate and inducers in minimal medium. This method for the incorporation of single NCAA into two different proteins using a single expression host system saves the production cost, manpower and time substantially.
•The recombinant strain to express dual protein was constructed.•Development of a novel protein expression and purification approach for dual expression of congener proteins.•Reports the effect of different plasmid vector for expression of unnatural amino acid containing proteins.</description><subject>Annexin V</subject><subject>Dual protein expression</subject><subject>Escherichia coli</subject><subject>Gene Expression</subject><subject>Green fluorescent protein (GFP)</subject><subject>Non canonical amino acid</subject><subject>Protein engineering</subject><subject>Protein Engineering - methods</subject><subject>Protein Processing, Post-Translational</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE1r3DAQhkVpaD5_QC9Fx17szkgrf9BTCGkbCPTSnIUsjXe12JIreUPz76tl09JTQTA6PO_LzMPYe4QaAZtP-3qhpRaAXY2iBpBv2AVC31Qg2v7t8b9pKtWL7pxd5rwHQGxAvWPnElQjVCsv2HLLR2P9RHymdRcdH2PiO7_d8YmeaeLuYCZOv5ZEOfsYeBx5IhvnwQcTVm6C4zaGLQVKfElxJR94eYZnH7al9Z9ofskrzdfsbDRTppvXecWevtz_uPtWPX7_-nB3-1hZqeRaoW17agfRWDFACzCoTjpnHKkNSiu7EZUYOqfcKFEOjpyUjWoM9D1uesBRXrGPp96y1c8D5VXPPluaJhMoHrIWqLoiQUgoKJ5Qm2LOiUa9JD-b9KIR9FG03usiWh9FaxS6iC6ZD6_1h2Em9zfxx2wBPp8AKkc-e0o6W0_BkvNF4Kpd9P-p_w1udY8j</recordid><startdate>201904</startdate><enddate>201904</enddate><creator>Ilamaran, M.</creator><creator>Sriram Raghavan, S.</creator><creator>Karthik, S.</creator><creator>Sanjay Nalawade, Ketaki</creator><creator>Samvedna, S.</creator><creator>Routray, W.</creator><creator>Kamini, N.R.</creator><creator>Saravanan, P.</creator><creator>Ayyadurai, N.</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><orcidid>https://orcid.org/0000-0002-8302-6411</orcidid></search><sort><creationdate>201904</creationdate><title>A facile method for high level dual expression of recombinant and congener protein in a single expression system</title><author>Ilamaran, M. ; Sriram Raghavan, S. ; Karthik, S. ; Sanjay Nalawade, Ketaki ; Samvedna, S. ; Routray, W. ; Kamini, N.R. ; Saravanan, P. ; Ayyadurai, N.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c353t-1c79e7b26c2b0700b583ddade5413c38f152b8d5df313bded33656a09914901f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Annexin V</topic><topic>Dual protein expression</topic><topic>Escherichia coli</topic><topic>Gene Expression</topic><topic>Green fluorescent protein (GFP)</topic><topic>Non canonical amino acid</topic><topic>Protein engineering</topic><topic>Protein Engineering - methods</topic><topic>Protein Processing, Post-Translational</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ilamaran, M.</creatorcontrib><creatorcontrib>Sriram Raghavan, S.</creatorcontrib><creatorcontrib>Karthik, S.</creatorcontrib><creatorcontrib>Sanjay Nalawade, Ketaki</creatorcontrib><creatorcontrib>Samvedna, S.</creatorcontrib><creatorcontrib>Routray, W.</creatorcontrib><creatorcontrib>Kamini, N.R.</creatorcontrib><creatorcontrib>Saravanan, P.</creatorcontrib><creatorcontrib>Ayyadurai, N.</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><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ilamaran, M.</au><au>Sriram Raghavan, S.</au><au>Karthik, S.</au><au>Sanjay Nalawade, Ketaki</au><au>Samvedna, S.</au><au>Routray, W.</au><au>Kamini, N.R.</au><au>Saravanan, P.</au><au>Ayyadurai, N.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A facile method for high level dual expression of recombinant and congener protein in a single expression system</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2019-04</date><risdate>2019</risdate><volume>156</volume><spage>1</spage><epage>7</epage><pages>1-7</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>Protein engineering is an emerging field for developing novel therapeutic proteins and commercial enzymes, along with a major impact on the global market. In recent decades, advanced methods employing protein modification through expansion of the genetic code have led to the development of proteins with new biochemical and physical properties. These techniques have produced engineered proteins with improved attribute comprising substrate relaxation, protein drug conjugation and high stability under extreme conditions of high temperatures, pH and organic solvents. Furthermore, residue specific incorporation is the simplest method for the global incorporation of non-canonical amino acid (NCAA) for protein modification; however it has the major drawbacks of high production cost and manpower requirement. In the present study, we developed a method for the incorporation of single NCAA in two different proteins by using Escherichia coli (E. coli) expression system. For that, the dual protein expressing Escherichia coli JW2581 strain was constructed by transforming pQE80L and pD881-PpiBT vectors with different promoters, selectable markers and AnnexinV, GFPHS gene. To modify the protein, the 3,4 dihydroxy phenyl alanine (DOPA) was globally incorporated into the GFPHS and Annexin V protein using dual protein expression system. The incorporation efficiency during the dual protein expression was achieved through optimized concentrations of amino acids, carbohydrate and inducers in minimal medium. This method for the incorporation of single NCAA into two different proteins using a single expression host system saves the production cost, manpower and time substantially.
•The recombinant strain to express dual protein was constructed.•Development of a novel protein expression and purification approach for dual expression of congener proteins.•Reports the effect of different plasmid vector for expression of unnatural amino acid containing proteins.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>30562573</pmid><doi>10.1016/j.pep.2018.12.003</doi><tpages>7</tpages><orcidid>https://orcid.org/0000-0002-8302-6411</orcidid></addata></record> |
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subjects | Annexin V Dual protein expression Escherichia coli Gene Expression Green fluorescent protein (GFP) Non canonical amino acid Protein engineering Protein Engineering - methods Protein Processing, Post-Translational Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - genetics |
title | A facile method for high level dual expression of recombinant and congener protein in a single expression system |
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