Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion
► We produced an improved system for production of recombinant phosphoproteins in Escherichia coli. ► Genome modification and strain engineering of E. coli enabled RF1 deletion. ► Single and double phosphoserine residues were incorporated into recombinant proteins at enhanced efficiency. ► Phosphose...
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| Veröffentlicht in: | FEBS letters 2012-10, Vol.586 (20), p.3716-3722 |
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| creator | Heinemann, Ilka U. Rovner, Alexis J. Aerni, Hans R. Rogulina, Svetlana Cheng, Laura Olds, William Fischer, Jonathan T. Söll, Dieter Isaacs, Farren J. Rinehart, Jesse |
| description | ► We produced an improved system for production of recombinant phosphoproteins in Escherichia coli. ► Genome modification and strain engineering of E. coli enabled RF1 deletion. ► Single and double phosphoserine residues were incorporated into recombinant proteins at enhanced efficiency. ► Phosphoserine insertion at native UAG sites reduces cellular fitness and viability.
Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151) [2]. However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability. |
| doi_str_mv | 10.1016/j.febslet.2012.08.031 |
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Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151) [2]. However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.</description><identifier>ISSN: 0014-5793</identifier><identifier>EISSN: 1873-3468</identifier><identifier>DOI: 10.1016/j.febslet.2012.08.031</identifier><identifier>PMID: 22982858</identifier><language>eng</language><publisher>England: Elsevier B.V</publisher><subject>Amino Acid Sequence ; Base Sequence ; Codon, Terminator - genetics ; codons ; Escherichia coli ; Escherichia coli - cytology ; Escherichia coli - genetics ; Escherichia coli - growth & development ; Escherichia coli - metabolism ; Escherichia coli Proteins - genetics ; essential genes ; Gene Deletion ; Genetic code ; Genetic code expansion ; Genome engineering ; Genome, Bacterial - genetics ; GFP ; green fluorescent protein ; MAGE ; Molecular Sequence Data ; multiplex automated genome engineering ; Peptide Termination Factors - deficiency ; Peptide Termination Factors - genetics ; Phenotype ; Phosphoproteomics ; Phosphoserine ; Phosphoserine - metabolism ; phosphoserine phosphatase ; phosphoseryl-tRNA synthetase ; Protein Biosynthesis - genetics ; protein synthesis ; Proteome - genetics ; release factor-1 (prfA) ; release factor-2 (prfB) ; RF-1 ; RF-2 ; Sep ; SepRS ; serine/threonine-protein kinase WNK4 ; Synthetic biology ; translation (genetics) ; viability ; WNK4</subject><ispartof>FEBS letters, 2012-10, Vol.586 (20), p.3716-3722</ispartof><rights>2012 Federation of European Biochemical Societies</rights><rights>FEBS Letters 356 (1994) 1873-3468 © 2015 Federation of European Biochemical Societies</rights><rights>Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.</rights><rights>2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c6646-6010b5c04b46916c759d38fc0bff928c3fdaaa1c4392bd71eccd3b25d39407873</citedby><cites>FETCH-LOGICAL-c6646-6010b5c04b46916c759d38fc0bff928c3fdaaa1c4392bd71eccd3b25d39407873</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1016%2Fj.febslet.2012.08.031$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0014579312007016$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,776,780,881,1411,1427,3537,27901,27902,45550,45551,46384,46808,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22982858$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Heinemann, Ilka U.</creatorcontrib><creatorcontrib>Rovner, Alexis J.</creatorcontrib><creatorcontrib>Aerni, Hans R.</creatorcontrib><creatorcontrib>Rogulina, Svetlana</creatorcontrib><creatorcontrib>Cheng, Laura</creatorcontrib><creatorcontrib>Olds, William</creatorcontrib><creatorcontrib>Fischer, Jonathan T.</creatorcontrib><creatorcontrib>Söll, Dieter</creatorcontrib><creatorcontrib>Isaacs, Farren J.</creatorcontrib><creatorcontrib>Rinehart, Jesse</creatorcontrib><title>Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion</title><title>FEBS letters</title><addtitle>FEBS Lett</addtitle><description>► We produced an improved system for production of recombinant phosphoproteins in Escherichia coli. ► Genome modification and strain engineering of E. coli enabled RF1 deletion. ► Single and double phosphoserine residues were incorporated into recombinant proteins at enhanced efficiency. ► Phosphoserine insertion at native UAG sites reduces cellular fitness and viability.
Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151) [2]. However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.</description><subject>Amino Acid Sequence</subject><subject>Base Sequence</subject><subject>Codon, Terminator - genetics</subject><subject>codons</subject><subject>Escherichia coli</subject><subject>Escherichia coli - cytology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - growth & development</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - genetics</subject><subject>essential genes</subject><subject>Gene Deletion</subject><subject>Genetic code</subject><subject>Genetic code expansion</subject><subject>Genome engineering</subject><subject>Genome, Bacterial - genetics</subject><subject>GFP</subject><subject>green fluorescent protein</subject><subject>MAGE</subject><subject>Molecular Sequence Data</subject><subject>multiplex automated genome engineering</subject><subject>Peptide Termination Factors - deficiency</subject><subject>Peptide Termination Factors - genetics</subject><subject>Phenotype</subject><subject>Phosphoproteomics</subject><subject>Phosphoserine</subject><subject>Phosphoserine - metabolism</subject><subject>phosphoserine phosphatase</subject><subject>phosphoseryl-tRNA synthetase</subject><subject>Protein Biosynthesis - genetics</subject><subject>protein synthesis</subject><subject>Proteome - genetics</subject><subject>release factor-1 (prfA)</subject><subject>release factor-2 (prfB)</subject><subject>RF-1</subject><subject>RF-2</subject><subject>Sep</subject><subject>SepRS</subject><subject>serine/threonine-protein kinase WNK4</subject><subject>Synthetic biology</subject><subject>translation (genetics)</subject><subject>viability</subject><subject>WNK4</subject><issn>0014-5793</issn><issn>1873-3468</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNUk1v1DAQjRCILoWfAPKRS4I_8uFcQKXatkiVOEDPlmNPNl5l7WBnF-1v4E8z0S4VnMrBsj3z3vOM32TZW0YLRln9YVv00KUR5oJTxgsqCyrYs2zFZCNyUdbyebailJV51bTiInuV0pbiXbL2ZXbBeSu5rOQq-7X2g_YGLJmGkJYF0XkgzuNhdsETu8fAhqyTGTBlBqeJCaMjUwwzOE_S0c8DJJfIAVOTRpYeycPVLcIs8iPolNzG78DPRHuLgRFDQHpt5hAJIxYDy1Ovsxe9HhO8Oe-X2cPN-vv1XX7_9fbL9dV9buq6rPOaMtpVhpZdWbesNk3VWiF7Q7u-b7k0ordaa2ZK0fLONgyMsaLjlRVtSRv8nsvs40l32nc7sAYLi3pUU3Q7HY8qaKf-zXg3qE04KFE2gtUlCrw_C8TwYw9pVjuXDIyj9hD2SbFGiKpkVFZPQxmvuMC-FtXqBDUxpBShf6yIUbV4rrbq7LlaPFdUKvQcee_-bueR9cdkBNydAD_dCMf_U1U368_82zJAy_wwTmmDWJT6dJIC9OfgIKpkHCzz4yKYWdngnqj2N4uk2VE</recordid><startdate>20121019</startdate><enddate>20121019</enddate><creator>Heinemann, Ilka U.</creator><creator>Rovner, Alexis J.</creator><creator>Aerni, Hans R.</creator><creator>Rogulina, Svetlana</creator><creator>Cheng, Laura</creator><creator>Olds, William</creator><creator>Fischer, Jonathan T.</creator><creator>Söll, Dieter</creator><creator>Isaacs, Farren J.</creator><creator>Rinehart, Jesse</creator><general>Elsevier B.V</general><scope>6I.</scope><scope>AAFTH</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>C1K</scope><scope>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20121019</creationdate><title>Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion</title><author>Heinemann, Ilka U. ; Rovner, Alexis J. ; Aerni, Hans R. ; Rogulina, Svetlana ; Cheng, Laura ; Olds, William ; Fischer, Jonathan T. ; Söll, Dieter ; Isaacs, Farren J. ; Rinehart, Jesse</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c6646-6010b5c04b46916c759d38fc0bff928c3fdaaa1c4392bd71eccd3b25d39407873</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Amino Acid Sequence</topic><topic>Base Sequence</topic><topic>Codon, Terminator - genetics</topic><topic>codons</topic><topic>Escherichia coli</topic><topic>Escherichia coli - cytology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - growth & development</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins - genetics</topic><topic>essential genes</topic><topic>Gene Deletion</topic><topic>Genetic code</topic><topic>Genetic code expansion</topic><topic>Genome engineering</topic><topic>Genome, Bacterial - genetics</topic><topic>GFP</topic><topic>green fluorescent protein</topic><topic>MAGE</topic><topic>Molecular Sequence Data</topic><topic>multiplex automated genome engineering</topic><topic>Peptide Termination Factors - deficiency</topic><topic>Peptide Termination Factors - genetics</topic><topic>Phenotype</topic><topic>Phosphoproteomics</topic><topic>Phosphoserine</topic><topic>Phosphoserine - metabolism</topic><topic>phosphoserine phosphatase</topic><topic>phosphoseryl-tRNA synthetase</topic><topic>Protein Biosynthesis - genetics</topic><topic>protein synthesis</topic><topic>Proteome - genetics</topic><topic>release factor-1 (prfA)</topic><topic>release factor-2 (prfB)</topic><topic>RF-1</topic><topic>RF-2</topic><topic>Sep</topic><topic>SepRS</topic><topic>serine/threonine-protein kinase WNK4</topic><topic>Synthetic biology</topic><topic>translation (genetics)</topic><topic>viability</topic><topic>WNK4</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Heinemann, Ilka U.</creatorcontrib><creatorcontrib>Rovner, Alexis J.</creatorcontrib><creatorcontrib>Aerni, Hans R.</creatorcontrib><creatorcontrib>Rogulina, Svetlana</creatorcontrib><creatorcontrib>Cheng, Laura</creatorcontrib><creatorcontrib>Olds, William</creatorcontrib><creatorcontrib>Fischer, Jonathan T.</creatorcontrib><creatorcontrib>Söll, Dieter</creatorcontrib><creatorcontrib>Isaacs, Farren J.</creatorcontrib><creatorcontrib>Rinehart, Jesse</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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>Environmental Sciences and Pollution Management</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>FEBS letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Heinemann, Ilka U.</au><au>Rovner, Alexis J.</au><au>Aerni, Hans R.</au><au>Rogulina, Svetlana</au><au>Cheng, Laura</au><au>Olds, William</au><au>Fischer, Jonathan T.</au><au>Söll, Dieter</au><au>Isaacs, Farren J.</au><au>Rinehart, Jesse</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion</atitle><jtitle>FEBS letters</jtitle><addtitle>FEBS Lett</addtitle><date>2012-10-19</date><risdate>2012</risdate><volume>586</volume><issue>20</issue><spage>3716</spage><epage>3722</epage><pages>3716-3722</pages><issn>0014-5793</issn><eissn>1873-3468</eissn><abstract>► We produced an improved system for production of recombinant phosphoproteins in Escherichia coli. ► Genome modification and strain engineering of E. coli enabled RF1 deletion. ► Single and double phosphoserine residues were incorporated into recombinant proteins at enhanced efficiency. ► Phosphoserine insertion at native UAG sites reduces cellular fitness and viability.
Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151) [2]. However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.</abstract><cop>England</cop><pub>Elsevier B.V</pub><pmid>22982858</pmid><doi>10.1016/j.febslet.2012.08.031</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Base Sequence Codon, Terminator - genetics codons Escherichia coli Escherichia coli - cytology Escherichia coli - genetics Escherichia coli - growth & development Escherichia coli - metabolism Escherichia coli Proteins - genetics essential genes Gene Deletion Genetic code Genetic code expansion Genome engineering Genome, Bacterial - genetics GFP green fluorescent protein MAGE Molecular Sequence Data multiplex automated genome engineering Peptide Termination Factors - deficiency Peptide Termination Factors - genetics Phenotype Phosphoproteomics Phosphoserine Phosphoserine - metabolism phosphoserine phosphatase phosphoseryl-tRNA synthetase Protein Biosynthesis - genetics protein synthesis Proteome - genetics release factor-1 (prfA) release factor-2 (prfB) RF-1 RF-2 Sep SepRS serine/threonine-protein kinase WNK4 Synthetic biology translation (genetics) viability WNK4 |
| title | Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion |
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