High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids
We describe an E. coli‐based cell‐free system for the production of proteins with a non‐natural amino acid (nnAA) incorporated site‐specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl‐tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS expe...
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| Veröffentlicht in: | Biotechnology and bioengineering 2009-02, Vol.102 (2), p.400-416 |
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| description | We describe an E. coli‐based cell‐free system for the production of proteins with a non‐natural amino acid (nnAA) incorporated site‐specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl‐tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200–300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli‐based cell‐free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p‐azido‐L‐phenylalanine (pAz) incorporated site‐specifically at the amber nonsense codon. O‐methyl‐L‐tyrosine and p‐acetyl‐L‐phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell‐free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)‐catalyzed (3 + 2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400–416. © 2008 Wiley Periodicals, Inc. |
| doi_str_mv | 10.1002/bit.22070 |
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The mutant Methanococcus jannaschii tyrosyl‐tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200–300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli‐based cell‐free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p‐azido‐L‐phenylalanine (pAz) incorporated site‐specifically at the amber nonsense codon. O‐methyl‐L‐tyrosine and p‐acetyl‐L‐phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell‐free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)‐catalyzed (3 + 2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400–416. © 2008 Wiley Periodicals, Inc.</description><identifier>ISSN: 0006-3592</identifier><identifier>EISSN: 1097-0290</identifier><identifier>DOI: 10.1002/bit.22070</identifier><identifier>PMID: 18781689</identifier><identifier>CODEN: BIBIAU</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>(3 + 2) cycloaddition reaction ; Amino acids ; Amino Acids - metabolism ; Azides - metabolism ; Biochemistry ; Biological and medical sciences ; Biotechnology ; cell-free protein synthesis ; Cell-Free System - metabolism ; Cells ; Chemical synthesis ; click chemistry ; E coli ; E. coli inner membrane vesicles ; Escherichia coli - metabolism ; Fundamental and applied biological sciences. Psychology ; Methanococcus ; Methanococcus - enzymology ; Methanococcus jannaschii ; Mutation ; non-natural amino acid ; Phenylalanine - analogs & derivatives ; Phenylalanine - metabolism ; Protein Biosynthesis ; Proteins ; RNA, Transfer, Tyr - metabolism ; Tyrosine-tRNA Ligase - genetics ; Tyrosine-tRNA Ligase - metabolism ; unnatural amino acid</subject><ispartof>Biotechnology and bioengineering, 2009-02, Vol.102 (2), p.400-416</ispartof><rights>Copyright © 2008 Wiley Periodicals, Inc.</rights><rights>2009 INIST-CNRS</rights><rights>Copyright John Wiley and Sons, Limited Feb 1, 2009</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5460-4b3832e5969f7aa367d6bb6d8d3581aca2866f87d5aac1fc4d03cdf86f4f5f293</citedby><cites>FETCH-LOGICAL-c5460-4b3832e5969f7aa367d6bb6d8d3581aca2866f87d5aac1fc4d03cdf86f4f5f293</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.22070$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbit.22070$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,777,781,1413,27906,27907,45556,45557</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=21073148$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18781689$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Goerke, Aaron R.</creatorcontrib><creatorcontrib>Swartz, James R.</creatorcontrib><title>High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol. Bioeng</addtitle><description>We describe an E. coli‐based cell‐free system for the production of proteins with a non‐natural amino acid (nnAA) incorporated site‐specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl‐tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200–300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli‐based cell‐free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p‐azido‐L‐phenylalanine (pAz) incorporated site‐specifically at the amber nonsense codon. O‐methyl‐L‐tyrosine and p‐acetyl‐L‐phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell‐free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)‐catalyzed (3 + 2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400–416. © 2008 Wiley Periodicals, Inc.</description><subject>(3 + 2) cycloaddition reaction</subject><subject>Amino acids</subject><subject>Amino Acids - metabolism</subject><subject>Azides - metabolism</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>cell-free protein synthesis</subject><subject>Cell-Free System - metabolism</subject><subject>Cells</subject><subject>Chemical synthesis</subject><subject>click chemistry</subject><subject>E coli</subject><subject>E. coli inner membrane vesicles</subject><subject>Escherichia coli - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Methanococcus</subject><subject>Methanococcus - enzymology</subject><subject>Methanococcus jannaschii</subject><subject>Mutation</subject><subject>non-natural amino acid</subject><subject>Phenylalanine - analogs & derivatives</subject><subject>Phenylalanine - metabolism</subject><subject>Protein Biosynthesis</subject><subject>Proteins</subject><subject>RNA, Transfer, Tyr - metabolism</subject><subject>Tyrosine-tRNA Ligase - genetics</subject><subject>Tyrosine-tRNA Ligase - metabolism</subject><subject>unnatural amino acid</subject><issn>0006-3592</issn><issn>1097-0290</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0VtrFDEYBuAgil2rF_4BCYKCF2lzmBzmUos9wHq4qPQyZDJJmzqbWfPNqPvvzbprBUF6FQLPd-JF6DmjR4xSftyl6YhzqukDtGC01YTylj5EC0qpIkK2_AA9AbitX22UeowOmNGGKdMuUHeerm_IEL6HAfswDCSWEDBs8nQTIAHepDD0gMeI12WcQsqA_Zgnl3LK1xjSFAisg08xeZzHTLKb5uIG7FYpj9j51MNT9Ci6AcKz_XuIvpy-vzw5J8tPZxcnb5fEy0ZR0nTCCB5kq9qonRNK96rrVG96IQ1z3vG6ezS6l855Fn3TU-H7aFRsooy8FYfo9a5v3fTbHGCyqwTbm1wO4wxWKa21kM29sBrKBVP3Qk6Z0ExvO778B96Oc8n1WssrkW2jt_u92SFfRoASol2XtHJlYxm12xxtzdH-zrHaF_uGc7cK_V-5D66CV3vgwLshFpd9gjvHGdWCNaa64537kYaw-f9E--7i8s9osqtIMIWfdxWufLVK11vs1ccze_XBfJannNul-AWwlMMC</recordid><startdate>20090201</startdate><enddate>20090201</enddate><creator>Goerke, Aaron R.</creator><creator>Swartz, James R.</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><general>Wiley</general><general>Wiley Subscription Services, Inc</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>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20090201</creationdate><title>High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids</title><author>Goerke, Aaron R. ; Swartz, James R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5460-4b3832e5969f7aa367d6bb6d8d3581aca2866f87d5aac1fc4d03cdf86f4f5f293</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>(3 + 2) cycloaddition reaction</topic><topic>Amino acids</topic><topic>Amino Acids - metabolism</topic><topic>Azides - metabolism</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>cell-free protein synthesis</topic><topic>Cell-Free System - metabolism</topic><topic>Cells</topic><topic>Chemical synthesis</topic><topic>click chemistry</topic><topic>E coli</topic><topic>E. coli inner membrane vesicles</topic><topic>Escherichia coli - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Methanococcus</topic><topic>Methanococcus - enzymology</topic><topic>Methanococcus jannaschii</topic><topic>Mutation</topic><topic>non-natural amino acid</topic><topic>Phenylalanine - analogs & derivatives</topic><topic>Phenylalanine - metabolism</topic><topic>Protein Biosynthesis</topic><topic>Proteins</topic><topic>RNA, Transfer, Tyr - metabolism</topic><topic>Tyrosine-tRNA Ligase - genetics</topic><topic>Tyrosine-tRNA Ligase - metabolism</topic><topic>unnatural amino acid</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Goerke, Aaron R.</creatorcontrib><creatorcontrib>Swartz, James R.</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>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology and bioengineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Goerke, Aaron R.</au><au>Swartz, James R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids</atitle><jtitle>Biotechnology and bioengineering</jtitle><addtitle>Biotechnol. Bioeng</addtitle><date>2009-02-01</date><risdate>2009</risdate><volume>102</volume><issue>2</issue><spage>400</spage><epage>416</epage><pages>400-416</pages><issn>0006-3592</issn><eissn>1097-0290</eissn><coden>BIBIAU</coden><abstract>We describe an E. coli‐based cell‐free system for the production of proteins with a non‐natural amino acid (nnAA) incorporated site‐specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl‐tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200–300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli‐based cell‐free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p‐azido‐L‐phenylalanine (pAz) incorporated site‐specifically at the amber nonsense codon. O‐methyl‐L‐tyrosine and p‐acetyl‐L‐phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell‐free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)‐catalyzed (3 + 2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400–416. © 2008 Wiley Periodicals, Inc.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>18781689</pmid><doi>10.1002/bit.22070</doi><tpages>17</tpages></addata></record> |
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| ispartof | Biotechnology and bioengineering, 2009-02, Vol.102 (2), p.400-416 |
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| subjects | (3 + 2) cycloaddition reaction Amino acids Amino Acids - metabolism Azides - metabolism Biochemistry Biological and medical sciences Biotechnology cell-free protein synthesis Cell-Free System - metabolism Cells Chemical synthesis click chemistry E coli E. coli inner membrane vesicles Escherichia coli - metabolism Fundamental and applied biological sciences. Psychology Methanococcus Methanococcus - enzymology Methanococcus jannaschii Mutation non-natural amino acid Phenylalanine - analogs & derivatives Phenylalanine - metabolism Protein Biosynthesis Proteins RNA, Transfer, Tyr - metabolism Tyrosine-tRNA Ligase - genetics Tyrosine-tRNA Ligase - metabolism unnatural amino acid |
| title | High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids |
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