Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene
Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine synthetase / tRNA CUA...
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| Veröffentlicht in: | Scientific reports 2018-09, Vol.8 (1), p.14527-11, Article 14527 |
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| creator | Aloush, Noa Schvartz, Tomer König, Andres I. Cohen, Sarit Brozgol, Eugene Tam, Benjamin Nachmias, Dikla Ben-David, Oshrit Garini, Yuval Elia, Natalie Arbely, Eyal |
| description | Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine
synthetase
/
tRNA
CUA
Pyl
pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNA
Pyl
gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNA
Pyl
on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNA
Pyl
population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application. |
| doi_str_mv | 10.1038/s41598-018-32824-1 |
| format | Article |
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synthetase
/
tRNA
CUA
Pyl
pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNA
Pyl
gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNA
Pyl
on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNA
Pyl
population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/s41598-018-32824-1</identifier><identifier>PMID: 30267004</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>13/31 ; 14/19 ; 14/34 ; 14/63 ; 631/1647/245/2225 ; 631/45/2783 ; 631/80/2373/2238 ; 631/92/96 ; 639/638/92/552 ; Amino acids ; Animals ; Chlorocebus aethiops ; Codon, Terminator ; COS Cells ; Fluorescence ; Genetic Code ; Humanities and Social Sciences ; Intracellular ; Intracellular levels ; Lysine - analogs & derivatives ; Lysine - genetics ; Mammalian cells ; multidisciplinary ; Optical Imaging ; Protein Biosynthesis ; Proteins ; Proteins - genetics ; RNA, Transfer - genetics ; Science ; Science (multidisciplinary) ; Stop codon ; Transfection ; tRNA</subject><ispartof>Scientific reports, 2018-09, Vol.8 (1), p.14527-11, Article 14527</ispartof><rights>The Author(s) 2018</rights><rights>2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c511t-1f7e72230ebeee4135d01bc300ff57141910988dd0334efb40a664bab0522d283</citedby><cites>FETCH-LOGICAL-c511t-1f7e72230ebeee4135d01bc300ff57141910988dd0334efb40a664bab0522d283</cites><orcidid>0000-0002-2537-6173 ; 0000-0002-0284-0092</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162220/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162220/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27903,27904,41099,42168,51555,53770,53772</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30267004$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Aloush, Noa</creatorcontrib><creatorcontrib>Schvartz, Tomer</creatorcontrib><creatorcontrib>König, Andres I.</creatorcontrib><creatorcontrib>Cohen, Sarit</creatorcontrib><creatorcontrib>Brozgol, Eugene</creatorcontrib><creatorcontrib>Tam, Benjamin</creatorcontrib><creatorcontrib>Nachmias, Dikla</creatorcontrib><creatorcontrib>Ben-David, Oshrit</creatorcontrib><creatorcontrib>Garini, Yuval</creatorcontrib><creatorcontrib>Elia, Natalie</creatorcontrib><creatorcontrib>Arbely, Eyal</creatorcontrib><title>Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine
synthetase
/
tRNA
CUA
Pyl
pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNA
Pyl
gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNA
Pyl
on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNA
Pyl
population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.</description><subject>13/31</subject><subject>14/19</subject><subject>14/34</subject><subject>14/63</subject><subject>631/1647/245/2225</subject><subject>631/45/2783</subject><subject>631/80/2373/2238</subject><subject>631/92/96</subject><subject>639/638/92/552</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Chlorocebus aethiops</subject><subject>Codon, Terminator</subject><subject>COS Cells</subject><subject>Fluorescence</subject><subject>Genetic Code</subject><subject>Humanities and Social Sciences</subject><subject>Intracellular</subject><subject>Intracellular levels</subject><subject>Lysine - analogs & derivatives</subject><subject>Lysine - genetics</subject><subject>Mammalian cells</subject><subject>multidisciplinary</subject><subject>Optical Imaging</subject><subject>Protein Biosynthesis</subject><subject>Proteins</subject><subject>Proteins - genetics</subject><subject>RNA, Transfer - genetics</subject><subject>Science</subject><subject>Science (multidisciplinary)</subject><subject>Stop codon</subject><subject>Transfection</subject><subject>tRNA</subject><issn>2045-2322</issn><issn>2045-2322</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kU1P3DAQhi1EBYjyBzggS71wSTtjO1-XSrBqKdKqRYWqR8tJJlmjrE3tLNL--5pdvsqhvtjyPPN6Xr-MHSN8RJDVp6gwr6sMsMqkqITKcIcdCFB5JqQQu6_O--woxltIKxe1wnqP7UsQRQmgDlg3t_fEZzSO_HJpBusG7nt-br0P08IP3plxXPO5aRJBHb8KfiLrIr8gR8FM6eq3nRbc8OvUOhK_Wofgx3W0jvj08_vZBnzP3vVmjHT0uB-yX1-_3My-ZfMfF5ezs3nW5ohThn1JpRASqCEihTLvAJtWAvR9XmIaHaGuqq4DKRX1jQJTFKoxTTImOlHJQ_Z5q3u3apbUteSmYEZ9F-zShLX2xup_K84u9ODvdYGFEAKSwOmjQPB_VhQnvbSxTdaNI7-KWiCqoq5VJRP64Q1661chfdeGkmUJNT5MJLZUG3yMgfrnYRD0Q456m6NOOepNjhpT08lrG88tT6klQG6BmEpuoPDy9n9k_wI6d6f1</recordid><startdate>20180928</startdate><enddate>20180928</enddate><creator>Aloush, Noa</creator><creator>Schvartz, Tomer</creator><creator>König, Andres I.</creator><creator>Cohen, Sarit</creator><creator>Brozgol, Eugene</creator><creator>Tam, Benjamin</creator><creator>Nachmias, Dikla</creator><creator>Ben-David, Oshrit</creator><creator>Garini, Yuval</creator><creator>Elia, Natalie</creator><creator>Arbely, Eyal</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>C6C</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>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-2537-6173</orcidid><orcidid>https://orcid.org/0000-0002-0284-0092</orcidid></search><sort><creationdate>20180928</creationdate><title>Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene</title><author>Aloush, Noa ; Schvartz, Tomer ; König, Andres I. ; Cohen, Sarit ; Brozgol, Eugene ; Tam, Benjamin ; Nachmias, Dikla ; Ben-David, Oshrit ; Garini, Yuval ; Elia, Natalie ; Arbely, Eyal</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c511t-1f7e72230ebeee4135d01bc300ff57141910988dd0334efb40a664bab0522d283</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>13/31</topic><topic>14/19</topic><topic>14/34</topic><topic>14/63</topic><topic>631/1647/245/2225</topic><topic>631/45/2783</topic><topic>631/80/2373/2238</topic><topic>631/92/96</topic><topic>639/638/92/552</topic><topic>Amino acids</topic><topic>Animals</topic><topic>Chlorocebus aethiops</topic><topic>Codon, Terminator</topic><topic>COS Cells</topic><topic>Fluorescence</topic><topic>Genetic Code</topic><topic>Humanities and Social Sciences</topic><topic>Intracellular</topic><topic>Intracellular levels</topic><topic>Lysine - analogs & derivatives</topic><topic>Lysine - genetics</topic><topic>Mammalian cells</topic><topic>multidisciplinary</topic><topic>Optical Imaging</topic><topic>Protein Biosynthesis</topic><topic>Proteins</topic><topic>Proteins - genetics</topic><topic>RNA, Transfer - genetics</topic><topic>Science</topic><topic>Science (multidisciplinary)</topic><topic>Stop codon</topic><topic>Transfection</topic><topic>tRNA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Aloush, Noa</creatorcontrib><creatorcontrib>Schvartz, Tomer</creatorcontrib><creatorcontrib>König, Andres I.</creatorcontrib><creatorcontrib>Cohen, Sarit</creatorcontrib><creatorcontrib>Brozgol, Eugene</creatorcontrib><creatorcontrib>Tam, Benjamin</creatorcontrib><creatorcontrib>Nachmias, Dikla</creatorcontrib><creatorcontrib>Ben-David, Oshrit</creatorcontrib><creatorcontrib>Garini, Yuval</creatorcontrib><creatorcontrib>Elia, Natalie</creatorcontrib><creatorcontrib>Arbely, Eyal</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Scientific reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Aloush, Noa</au><au>Schvartz, Tomer</au><au>König, Andres I.</au><au>Cohen, Sarit</au><au>Brozgol, Eugene</au><au>Tam, Benjamin</au><au>Nachmias, Dikla</au><au>Ben-David, Oshrit</au><au>Garini, Yuval</au><au>Elia, Natalie</au><au>Arbely, Eyal</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene</atitle><jtitle>Scientific reports</jtitle><stitle>Sci Rep</stitle><addtitle>Sci Rep</addtitle><date>2018-09-28</date><risdate>2018</risdate><volume>8</volume><issue>1</issue><spage>14527</spage><epage>11</epage><pages>14527-11</pages><artnum>14527</artnum><issn>2045-2322</issn><eissn>2045-2322</eissn><abstract>Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine
synthetase
/
tRNA
CUA
Pyl
pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNA
Pyl
gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNA
Pyl
on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNA
Pyl
population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>30267004</pmid><doi>10.1038/s41598-018-32824-1</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0002-2537-6173</orcidid><orcidid>https://orcid.org/0000-0002-0284-0092</orcidid><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Springer Nature OA Free Journals; Nature Free; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | 13/31 14/19 14/34 14/63 631/1647/245/2225 631/45/2783 631/80/2373/2238 631/92/96 639/638/92/552 Amino acids Animals Chlorocebus aethiops Codon, Terminator COS Cells Fluorescence Genetic Code Humanities and Social Sciences Intracellular Intracellular levels Lysine - analogs & derivatives Lysine - genetics Mammalian cells multidisciplinary Optical Imaging Protein Biosynthesis Proteins Proteins - genetics RNA, Transfer - genetics Science Science (multidisciplinary) Stop codon Transfection tRNA |
| title | Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene |
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