Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging
A luciferase from the railroad worm (Phrixothrix hirtus) is the only red‐emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low acti...
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| Veröffentlicht in: | Protein science 2010-01, Vol.19 (1), p.26-33 |
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| creator | Li, Xueyan Nakajima, Yoshihiro Niwa, Kazuki Viviani, Vadim R. Ohmiya, Yoshihiro |
| description | A luciferase from the railroad worm (Phrixothrix hirtus) is the only red‐emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site‐directed mutagenesis, we produced red‐emitting mutants with higher activity and better stability. Compared with the wild‐type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8‐fold in I212L/N351K, 8.4‐fold in I212L, and 7.8‐fold in I212L/S463R; and the cell‐based activities were 3.6‐fold in I212L/N351K and 3.4‐fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell‐based BLI was performed, and the luminescence signal was 3.6‐fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI. |
| doi_str_mv | 10.1002/pro.279 |
| format | Article |
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However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site‐directed mutagenesis, we produced red‐emitting mutants with higher activity and better stability. Compared with the wild‐type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8‐fold in I212L/N351K, 8.4‐fold in I212L, and 7.8‐fold in I212L/S463R; and the cell‐based activities were 3.6‐fold in I212L/N351K and 3.4‐fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell‐based BLI was performed, and the luminescence signal was 3.6‐fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.</description><identifier>ISSN: 0961-8368</identifier><identifier>EISSN: 1469-896X</identifier><identifier>DOI: 10.1002/pro.279</identifier><identifier>PMID: 19866487</identifier><identifier>CODEN: PRCIEI</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>Animals ; bioassays ; bioimaging ; Biological Assay - methods ; Coleoptera - enzymology ; Coleoptera - genetics ; enhanced activity ; Gene Expression ; Luciferases - chemistry ; Luciferases - genetics ; Luciferases - metabolism ; Mice ; Mutagenesis, Site-Directed ; Mutation ; NIH 3T3 Cells ; Protein Stability ; red‐emitting luciferase ; Spectrum Analysis ; thermostable mutant</subject><ispartof>Protein science, 2010-01, Vol.19 (1), p.26-33</ispartof><rights>Copyright © 2009 The Protein Society</rights><rights>Copyright © 2010 The Protein Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4989-8cffbbde63cda213ed456d5bed67ed86b85647b44fbbe0704b85ff2d2ab31aa53</citedby><cites>FETCH-LOGICAL-c4989-8cffbbde63cda213ed456d5bed67ed86b85647b44fbbe0704b85ff2d2ab31aa53</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817836/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817836/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,1411,1427,27903,27904,45553,45554,46387,46811,53769,53771</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19866487$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Xueyan</creatorcontrib><creatorcontrib>Nakajima, Yoshihiro</creatorcontrib><creatorcontrib>Niwa, Kazuki</creatorcontrib><creatorcontrib>Viviani, Vadim R.</creatorcontrib><creatorcontrib>Ohmiya, Yoshihiro</creatorcontrib><title>Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging</title><title>Protein science</title><addtitle>Protein Sci</addtitle><description>A luciferase from the railroad worm (Phrixothrix hirtus) is the only red‐emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site‐directed mutagenesis, we produced red‐emitting mutants with higher activity and better stability. Compared with the wild‐type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8‐fold in I212L/N351K, 8.4‐fold in I212L, and 7.8‐fold in I212L/S463R; and the cell‐based activities were 3.6‐fold in I212L/N351K and 3.4‐fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell‐based BLI was performed, and the luminescence signal was 3.6‐fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.</description><subject>Animals</subject><subject>bioassays</subject><subject>bioimaging</subject><subject>Biological Assay - methods</subject><subject>Coleoptera - enzymology</subject><subject>Coleoptera - genetics</subject><subject>enhanced activity</subject><subject>Gene Expression</subject><subject>Luciferases - chemistry</subject><subject>Luciferases - genetics</subject><subject>Luciferases - metabolism</subject><subject>Mice</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>NIH 3T3 Cells</subject><subject>Protein Stability</subject><subject>red‐emitting luciferase</subject><subject>Spectrum Analysis</subject><subject>thermostable mutant</subject><issn>0961-8368</issn><issn>1469-896X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kN1KwzAYhoMobk7xDqTggQfSmaRpmp4IMuYPTCai4FlImnTL6JqZtI6deQleo1dixoY_Bx6FL3nyfC8vAMcI9hGE-GLhbB9n-Q7oIkLzmOX0ZRd0YU5RzBLKOuDA-xmEkCCc7IMOyhmlhGVdcD-sp6IutIqcVp_vH3pumsbUk8gJUzkrVLS0bh5VbWFK7YTXUWldJI0V3ouVj0St1pOZi0n4dQj2SlF5fbQ9e-D5evg0uI1H45u7wdUoLkjOQryiLKVUmiaFEhglWpGUqlRqRTOtGJUspSSThARKwwyScFGWWGEhEyREmvTA5ca7aOVcq0LXjRMVX7iQw624FYb_fanNlE_sG8cMZaGQIDjdCpx9bbVv-My2rg6ZOcooZQQhjAN1tqEKZ713uvzegCBf9x5my0PvgTz5HeiH2xYdgPMNsDSVXv3n4Q-P47XuC8iKj_I</recordid><startdate>201001</startdate><enddate>201001</enddate><creator>Li, Xueyan</creator><creator>Nakajima, Yoshihiro</creator><creator>Niwa, Kazuki</creator><creator>Viviani, Vadim R.</creator><creator>Ohmiya, Yoshihiro</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><general>Wiley Subscription Services, 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>7QO</scope><scope>7T5</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>201001</creationdate><title>Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging</title><author>Li, Xueyan ; Nakajima, Yoshihiro ; Niwa, Kazuki ; Viviani, Vadim R. ; Ohmiya, Yoshihiro</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4989-8cffbbde63cda213ed456d5bed67ed86b85647b44fbbe0704b85ff2d2ab31aa53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Animals</topic><topic>bioassays</topic><topic>bioimaging</topic><topic>Biological Assay - methods</topic><topic>Coleoptera - enzymology</topic><topic>Coleoptera - genetics</topic><topic>enhanced activity</topic><topic>Gene Expression</topic><topic>Luciferases - chemistry</topic><topic>Luciferases - genetics</topic><topic>Luciferases - metabolism</topic><topic>Mice</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutation</topic><topic>NIH 3T3 Cells</topic><topic>Protein Stability</topic><topic>red‐emitting luciferase</topic><topic>Spectrum Analysis</topic><topic>thermostable mutant</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Xueyan</creatorcontrib><creatorcontrib>Nakajima, Yoshihiro</creatorcontrib><creatorcontrib>Niwa, Kazuki</creatorcontrib><creatorcontrib>Viviani, Vadim R.</creatorcontrib><creatorcontrib>Ohmiya, Yoshihiro</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Protein science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Xueyan</au><au>Nakajima, Yoshihiro</au><au>Niwa, Kazuki</au><au>Viviani, Vadim R.</au><au>Ohmiya, Yoshihiro</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging</atitle><jtitle>Protein science</jtitle><addtitle>Protein Sci</addtitle><date>2010-01</date><risdate>2010</risdate><volume>19</volume><issue>1</issue><spage>26</spage><epage>33</epage><pages>26-33</pages><issn>0961-8368</issn><eissn>1469-896X</eissn><coden>PRCIEI</coden><abstract>A luciferase from the railroad worm (Phrixothrix hirtus) is the only red‐emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site‐directed mutagenesis, we produced red‐emitting mutants with higher activity and better stability. Compared with the wild‐type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8‐fold in I212L/N351K, 8.4‐fold in I212L, and 7.8‐fold in I212L/S463R; and the cell‐based activities were 3.6‐fold in I212L/N351K and 3.4‐fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell‐based BLI was performed, and the luminescence signal was 3.6‐fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>19866487</pmid><doi>10.1002/pro.279</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals bioassays bioimaging Biological Assay - methods Coleoptera - enzymology Coleoptera - genetics enhanced activity Gene Expression Luciferases - chemistry Luciferases - genetics Luciferases - metabolism Mice Mutagenesis, Site-Directed Mutation NIH 3T3 Cells Protein Stability red‐emitting luciferase Spectrum Analysis thermostable mutant |
| title | Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging |
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