Characterization of recombinant photoconverting green fluorescent Akanes
Summary Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes mature...
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| Veröffentlicht in: | Journal of biochemistry (Tokyo) 2024-01, Vol.175 (1), p.25-34 |
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| container_title | Journal of biochemistry (Tokyo) |
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| creator | Jimbo, Mitsuru Otake, Mayumi Amano, Haruna Yasumoto, Ko Watabe, Shugo Okada, Daisuke Kumagai, Hiroshi |
| description | Summary
Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.
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Graphical Abstract |
| doi_str_mv | 10.1093/jb/mvad078 |
| format | Article |
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Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.
Graphical Abstract
Graphical Abstract</description><identifier>ISSN: 0021-924X</identifier><identifier>EISSN: 1756-2651</identifier><identifier>DOI: 10.1093/jb/mvad078</identifier><identifier>PMID: 37812399</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Amino Acid Sequence ; Green Fluorescent Proteins - chemistry ; Light ; Luminescent Proteins - chemistry ; Luminescent Proteins - genetics ; Luminescent Proteins - metabolism ; Tandem Mass Spectrometry</subject><ispartof>Journal of biochemistry (Tokyo), 2024-01, Vol.175 (1), p.25-34</ispartof><rights>The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. 2023</rights><rights>The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c303t-4a977072e32672ed6b192a25ffcdd182ca66de7a538e4acb092cd2f1c3cb14da3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,1578,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37812399$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jimbo, Mitsuru</creatorcontrib><creatorcontrib>Otake, Mayumi</creatorcontrib><creatorcontrib>Amano, Haruna</creatorcontrib><creatorcontrib>Yasumoto, Ko</creatorcontrib><creatorcontrib>Watabe, Shugo</creatorcontrib><creatorcontrib>Okada, Daisuke</creatorcontrib><creatorcontrib>Kumagai, Hiroshi</creatorcontrib><title>Characterization of recombinant photoconverting green fluorescent Akanes</title><title>Journal of biochemistry (Tokyo)</title><addtitle>J Biochem</addtitle><description>Summary
Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.
Graphical Abstract
Graphical Abstract</description><subject>Amino Acid Sequence</subject><subject>Green Fluorescent Proteins - chemistry</subject><subject>Light</subject><subject>Luminescent Proteins - chemistry</subject><subject>Luminescent Proteins - genetics</subject><subject>Luminescent Proteins - metabolism</subject><subject>Tandem Mass Spectrometry</subject><issn>0021-924X</issn><issn>1756-2651</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kM1LwzAchoMobk4v_gHSiyBCXT7apD2OMZ0w8KLgLaTJr1tn29SkHehfb8amRy-_D3h4eXkQuib4geCcTbfFtNkpg0V2gsZEpDymPCWnaIwxJXFOk_cRuvB-u38pY-doxEQWrjwfo-V8o5zSPbjqW_WVbSNbRg60bYqqVW0fdRvbW23bHbi-atfR2gG0UVkP1oHXEIjZh2rBX6KzUtUero57gt4eF6_zZbx6eXqez1axZpj1caJyIbCgwCgP0_CC5FTRtCy1MSSjWnFuQKiUZZAoXeCcakNLopkuSGIUm6C7Q27n7OcAvpdNFXrUdShhBy9pJpKMJRyLgN4fUO2s9w5K2bmqUe5LEiz35uS2kEdzAb455g5FA-YP_VUVgNsDYIfuv6AfYdx4ig</recordid><startdate>20240101</startdate><enddate>20240101</enddate><creator>Jimbo, Mitsuru</creator><creator>Otake, Mayumi</creator><creator>Amano, Haruna</creator><creator>Yasumoto, Ko</creator><creator>Watabe, Shugo</creator><creator>Okada, Daisuke</creator><creator>Kumagai, Hiroshi</creator><general>Oxford University Press</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></search><sort><creationdate>20240101</creationdate><title>Characterization of recombinant photoconverting green fluorescent Akanes</title><author>Jimbo, Mitsuru ; Otake, Mayumi ; Amano, Haruna ; Yasumoto, Ko ; Watabe, Shugo ; Okada, Daisuke ; Kumagai, Hiroshi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c303t-4a977072e32672ed6b192a25ffcdd182ca66de7a538e4acb092cd2f1c3cb14da3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>Amino Acid Sequence</topic><topic>Green Fluorescent Proteins - chemistry</topic><topic>Light</topic><topic>Luminescent Proteins - chemistry</topic><topic>Luminescent Proteins - genetics</topic><topic>Luminescent Proteins - metabolism</topic><topic>Tandem Mass Spectrometry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jimbo, Mitsuru</creatorcontrib><creatorcontrib>Otake, Mayumi</creatorcontrib><creatorcontrib>Amano, Haruna</creatorcontrib><creatorcontrib>Yasumoto, Ko</creatorcontrib><creatorcontrib>Watabe, Shugo</creatorcontrib><creatorcontrib>Okada, Daisuke</creatorcontrib><creatorcontrib>Kumagai, Hiroshi</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>Journal of biochemistry (Tokyo)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jimbo, Mitsuru</au><au>Otake, Mayumi</au><au>Amano, Haruna</au><au>Yasumoto, Ko</au><au>Watabe, Shugo</au><au>Okada, Daisuke</au><au>Kumagai, Hiroshi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of recombinant photoconverting green fluorescent Akanes</atitle><jtitle>Journal of biochemistry (Tokyo)</jtitle><addtitle>J Biochem</addtitle><date>2024-01-01</date><risdate>2024</risdate><volume>175</volume><issue>1</issue><spage>25</spage><epage>34</epage><pages>25-34</pages><issn>0021-924X</issn><eissn>1756-2651</eissn><abstract>Summary
Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.
Graphical Abstract
Graphical Abstract</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>37812399</pmid><doi>10.1093/jb/mvad078</doi><tpages>10</tpages></addata></record> |
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| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Green Fluorescent Proteins - chemistry Light Luminescent Proteins - chemistry Luminescent Proteins - genetics Luminescent Proteins - metabolism Tandem Mass Spectrometry |
| title | Characterization of recombinant photoconverting green fluorescent Akanes |
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