{"refrec":{"BRefID":308016,"RR":"<b>Horiuchi, Y.; Laskaratou, D.; Sliwa, M.; Ruckebusch, C.; Hatori, K.; Mizuno, H.; Hotta, J.-I.</b> (2018). Frame-insensitive expression cloning of fluorescent protein from <i>Scolionema suvaense</i>. <i>International Journal of Molecular Sciences 19(2)</i>: 371. <a href=\"https://dx.doi.org/10.3390/ijms19020371\" target=\"_blank\">https://dx.doi.org/10.3390/ijms19020371</a>","BEntID":300342,"PublicFlag":1,"CheckedFlag":1,"wosflag":1,"vabbflag":0,"RefStringPartII":". <i>International Journal of Molecular Sciences 19(2)</i>: 371. <a href=\"https://dx.doi.org/10.3390/ijms19020371\" target=\"_blank\">https://dx.doi.org/10.3390/ijms19020371</a>","DocTypID":8,"DocType":"Journal article","MarineFlag":0,"FreshFlag":0,"BrackishFlag":0,"TerrestrialFlag":0,"Authorstring":"Horiuchi, Y.; Laskaratou, D.; Sliwa, M.; Ruckebusch, C.; Hatori, K.; Mizuno, H.; Hotta, J.-I.","OrigTitleTranslFlag":0,"Authorstringtrunc":"Horiuchi, Y. <i>et al.</i>","Englishabstract":"Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)<sub>14</sub> next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, <span class=\"html-italic\">E. coli</span> cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from <span class=\"html-italic\">Scolionema suvaense</span>. We screened 3658 <span class=\"html-italic\">E. coli</span> colonies transformed with pRSET-TriEX containing <span class=\"html-italic\">Scolionema suvaense</span> cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10<sup>5</sup> M<sup>−1</sup>·cm<sup>−1</sup>. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.","AbstractOtherLang":null,"BibLvlCode":"AS","StandardTitle":"Frame-insensitive expression cloning of fluorescent protein from <i>Scolionema suvaense</i>","OrigTitleLangCode":"en","OrigTitleLangCodeExtended":"eng","OrigTitleLangID":15,"DateLastModified":{"date":"2024-12-10 01:33:17.368041","timezone_type":1,"timezone":"+01:00"},"UserAccessRight":null,"UserAccID":null,"AuthorKeywords":"fluorescent protein; expression cloning; reading frame; frameshift","OtherDescriptors":null,"Notes":null,"AnaPub":2018,"MonPub":null,"DateUpdate":"2019-03-19","DateCreate":"2019-03-13","SecASFANote":null,"ConfID":null,"PeerRev":1,"VlizCoreFlag":1,"WoScode":"WOS:000427527400055","VABBcode":null,"OpenAcc":1,"DOI":"10.3390/ijms19020371"},"refs":null,"anarec":{"AnaID":308016,"PubliDate":2018,"Pagination":"371","XtraPublOfAnaID":null,"ISBN":null,"Volume":"19","Issue":"2","BRefMon":null,"BRefMonRR":null,"BRefXtra":null,"BRefXtraRR":null,"SerBRefID":270393,"SerRR":"International Journal of Molecular Sciences. 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