Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Lin, Guang-Zhe | - |
dc.contributor.author | Lian, Yu-Ji | - |
dc.contributor.author | Ryu, Jae-Ha | - |
dc.contributor.author | Sung, Mi-Kyung | - |
dc.contributor.author | Park, Jong-Sug | - |
dc.contributor.author | Park, Hong-Jae | - |
dc.contributor.author | Park, Byung Ki | - |
dc.contributor.author | Shin, Jong-Suh | - |
dc.contributor.author | Lee, Myeong-Sok | - |
dc.contributor.author | Cheon, Choong-Ill | - |
dc.date.available | 2021-02-22T15:01:55Z | - |
dc.date.issued | 2007-10 | - |
dc.identifier.issn | 1046-5928 | - |
dc.identifier.issn | 1096-0279 | - |
dc.identifier.uri | https://scholarworks.sookmyung.ac.kr/handle/2020.sw.sookmyung/14625 | - |
dc.description.abstract | Flavonols, a class of bioactive polyphenols present in plants, are the products of flavonol desaturation catalyzed by flavonol synthase (FLS). We cloned the cDNA coding for the enzyme FLS from Camellia sinensis (CsFLS) by end-to-end PCR followed by 5'- and 3'-RACE. The putative CsFLS had 333 amino acid residues, displayed identities to the FLSs of Arabidopsis and Ginkgo of 53% and 52.5%, respectively, and contained several conserved elements found in the 2-oxoglutarate-Fe(II)-dioxygenase superfamily. The cDNA of CYFLS was subcloned into pET28a(+) and introduced into Escherichia coli (BL21 -CodonPlus-RIL). Induction with 0. 1 mM IPTG at low temperature (20 degrees C) led to higher amounts of CsFLS in the soluble fraction than induction at 30 degrees C. The enzyme aggregated into inclusion bodies could be rescued by denaturation with 6 M urea and purification with a His-Bind purification kit. The purified protein was desalted by Amicon Ultra-15 centrifugal filter unit, and the His-tag was removed with thrombin. The finally purified protein was assayed with dihydroquercetin as substrate and the products were analyzed by HPLC. The addition of FeSO4 to the buffers used in the CsFLS purification significantly increased the recovery of active enzyme. The CsFLS obtained in this study was found to have higher specific activity and lower K,, than previously reported FLSs. (C) 2007 Elsevier Inc. All rights reserved. | - |
dc.format.extent | 6 | - |
dc.language | 영어 | - |
dc.language.iso | ENG | - |
dc.publisher | ACADEMIC PRESS INC ELSEVIER SCIENCE | - |
dc.title | Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli | - |
dc.type | Article | - |
dc.publisher.location | 미국 | - |
dc.identifier.doi | 10.1016/j.pep.2007.05.013 | - |
dc.identifier.scopusid | 2-s2.0-34548401236 | - |
dc.identifier.wosid | 000250070400009 | - |
dc.identifier.bibliographicCitation | PROTEIN EXPRESSION AND PURIFICATION, v.55, no.2, pp 287 - 292 | - |
dc.citation.title | PROTEIN EXPRESSION AND PURIFICATION | - |
dc.citation.volume | 55 | - |
dc.citation.number | 2 | - |
dc.citation.startPage | 287 | - |
dc.citation.endPage | 292 | - |
dc.type.docType | Article | - |
dc.description.isOpenAccess | N | - |
dc.description.journalRegisteredClass | scie | - |
dc.description.journalRegisteredClass | scopus | - |
dc.relation.journalResearchArea | Biochemistry & Molecular Biology | - |
dc.relation.journalResearchArea | Biotechnology & Applied Microbiology | - |
dc.relation.journalWebOfScienceCategory | Biochemical Research Methods | - |
dc.relation.journalWebOfScienceCategory | Biochemistry & Molecular Biology | - |
dc.relation.journalWebOfScienceCategory | Biotechnology & Applied Microbiology | - |
dc.subject.keywordPlus | PETUNIA-HYBRIDA | - |
dc.subject.keywordPlus | FLAVANONE 3-BETA-HYDROXYLASE | - |
dc.subject.keywordPlus | CITRUS-UNSHIU | - |
dc.subject.keywordPlus | ACTIVE-SITE | - |
dc.subject.keywordPlus | TEA | - |
dc.subject.keywordPlus | ARABIDOPSIS | - |
dc.subject.keywordPlus | DIOXYGENASE | - |
dc.subject.keywordPlus | CONVERSION | - |
dc.subject.keywordPlus | OXYGENASES | - |
dc.subject.keywordPlus | APOPTOSIS | - |
dc.subject.keywordAuthor | Camellia sinensis | - |
dc.subject.keywordAuthor | flavonol synthase | - |
dc.subject.keywordAuthor | protein purification | - |
dc.identifier.url | https://www.sciencedirect.com/science/article/abs/pii/S1046592807001490?via%3Dihub | - |
Items in ScholarWorks are protected by copyright, with all rights reserved, unless otherwise indicated.
Sookmyung Women's University. Cheongpa-ro 47-gil 100 (Cheongpa-dong 2ga), Yongsan-gu, Seoul, 04310, Korea02-710-9127
Copyright©Sookmyung Women's University. All Rights Reserved.
Certain data included herein are derived from the © Web of Science of Clarivate Analytics. All rights reserved.
You may not copy or re-distribute this material in whole or in part without the prior written consent of Clarivate Analytics.