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Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli

Authors
Lin, Guang-ZheLian, Yu-JiRyu, Jae-HaSung, Mi-KyungPark, Jong-SugPark, Hong-JaePark, Byung KiShin, Jong-SuhLee, Myeong-SokCheon, Choong-Ill
Issue Date
Oct-2007
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Keywords
Camellia sinensis; flavonol synthase; protein purification
Citation
PROTEIN EXPRESSION AND PURIFICATION, v.55, no.2, pp.287 - 292
Journal Title
PROTEIN EXPRESSION AND PURIFICATION
Volume
55
Number
2
Start Page
287
End Page
292
URI
https://scholarworks.sookmyung.ac.kr/handle/2020.sw.sookmyung/14625
DOI
10.1016/j.pep.2007.05.013
ISSN
1046-5928
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.
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이과대학 > 생명시스템학부 > 1. Journal Articles
생활과학대학 > 식품영양학과 > 1. Journal Articles
약학대학 > 약학부 > 1. Journal Articles

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생활과학대학 (식품영양학과)
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