title: Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli.
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 CsFLS was subcloned into pET28a(+) and introduced into Escherichia coli (BL21-CodonPlus-RIL). Induction with 0.1mM 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 6M 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 FeSO(4) 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(m) than previously reported FLSs.
authors.affiliation: Department of Biological Science, Sookmyung Women's University, Hyochangwon-gil 52, Yongsan-gu, Seoul 140-742, Republic of Korea.
authors.forename: Guang-Zhe
authors.initials: GZ
authors.lastname: Lin
authors.forename: Yu-Ji
authors.initials: YJ
authors.lastname: Lian
authors.forename: Jae-Ha
authors.initials: JH
authors.lastname: Ryu
authors.forename: Mi-Kyung
authors.initials: MK
authors.lastname: Sung
authors.forename: Jong-Sug
authors.initials: JS
authors.lastname: Park
authors.forename: Hong-Jae
authors.initials: HJ
authors.lastname: Park
authors.forename: Byung Ki
authors.initials: BK
authors.lastname: Park
authors.forename: Jong-Suh
authors.initials: JS
authors.lastname: Shin
authors.forename: Myeong-Sok
authors.initials: MS
authors.lastname: Lee
authors.forename: Choong-Ill
authors.initials: CI
authors.lastname: Cheon
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D028241:Camellia sinensis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D010088:Oxidoreductases; C085313:flavonol synthase