Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli.
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.
Lin
Guang-Zhe
GZ
Department of Biological Science, Sookmyung Women's University, Hyochangwon-gil 52, Yongsan-gu, Seoul 140-742, Republic of Korea.
Lian
Yu-Ji
YJ
Ryu
Jae-Ha
JH
Sung
Mi-Kyung
MK
Park
Jong-Sug
JS
Park
Hong-Jae
HJ
Park
Byung Ki
BK
Shin
Jong-Suh
JS
Lee
Myeong-Sok
MS
Cheon
Choong-Ill
CI
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
D017931:DNA Primers; D018076:DNA, Complementary; D010940:Plant Proteins; D010088:Oxidoreductases; C085313:flavonol synthase