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 0.1 mM IPTG at low temperature (20 °C) led to higher amounts of CsFLS in the soluble fraction than induction at 30 °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 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.