Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli Guang-Zhe Lin a,b , Yu-Ji Lian a,b , Jae-Ha Ryu c , Mi-Kyung Sung d , Jong-Sug Park e , Hong-Jae Park e , Byung Ki Park f , Jong-Suh Shin f , Myeong-Sok Lee a , Choong-Ill Cheon a, * a Department of Biological Science, Sookmyung Women’s University, Hyochangwon-gil 52, Yongsan-gu, Seoul 140-742, Republic of Korea b College of Life Science, Linyi Normal University, Linyi, Shandong, China c College of Pharmacy, Sookmyung Women’s University, Seoul, Republic of Korea d Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Republic of Korea e National Institute of Agricultural Biotechnology, RDA, Suwon, Republic of Korea f Division of Animal Resource Science, Kangwon National University, Chuncheon, Republic of Korea Received 13 March 2007, and in revised form 21 May 2007 Available online 2 June 2007 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 0 - and 3 0 -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 HisÆBind 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. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Camellia sinensis; Flavonol synthase; Protein purification Flavonoids have been widely studied, as their consump- tion in vegetables and fruits is beneficial for health on account of their anti-oxidative, anti-inflammatory, and anti-carcinogenic activities [1,2]. Flavonoids are divided into several classes, such as flavonols, anthocyanidins, catechins, and flavones, depending on the modifications to their benzene rings. Flavonols abundant in green tea (Camellia sinensis), have benefits in several chronic diseases [3]. The contents of the flavonols, quercetin, and kaempferol, in tea were reported to be higher than in tomato juice and red wine [4]. Quercetin, the most abundant flavonoid in the human diet, has been reported to have anti-proliferative effects on human cancer cell lines and to induce apoptosis of leukemic cells. It appears to induce apoptosis by affecting many key signaling components including caspases, anti-apoptotic proteins, and members of the MAP kinase family [5,6]. Flavonol synthase (FLS) 1 , a dioxygenase converting dihyroflavonols into flavonols, was found initially in 1046-5928/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.05.013 * Corresponding author. Fax: +82 2 2077 7322. E-mail address: ccheon@sookmyung.ac.kr (C.-I. Cheon). 1 Abbreviations used: FLS, flavonol synthase; EST, expressed sequence tag; TES, Tea Experimental Station; GSPs, gene-specific primers; CsFLS, cDNA encoding flavonol synthase. www.elsevier.com/locate/yprep Protein Expression and Purification 55 (2007) 287–292