title: Cloning, expression and purification of isoflavone-2'-hydroxylase from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao.
abstract: Plant cytochrome P450 enzymes play vital roles in the biosynthesis of plant secondary metabolites, including phenylpropanoids and phytoalexins. Isoflavone-2'-hydroxylase (AmI2'H) from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao is a membrane protein and an eukaryotic cytochrome P450 enzyme involved in isoflavonoid biosynthesis. We cloned the AmI2'H gene by employing RACE methods and modified the gene sequence to facilitate protein expression and increase protein solubility. Two vectors, pET-28a(+) and pCW ori(+), were used to express AmI2'H in Escherichia coli. The expression efficiency and purity of target protein were analyzed and demonstrated that pET-28a(+) vector containing the AmI2'H gene could produce larger amounts of target proteins with higher purity than pCWori(+). The purified proteins were identified as AmI2'H by LC-ESI-MS/MS analysis and their proper folding was assessed by CO difference spectrum.
authors.affiliation: Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Electronic address: sunnytt91@gmail.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Electronic address: 09210700078@fudan.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Electronic address: linnezhang@gmail.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA. Electronic address: hypan@126.com.
authors.forename: Haiyun
authors.initials: H
authors.lastname: Pan
authors.affiliation: Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Electronic address: ryxu822@fudan.edu.cn.
authors.forename: Rongyan
authors.initials: R
authors.lastname: Xu
authors.affiliation: Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China; Institute of Biodiversity Science and Geobiology, Tibet University, Lhasa 850000, China. Electronic address: yangzhong@fudan.edu.cn.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Electronic address: jkchen@fudan.edu.cn.
authors.forename: Jiakuan
authors.initials: J
authors.lastname: Chen
authors.affiliation: Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Electronic address: nanpeng@fudan.edu.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Nan
mesh_terms: D000595:Amino Acid Sequence; D027885:Astragalus propinquus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C115412:cytochrome P-450 CYP81E1 (Glycyrrhiza echinata)
keywords: CO difference spectrum; Cytochrome P450; Expression vector; Isoflavone-2′-hydroxylase; LC–ESI-MS/MS