Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8360353 | Protein Expression and Purification | 2015 | 7 Pages |
Abstract
Plant cytochrome P450 enzymes play vital roles in the biosynthesis of plant secondary metabolites, including phenylpropanoids and phytoalexins. Isoflavone-2â²-hydroxylase (AmI2â²H) 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 AmI2â²H 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 AmI2â²H in Escherichia coli. The expression efficiency and purity of target protein were analyzed and demonstrated that pET-28a(+) vector containing the AmI2â²H gene could produce larger amounts of target proteins with higher purity than pCWori(+). The purified proteins were identified as AmI2â²H by LC-ESI-MS/MS analysis and their proper folding was assessed by CO difference spectrum.
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Authors
Jing Chen, Hui Yuan, Lin Zhang, Haiyun Pan, Rongyan Xu, Yang Zhong, Jiakuan Chen, Peng Nan,