Cloning, expression and purification of isoflavone-2′-hydroxylase from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao

•We succeeded in cloning the AmI2′H gene.•We modified the ORF sequence of this plant P450 protein to facilitate its expression and solubility using gene engineering techniques.•The pET 28a_AmI2′H was better than pCW_AmI2′H in both expression efficiency and purity. 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.