牛大力CsMYB36转录因子的原核表达、蛋白纯化及分子对接分析OA

[Objective]Recombinant protein of the isoflavonoid biosynthesis-regulating transcription factor CsMYB36 from Callerya speciosa was obtained to predict the promoters of interacting target proteins.[Methods]CsMYB36 was cloned to perform heterologous expression and construct the recombinant expression vector pET32a-CsMYB36 for expression in Escherichia coli BL21(DE3).After IPTG-induced expression,the target protein was isolated and purified with His affinity resin to optimize the operational conditions.Online software was employed to predict molecular docking between CsMYB36 and target gene promoters in the isoflavone biosynthesis pathway for initial identification.[Result]The optimized induction and expression for the recombinant CsMYB36 applied 0.5 mmol·L-1 IPTG at 37℃for 6 h.The soluble protein was expressed in the entire strain as well as the supernatant and precipitate.The highly purified protein was obtained with 350 mmol·L-1 imidazole elution,and its size correctly detected by SDS-PAGE at the concentration of 1.138 mg·mL-1.CsMYB36 could bind to the promoters of genes such as CsIFR2,CsIFS1,CsCHS4,and CsI3'H2 in the isoflavone biosynthesis pathway at sites depending upon the key residues of ARG-67 and THR-109.[Conclusion]The recombinant CsMYB36 was successfully secured with information on molecular docking that provided the basis for further EMSA in vitro functional verification studies.