牛大力CsMYB36转录因子的原核表达、蛋白纯化及分子对接分析

祝滢; 李斌; 陈柳萍; 廖海伦; 明如宏; 黄鼎; 姚绍嫦

牛大力CsMYB36转录因子的原核表达、蛋白纯化及分子对接分析

Prokaryotic Expression, Purification, and Molecular Docking of CsMYB36 in Callerya speciosa

摘要

摘要:
目的为获得牛大力异黄酮生物合成调控转录因子CsMYB36的重组蛋白，并预测其互作的靶蛋白启动子，本研究在成功克隆牛大力中转录因子CsMYB36的基础上，对CsMYB36进行异源表达，并对其产物蛋白进行纯化。
方法构建重组表达载体pET32a-CsMYB36，分析该蛋白在大肠杆菌BL21（DE3）中的表达情况，然后利用IPTG诱导蛋白表达、His亲和树脂分离纯化目标蛋白，确定表达与纯化条件；利用在线软件对CsMYB36蛋白与异黄酮生物合成途径的靶基因启动子进行分子对接预测，初步筛选出互作的启动子。
结果 CsMYB36重组蛋白最佳的诱导表达条件为0.5 mmol·L⁻¹ IPTG、37℃和6 h；重组蛋白分布在全菌、破碎上清以及沉淀中，表明该蛋白表达能够以可溶形式存在；经350 mM咪唑洗脱后蛋白纯度较好；经SDS-PAGE检测重组蛋白的大小正确，浓度为1.138 mg·mL⁻¹；CsMYB36可与CsIFR2、CsIFS1、CsCHS4、CsI3′H2异黄酮生物合成途径的靶基因启动子结合，结合位点依赖于精氨酸-67和苏氨酸-109关键残基。
结论成功获得牛大力CsMYB36重组蛋白，分子对接结果有助于了解该蛋白的基本特性和研究其与启动子的结合位点，两者为后续EMSA体外功能验证实验奠定研究基础。

Abstract:
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 6h. The soluble protein was expressed in the entire strain as well as the supernatant and precipitate. The highly purified protein was obtained with 350mM imidazole elution, and its size correctly detected by SDS-PAGE at the concentration of 1.138mg·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.

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