茉莉花<i>JsMYB</i>305转录因子的原核表达及蛋白纯化

万超; 张月; 胡莉; 伍炳华; 袁媛

doi:10.19303/j.issn.1008-0384.2022.002.005

茉莉花JsMYB305转录因子的原核表达及蛋白纯化

Prokaryotic Expression and Purification of JsMYB305 Transcription Factor in Jasminum sambac

摘要

摘要:
目的获得茉莉花香气调控转录因子JsMYB305的重组蛋白，为深入研究JsMYB305调控茉莉萜类香气代谢的分子机理及筛选其他互作蛋白提供基础。
方法通过酶切连接的方式，将JsMYB305的编码序列构建到原核表达载体pGEX-4T-1，转化BL21（DE3）表达菌株，通过IPTG诱导蛋白表达、GST亲和树脂分离纯化，最后Western Blot鉴定重组蛋白。
结果重组蛋白的诱导条件为0.2 mmol·L⁻¹ IPTG，最适宜纯化的温度和时间为28 ℃诱导4 h，经20 mmol·L⁻¹ GSH洗脱的蛋白纯度较好，Western Blot结果表明重组蛋白的大小正确。
结论成功获得了JsMYB305重组蛋白，为后期利用GST-pull down技术筛选互作蛋白及EMSA研究JsMYB305对特定启动子位点的结合提供基础。

Abstract:
Objective The recombinant protein of fragrance-regulating transcription factor JsMYB305 was obtained in preparation for studies on the molecular mechanism of terpenoid-regulation and interacting proteins of the gene in Jasminum sambac.
Methods Coding sequence of JsMYB305 was constructed into prokaryotic expression vector pGEX-4T-1 by enzyme digestion and ligation, then, transformed into the BL21 (DE3) expressing strain. The protein expression was induced by IPTG, and the recombinant protein separated and purified using GST affinity resin followed by western blot for positive identification.
Result After induction by 0.2 mmol·L⁻¹ IPTG, the recombinant protein was purified at the optimized temperature of 28 ℃ for 4 h and eluted with 20 mmol·L⁻¹ GSH for purification. The western blot confirmed the protein weight to be as expected.
Conclusion The recombinant protein of JsMYB305 was successfully obtained to pave the way for further studies on the search for the interacting proteins by GST-pull down technique and the binding of the gene to specific promoter sites by EMSA.

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