Grb7蛋白SH2结构域的表达及其纯化的方法

The expression and purification of Grb7 SH2 domain

目的:探讨Grb7蛋白SH2结构域的表达及其纯化的方法。方法:将Grb7蛋白SH2结构域编码基因与谷胱甘肽巯基转移酶(GST)标签编码基因构建为融合基因,插入原核表达载体pGEX-4T-1的多克隆酶切位点EcoRⅠ与XhoⅠ之间,挑选测序正确的质粒将其转染至BL 21感受态细胞内,感受态细胞于37℃摇培至其OD值达到0.6～0.8后,经0.2 mmol/L IPTG 25℃过夜诱导表达融合蛋白,MALDI-TOF质谱检测蛋白纯化产物,SPR生物传感器(Biacore 3000)检测纯化蛋白与天然配体磷酸化肽段的相互作用。结果:GST-Grb7 SH2结构域融合蛋白在裂解菌液的上清液中存在,500 ml LB/Amp菌液可纯化得到415μg GST-Grb7 SH2结构域融合蛋白,MALDI-TOF质谱图上可见明显目的蛋白峰,Biacore 3000检测到纯化蛋白可结合不同浓度的磷酸化肽段配体。结论:运用改进的GST融合蛋白纯化方法可得到具有一定纯度及生物学活性的GST-Grb7 SH2结构域融合蛋白,为该蛋白结构域后续功能研究奠定了基础。

Objective：To explore the method which to express and purify the bioactive Grb7 SH2 domain.Methods：The fusion gene of GST-Grb7 SH2,which was constructed into the site between EcoRⅠ and XhoⅠ in the pEGX-4T-1,was induced by the 0.2 mmol/L IPTG to express in the BL 21 competent cells,at 25 ℃ for overnight,and the MALDI-TOF and Biacore3000 were used to identify the purity and bioactivity of the recombinant protein.Results：GST-Grb7 SH2 domain fusion protein（415 μg） was purified from 500 ml LB bacterial liquid,and protein peaks were observed on the map of MALDI-TOF,and interactions between GST-Grb7 SH2 domain and phosphorylated peptide pY（1180） from the ErbB3,with concentrations of 10 nmol/L and 100 nmol/L,were measured by Biacore 3000.Conclusions：The recombinant GST-Grb7 SH2 domain can be obtained through a improved method,which may help to furtherly investigate the functions of Grb7 SH2 domain.