基于GST-pull down的小G蛋白Rac1活性检测体系的建立

目的:根据人PAK1基因的p21结合结构域(PBD)能够特异结合GTP-Rac1的特性,构建GST-PBD原核表达质粒,并利用GST-pull down方法检测真核细胞内小G蛋白Rac1的活性。方法:将人PAK1基因的PBD结构域克隆到pGEX原核表达载体上,经诱导纯化得到GST-PBD融合蛋白,并通过GST-pull down实验评估该方法的特异性和准确性。结果:pGEX-PBD质粒构建成功;纯化得到的GST-PBD融合蛋白能够特异性地与激活形式的Rac1(GTP-Rac1)结合,并且能够准确反映血小板衍生生长因子刺激下细胞内Rac1的激活过程。结论:GST-PBD融合蛋白及其整套GST-pull down检测体系能方便有效地检测细胞内Rac1的活性。

Construction of Small G Protein Rac1 Activity Analysis System Based on GST-Pull Down

Objective： According to the character of human PAK1 gene whose p21 binding domain（PBD） can bind to GTP-Rac1,we plan to built the GST-PBD prokaryotic expression plasmid,and detect the activity of Rac1 in eukaryocytes through GST-pull down.Methods： The PBD domain of human PAK1 gene was cloned into pGEX prokaryotic expression vector and the GST-PBD fusion protein was induced and purified.Then GST-pull down experiments were performed to evaluate the specificity and accurance of this method.Results： The pGEX-PBD clone was successfully constructed.The purified GST-PBD fusion protein can specifically bind to the active form of Rac1（GTP-Rac1）,and correctly reflect the procedure of Rac1 activation when cells were treated with platelet-derived growth factor.Conclusion： The GST-PBD fusion protein and the whole GST-pull down experiment system are convenient and effective for Rac1 activity analysis.