带6个组氨酸尾的可溶性GDNF第1受体的重组表达和活性分析

RECOMBINANT EXPRESSION AND ACTIVITY ANALYSIS OF C-TERMINALLY HEXAHISTIDINE-TAGGED SOLUBLE GFRα-1

目的 用DNA重组方法去除胶质细胞源性神经营养因子 (GDNF)受体 (GFRα 1)基因 3’ 端的与细胞膜糖基化磷脂酰肌醇 (GPI)相连的信号序列 ,使表达的GFRα 1以可溶性形式介导GDNF的作用。 方法 设计一对引物 ,其中下游引物含有 6个组氨酸密码子序列 ,以人GFRα 1全长cDNA为模板 ,PCR扩增编码可溶性GFRα 1序列并亚克隆于pBK RSV真核表达载体 ,转染COS 7细胞 ,收获培养上清 ,进行SDS PAGE及Western免疫印迹分析 ;使用钴离子金属螯合层析柱进行纯化 ;检测RET酪氨酸磷酸化以鉴定其生物学活性。 结果 Western免疫印迹证实培养上清液中有分子量为 6 0kD的特异阳性条带 ,与糖基化的GFRα 1分子量相符 ;金属螯合层析得到纯度达90 %以上的重组GFRα 1蛋白 ,每mlCOS 7培养上清中含有 0 5 μg。 结论 带 6个组氨酸尾的重组可溶性GFRα 1能够介导GDNF的作用 。

Objective In present study,the GPI anchoring signal at C terminal of GFRα l was deleted by using DNA recombination techniques to get soluble GFRα 1 which was expected to mediate the GDNF biological effects. Methods The specific primers for PCR were designed,with the downstream primer containing sequences encoding hexahistidine and stop codon.PCR amplification was performed to harvest the DNA fragment encoding for soluble GFRα 1 with C terminal hexahistidine tail by using the complete cDNA of human GFRα 1 as template.The PCR product was subcloned into eukaryotic expression vector pBK RSV which was then transfected into COS 7 cell line by electroporation.The culture supernatant was collected for purification,SDS PAGE and Western blot and biological activity analysis. Results Expression of the soluble GFRα 1 was confirmed from the culture supernatant by Western blot analysis as revealed by a molecular weight of 60 kDa,identical to that of the expected glycosylated GFRα 1.Using cobalt metali on affinity chromatography method,recombined soluble GFRα 1 protein was purified from the culture medium with a purity of up to 90% as well as a product output 0 5?μg/ml.Conclusion\ The purified soluble GFRα 1 with hexahistidine tail was shown to be able to mediate GDNF induced RET phosphorylation.\;[