DOC-1R缺失型原核表达载体的构建及其重组蛋白的纯化

Construction of deleted DOC-1R gene expression vector and purification of DOC-1R recombinant protein

目的构建DOC-1R(deleted in oral cancer-I related)缺失型原核表达载体并纯化其重组蛋白,用于进一步研究DOC-1R互作蛋白及DOC-1R与其相互作用区域。方法经基因重组技术构建分别缺失DOC-1R N端1/3序列及其C端1/3序列的原核表达载体,经测序鉴定、转化至E.coli BL21菌株后,诱导表达两种缺失型GSTDOC-1R重组蛋白,并获得纯化的重组蛋白。结果两载体克隆序列及开放阅读框架均正确,经IPTG诱导在35kD处获得重组蛋白,纯化后得到大量GST-DOC-1R缺失型重组蛋白。结论成功构建了两种pGEX-DOC-1R缺失型原核表达载体,表达并纯化出GST-DOC-1R delN42、GST-DOC-1R delC42融合蛋白。

Objective To construct the deleted prokaryotic expression vectors of DOC-1 R（deleted in oral cancer-1 related） and purify its recombinant proteins for further functional study. Methods The prokaryotic expression vectors, lacking the N-terminal 1/3 amino acid region or the C-terminal 1/3 amino acid region of DOC-1 R respectively, were constructed with recombinant techniques. After identification by DNA sequencing and transformation into E.coli BL21, the two deleted DOC-1 R vectors were induced by IPTG. Then the two fusion proteins were purified by GST-pull down.Results Both the sequences and the open reading frames of the vectors were correct. The fusion proteins showed specific bands at 35 kD, respectively. Large amounts of GST-DOC-1 R delN42 and GST-DOC-1 R delC42 fusion proteins were purified by pull-down. Conclusion The deleted pGEX-DOC-1 R prokaryotic expression plasmids were successfully constructed. The recombinant GST-DOC-1 R delN42 and GST-DOC-1 R delC42 fusion proteins could be expressed and purified.