乳腺癌易感基因BRCA1的BRCT结构域的融合表达及纯化

Purification of BRCT domain of BRCA1 and expression in E.coli DH5α

乳腺癌易感基因BRCA1在乳腺癌发生、发展中起着重要作用。在其C 末段有两个串联重复的BRCT(BRCT1和BRCT2 )结构域。它们与BRCA1的重要功能密切相关 ,BRCT结构域还存在于其它 5 0多种蛋白中。本文用PCR方法扩增了BRCT1和BRCT2结构域编码序列 ,并将其以正确相位与pGEX 2T载体中的GST编码序列融合 ,构建成重组质粒pGST BRCT1和pGST BRCT2。将这两种重组质粒分别转化E coliDH5α后 ,分别表达了GST BRCT1和GST BRCT2两种融合蛋白。Westernblot检测结果表明 ,两种融合蛋白均能与GST抗体反应。表达的GST BRCT1和GST BRCT2经GST Sepharose 4B亲和层析获得了纯化的融合蛋白 。

Breast cancer susceptibility gene (BRCA1) plays an important role in the development and progression of breast cancer. There are two repeats of BRCA1 C Teminal domain at its C temimus(BRCT1 and BRCT2) that are required for its function in both transcriptional activation and DNA repairing. BRCT domains have been identified in more than 50 other proteins. In this study, the coding sequences of BRCT1 and BRCT2 domains were amplified by PCR and fused into frame with the coding region of GST in the pGEX 2T vector to generate the pGST BRCT1 and pGST BRCT2 recombinant plasmids. In the optimal conditions, the fusion proteins GST BRCT1 and GST BRCT2 were expressed in E.coli DH5α at higher level. The western blot analysis showed that GST BRCT1 and GST BRCT2 interacted with GST antibody. The fusion proteins were purified by GST Sepharose 4B affinity chromatography.