Smad家族成员2/3/4原核表达载体构建和融合蛋白纯化

Construction of prokaryotic expression vectors of Smad gene family members 2/3/4 and purification of fusion proteins

目的构建针对Smad家族相关系列原核表达载体,观察其相应蛋白诱导表达纯化情况。方法 PCR扩增Smad2/3/4全长及截短片段,定向插入p GEX-6P-1载体中,构建原核表达重组质粒pGEX-6P-1-Smad2/3/4。经酶切和测序正确后,重组质粒转化表达菌株BL21(DE3),异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达;上清经GST-Sepharose4B亲和纯化,SDS-PAGE考马斯亮蓝染色鉴定。结果原核表达载体p GEX-6P-1-Smad2/3/4构建成功,SDS-PAGE证实Smad3A和Smad4存在包涵体,而上清中GST-Smad3(B-D)和GST-Smad2可被纯化。结论构建的融合蛋白原核表达载体通过诱导和纯化后得到GST-Smad3截断融合蛋白(B-D)和GST-Smad2。

Objective To construct the prokaryotic expression vectors of Smad gene family members 2/3/4 and observe their induced expression and purification. Methods Full-length and truncated Smad2/3/4 coding sequence were amplified by PCR and then cloned into pGEX-6P-1 vector to construct the recombinant plasmid pGEX-6P-1-Smad2/3/4. After identified by restriction enzyme digestion and sequencing, the recombinant plasmid was transformed into Ecoli BL21 （DE3） strain, followed by induced with IPTG. Supernatant was purified by GST-Sepharose 4B and identified by Coomassie brilliant blue stain and SDS-PAGE. Results The prokaryotic expression vector pGEX-6p-l-Smad2/3/4 was successfully constructed. SDS- PAGE confirmed that Smad3A and Smad4 were existent in the inclusion bodies, and GST-Smad3 （B-D） and GST- Smad2 were purified in the supernatant. Conclusion The truncated fusion protein GST-Smad3 （B-D） and GST-Smad2 were obtained from the prokaryotic expression vector of fusion protein.