人PPARγ-LBD cDNA的克隆及其在大肠杆菌中的表达纯化

Cloning and Expression of Human PPARγ-LBD cDNA in E. coli

从正常中国人的面部脂肪组织分离总RNA,采用RT-PCR获得人的PPARγ-LBD cDNA,然后克隆至原核表达载体pET28a,构建高效原核表达质粒pET28a-PPARγ-LBD,序列分析表明正常中国人的PPARγ-LBD cDNA序列与Gene Bank报道的序列一致。把构建的pET28a-PPARγ-LBD质粒转化大肠杆菌BL21(DE3),IPTG进行诱导表达,Western blot检测表达产物,在相对分子质量34kDa处有特异的蛋白表达条带,表达蛋白以可溶性和包涵体方式存在。在N末端融合6×His纯化标签的表达产物用Ni^(2+)-NTA离子交换树脂进行纯化,纯化蛋白进行SDS-PAGE纯度分析大于90％以上。因此,获得了正常中国人的PPARγ-LBD cDNA序列,并且在E.Coli中成功表达和纯化了PPARγ-LBD蛋白。

To obtain PPARγ-LBD cDNA, the total RNA was isolated from facil adipose tissue of Chinese and cDNA was synthesized by RT-PCR. The specific amplification product was cloned and sequenced. Then PPARγ-LBD cDNA was inserted into prokaryotic expression vector pET28a and made it expressed in E. coli. The expression product fused with 6 x His at N-terminal was analyzed by Western-blot, and purified by the Ni2+-NTA ion exchange resin and the purity of PPARγ-LBD was analyzed by SDS-PAGE. The results showed that human PPARγ-LBD cDNA has been obtained and was identical with Gene Bank reports. The molecular weight of expressed product analyzed by Western-blot was about 34 kDa. The purity of the recom-bined PPARγ-LBD was more than 90% after purified by the Ni2+ -NTA ion exchange resin. Thus, The sequence of Chinese PPARγ-LBD was achived, and PPARγ-LBD was expressed and purified successfully in E. coli.