人源EFA6A蛋白Sec7结构域的克隆表达与纯化

Cloning, Expression, Purification of Sec7 Domain of hEFA6A Protein

作为小GTP酶Arf6的鸟甘酸交换因子(GEF),人EFA6A蛋白主要包含PH和Sec7两个结构域,Sec7是行使GEF功能的核心区域。通过分析Jpred、Uniprot等生物信息学软件的预测结果,从全长1 024 aa中选取的重组Sec7结构域的边界为506-719,共214 aa。以人脑cDNA文库为模板,通过优化PCR程序成功扩增出Sec7基因,经NdeI和XhoI双酶切后亚克隆至原核表达载体p28a中,成功构建p28-Sec7重组子,测序结果与NCBI中公布的序列100%吻合。将重组质粒p28-Sec7转化至BL21-Gold(DE3)宿主菌中,终浓度0.3 mmol/L IPTG、16℃、24 h诱导表达,重组蛋白经过Ni柱和分子筛两步纯化。试验结果显示,重组Sec7成功表达,性质均一,纯度高于95%,表达量为70 mg/L。

As a GEF for the small GTPase Arf6,hEFA6A contains two domains which are pH domain and Sec7 domain.Sec7 domain is the key central domain that executes the function as a GEF.By analyzing the prediction results from Jpred and Uniprot,we chose the boundary contained 214 aa（506-719）from the full-length（1 024 aa）of hEFA6A.Sec7 gene fragment was cloned from homo brain cDNA library and ligated to p28a vector after Nde I and Xho I double digestion.Then the constructive vector p28a-Sec7 was transferred to BL21-Gold（DE3） competent cell for expression.The final concentration of IPTG was 0.3 mmol/L and E.coli cells were cultivated at 16℃ for 24 hours.The recombinant protein was then purified by Ni-NTA affinity chromatography and GE superdexS200 gel filtration column.Our results showed that Sec7 gene and the constructive vector p28a-Sec7 were acquired successfully.The sequence of Sec7 was completely matched with the sequence reported in NCBI.We had a high expression of 70 mg/L and harvested the 95% purity target protein successfully.