Abstract: 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.

Key words: Arf6 hEFA6A, Sec7, Expression, Ni-NTA column, Gel filtration