Soluble expression and purification of fusion protein GSTª²p60PLAD in E.coli

    CAO Xiaoª²Rui, ZHU Qingª²Sheng, ZHANG Daª²Wei, LI Liª²Wen, YANG Quanª²Sheng

    PLA Institute of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To produce the GSTª²p60PLAD fusion protein. METHODS: The designed gene sequence was obtained by backª²translating the amino acid sequence of human p60PLAD by using the preferred codons of E. coli. And 5 primers were designed to synthesize the gene by the method of overlap extension PCR. After the PCR product was cloned into pGEXª²4Tª²2 vector, the resulted plasmid pGEXª²4Tª²2ª²p60PLAD was confirmed by DNA sequencing and transformed into E.coli strain BLR(DE3)polys. The supernatant of lysates was collected after the resuspension and sonication of the bacteria which were collected after IPTG induction. Then the recombinant GSTª²p60PLAD fusion protein was purified to homogeneity by GSTrapFF affinity column, desalted by HiTrap desalting column and analyzed by SDSª²PAGE. RESULTS: The recombinant plasmid pGEXª²4Tª²2ª²p60PLAD was constructed successfully, which was confirmed by DNA sequencing.The soluble fusion protein GSTª²p60PLAD, whose Mr was about 34¡Á103, was expressed in E.coli with high efficiency. After purification by affinity column, the purity of the recombinant protein was very high, as SDSª²PAGE revealed. CONCLUSION: The highª²purity fusion protein GSTª²p60PLAD has been produced successfully,which provides a necessary foundation for investigating the structure and biological activity of p60PLAD protein. ......