Expression of Human GST-AWP1 Fusion Protein in E. coli and Preparation of Polyclonal Antibody Against This Protein

In order to further study the structure and biological function of a human novel protein that associates with serine/theronine kinase PRK1 (AWP1) and to investigate the proteins interacted with AWP1, GST-AWP1 fusion protein vector was constructed. GST-AWP1 fusion protein was expressed and purified in prokaryotic system, and its polyclonal antibody was prepared. AWP1 cDNA codon domain was amplified from human EC304 cell line by RT-PCR method and recombined into pGEX-KG plasmid expressing glutathione S-transferase (GST) fusion protein. After identified by the restriction enzyme digestion and sequencing, the recombinant clone was transformed into the competent expressive cells of E. coli BL21. GST-AWP1 fusion protein was induced by IPTG and further purified by GST agarose to obtain a fusion protein with molecular weight of 56 kD. Then New Zealand rabbits were injected with purified GST-AWP1 fusion protein to induce immunoreaction, and a highly reactive and specific antiserum was prepared by ELISA and Western blotting. The results showed that GST-AWP1 fusion protein was successfully highly expressed and its polyclonal antibody was prepared.