Overexpression, Purification And Polyclonal Antibody Preparation Of Immunophilin Fpr3

Immunophilins consist of a family of highly conserved proteins binding with immunosuppressive drugs such as FK506, rapamycin and cyclosporin A, many but not all of which possess peptidylprolyl cis-trans isomerase (PPIase) or rotamase activity. The immunophilins encompass two ubiquitous protein families: FKBPs and cyclophilins. The cyclophilins(Cyps)bind cyclosporin A(CsA), while the FK506-binding proteins(FKBPs) bind macrolides such as FK506(tacrolimus)and rapamycin. Fpr3 is a PPIase acting in conjunction with protein chaperones, accelerating the rate of conformational interconversions around proline residues in polypeptides.Fpr3 interacts with PP1 through its PPIase domain and serves to maintain recombination checkpoint activity, but the PPIase activity of Fpr3 is not required for the protein's checkpoint function.The expression vector pGEX-5X-1-Fpr3 was transformed into E.coli BL21, expressed and purified. The purified fusion protein was used to immunize rabbit for preparation of polyclonal antibody. The main results are as following:1. Expression and purification of the recombinant protein GST-Fpr3The constructed vector pGEX-5X-1-Fpr3 was transformed into E.coli BL21. Experiments on optimal induction time and optimal induction concentration of IPTG showed that the optimal induction concentration was 0.4mmol/L and the optimal induction time was 5h. The fusion protein was purified by GSTrap affinity chromatography .SDS-PAGE and Western blot analysis showed that molecular weight of the recombinant protein was about 90kDa. The GST tag was removed by the method of on-column cleavage with Factor Xa. The Fpr3 was assayed and identified by SDS-PAGE.2. Preparation and identification of polyclonal antibody against GST-Fpr3The purified fusion protein was used to immunize the Japanese rabbits to produce the polyclonal antibody. The polyclonal antibody was purified by ammonium sulphate precipitation followed by rProtein A affinity chromatography. Western blot analysis indicated that the polyclonal antibody could react with the target protein. ELISA showed that the titer of polyclonal antibody was 1:100000.