Expression of glutathione S-transferase fusion proteins of human CYP1A1 and preparation of anti CYP1A1 polyclonal antibody

Our aim is to get high amounts of pure antigens to raise specific antibodies and to perform quantifications. Fusion proteins constructed between glutathione S transferase(GST) and CYP1A1 was expressed in Escherichia coli DH5α. We identified recombinants pGEX/1A1 by direct expression screening. Insoluble proteins were isolated from the bacteria and the fusion proteins were purified from a preparative (2 mm) SDS PAGE. The polyacrylamide gel containing the fusion proteins GST 1A1 was used to immunize BALB/c mice from which polyclonal ascites fluid was prepared. Anti CYP1A1 polyclonal antibodies(1A1pAb) was purified by cross absorption with GST 2B6(GST fusion protein of CYP2B6 204-353 amino acid expressed in this laboratory, purified by preparative SDS PAGE)and then by passing through protein A Sepharose affinity column. The purified GST 2B6 and GST 1A1 were used to test the specificity of 1A1pAb. 1A1pAb is specific to GST 1A1 and is still weakly crossreactive with GST 2B6. But CYP1A1 can be discriminated from other CYP subtypes.