Exploring The "Big Hit" Variant Of Cascade Affinity Chromatography (Cac) For The Purification Of Prostate Specific Antigen (Psa)

1304 Prostate-specific antigen (PSA), a member of the kallikrein family of proteins, is a 30 kDa serine protease produced by the epithelium of the human prostate gland and secreted into seminal fluid. The concentration of PSA in the blood is used as a unique serum biomarker for the early detection of prostate cancer. Chromatography is a very powerful technique that is used to separate and purify biomolecules. Combination of chromatographic techniques termed “Cascade Affinity Cromatography” [CAC] paves the way for an efficient purification. We report here the big “HIT” variant of CAC for the purification of PSA. i) Heparin-Sepharose ii) IMAC (Immobilized Metal-ion Affinity Chromatography) & iii) TIC (Thiophilic Interaction Chromatography). Heparin-Sepharose® requires the presence, clustering, and exposure of basic residues, lysine and arginine, to generate an affinity of a protein. PSA and other kallikreins fulfill these requirements. Our results show that PSA and other kallikreins bind avidly to Heparin-Sepharose® gel. In addition, coupling Heparin-Sepharose® chromatography with immobilized metal-ion affinity chromatography (IMAC) allows further purification of PSA from biological samples. IMAC utilizes binding of histidine residues to transition metals ions chelated to immobilized iminodiacetic acid (IDA). PSA has several histidine residues on its surface as revealed by the tertiary structure of mouse and horse kallikreins. Our preliminary results show that PSA and other kallikreins bind to IDA-Ni2+. TIC utilizes the interaction of thiophilic ligands with aromatic residues exposed on a protein surface. Already, TIC has been successfully applied to separate PSA from its complexes with a variety of inhibitors. Isolation of the protein by TIC has used 2S and 3S gels. Our preliminary results show that purification of PSA is feasible in “HIT”. These investigations led us to develop protocols for the isolation, separation and purification of PSA from its inhibitors such as macroglobulins, anti-chymotrypsin, antitrypsin and their complexes with PSA. In conclusion, Heparin-Sepharose®, IMAC and TIC can be used as cascade affinity chromatography to further purify PSA from biological fluids [HIT of PSA].