Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography.
Prostate-specific membrane antigen (PSMA) is a type II membrane protein that has attracted significant attention as a target for immunioscintigraphic and radioimmunotherapeutic applications for prostate cancer. However, definitive studies on its substrate and inhibitor specificity as well as protein-protein interactions have been somewhat limited by difficulties in the purification of native PSMA. In this study, we optimized the purification of native PSMA from LNCaP cells using conformational epitope-specific antibody-affinity chromatography. Western blot analysis and an HPLC-based enzymatic activity assay were used to compare the yield and activity of PSMA purified by different methods. The ratio of purified PSMA in a native and active conformation was determined by quantifying the amount of non-native PSMA not retained in a second antibody-affinity isolation. The addition of both a neutralization step and the inclusion of Zn(2+) to the equilibration buffer in desalting step provides considerable enhancement in the yield of active PSMA from LNCaP cells.
Liu
Tiancheng
T
Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA.
Toriyabe
Yoko
Y
Berkman
Clifford E
CE
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007167:Immunotherapy; D008297:Male; D017430:Prostate-Specific Antigen / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000276:immunology / Q000628:therapy; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017430:Prostate-Specific Antigen