Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography |
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Authors: | Liu Tiancheng Toriyabe Yoko Berkman Clifford E |
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Affiliation: | Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA. |
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Abstract: | 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. |
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Keywords: | Prostate-specific membrane antigen, PSMA Conformational epitope Antibody-affinity chromatography |
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