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使用构象表位特异性抗体亲和色谱法纯化前列腺特异性膜抗原

Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography.

作者信息

Liu Tiancheng, Toriyabe Yoko, Berkman Clifford E

机构信息

Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA.

出版信息

Protein Expr Purif. 2006 Oct;49(2):251-5. doi: 10.1016/j.pep.2006.05.008. Epub 2006 May 22.

DOI:10.1016/j.pep.2006.05.008
PMID:16815035
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.

摘要

前列腺特异性膜抗原(PSMA)是一种II型膜蛋白,作为前列腺癌免疫闪烁显像和放射免疫治疗应用的靶点已引起广泛关注。然而,由于天然PSMA纯化困难,关于其底物和抑制剂特异性以及蛋白质-蛋白质相互作用的确定性研究受到一定限制。在本研究中,我们使用构象表位特异性抗体亲和色谱法优化了从LNCaP细胞中纯化天然PSMA的方法。采用蛋白质印迹分析和基于高效液相色谱的酶活性测定法比较不同方法纯化的PSMA的产量和活性。通过定量在第二次抗体亲和分离中未保留的非天然PSMA的量,确定天然和活性构象的纯化PSMA的比例。在脱盐步骤的平衡缓冲液中添加中和步骤和锌离子(Zn(2+))可显著提高从LNCaP细胞中获得活性PSMA的产量。

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