Baijal-Gupta M, Fraser J E, Clarke M W, Xuan J W, Finkelman M A
Procyon Biopharma Inc., London, Ontario, Canada.
Protein Expr Purif. 1996 Dec;8(4):483-8. doi: 10.1006/prep.1996.0128.
A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS-polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18-25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS-PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS-PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.
本文描述了一种从人精浆中纯化天然前列腺分泌蛋白（PSP94）的简单三步法。步骤包括硫酸铵分级沉淀，随后是Macro-Prep S支持物（阳离子）流通法，最后是使用两步梯度洗脱的Macro-Prep高Q支持物（阴离子交换）色谱法。该方法的优点在于其简单性、速度和相对廉价的材料，因此比先前报道的方案具有优势。通过SDS-聚丙烯酰胺凝胶电泳上的单条带判断，产物的纯度与通过分析型HPLC阴离子交换法获得的纯度相当。每50毫升精浆的高纯PSP94产量在18 - 25毫克范围内。对脱盐、冻干、纯化的PSP94样品进行了SDS-PAGE、蛋白质印迹和N端测序表征。所有测试参数均证实了其身份。初步数据表明该方法适用于大规模生产。描述了通过SDS-PAGE和密度图像分析在各个纯化步骤直接定量PSP94以评估PSP94回收率的方法。所得结果表明，这是制备高度纯化天然PSP94的有效策略。
