PURIFICATION OF SELENOPROTEIN-P FROM HUMAN PLASMA
PURIFICATION OF SELENOPROTEIN-P FROM HUMAN PLASMA
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DOI：
10.1016/0167-4838(94)90014-0
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发表时间：
1994-02-16
期刊：
影响因子：
--
通讯作者：
BURK, RF
中科院分区：
文献类型：
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作者：
AKESSON, B;BELLEW, T;BURK, RF
使用培养中的 HepG2 细胞分泌的 Se-75 标记的硒蛋白 P 作为标记物，通过四个色谱步骤从人血浆中部分纯化（>1000 倍）硒蛋白 P。将纯化的制剂注射到小鼠体内，产生单克隆抗体，沉淀标记的蛋白质。两种不同的单克隆抗体均不与来自五种动物的血浆发生交叉反应。将抗体与琼脂糖偶联，并通过免疫亲和层析和肝素琼脂糖层析从人血浆中纯化硒蛋白 P。使用两种不同的基质结合单克隆抗体，纯化程序在 SDS-PAGE 上产生两条迁移率分别为 61 和 55 kDa 的条带。两条带均对碳水化合物进行染色，并在酶促去糖基化后显示出电泳迁移率增加。免疫亲和层析除去约。血浆中硒的三分之一，即总硒浓度为 1.1 μmol/l 时为 0.4 μmol Se/l，表明在美国健康献血者中，硒蛋白 P 占血浆总硒的比例。
Selenoprotein P was partially purified (>1000-fold) from human plasma in four chromatographic steps using Se-75-labeled selenoprotein P secreted by HepG2 cells in culture as a marker. The purified preparation was injected into mice and monoclonal antibodies, which precipitated the labeled protein, were generated. Neither of two different monoclonal antibodies had cross-reactivity with plasma from five animal species. Antibodies were coupled to agarose, and selenoprotein P was purified from human plasma by immunoaffinity chromatography followed by chromatography on heparin agarose. With two different matrix-bound monoclonal antibodies, the purification procedure gave two bands on SDS-PAGE with mobilities corresponding to 61 and 55 kDa. Both bands stained for carbohydrate and showed increased electrophoretic mobility after enzymatic deglycosylation. Immunoaffinity chromatography removed approx. one-third of the selenium from plasma or 0.4 mu mol Se/l at a total selenium concentration of 1.1 mu mol/l, indicating that selenoprotein P constituted this proportion of total plasma selenium in healthy US blood donors.