Nyberg L, Jones I
Department of Clinical Chemistry, College of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden.
J Steroid Biochem. 1988 Mar;29(3):341-6. doi: 10.1016/0022-4731(88)90036-2.
A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.
开发了一种快速、可重复且分辨率高的两步法来纯化小鼠皮质类固醇结合球蛋白（CBG）。第一步是在Sephacryl-S-200柱上进行液相色谱，随后将含CBG的残余物通过快速蛋白质液相色谱（FPLC）进行色谱分离。这使我们能够快速获得高度纯化的蛋白质，并通过十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳（SDS-PAGE）和沉降平衡离心法对明显分离出的CBG进行纯度测试。估计怀孕小鼠血清中CBG浓度为0.78 g/l（占总蛋白的1.5%）。通过巯基乙醇处理证明了该蛋白质的单体结构。未检测到NH2末端氨基酸，可能是由于氨基酸被封闭。通过分析超速离心法测定小鼠CBG的分子量（Mr）为52,000，沉降常数S20°，w为3.9 S。该蛋白质在进行等电聚焦时显示出5条带：3条带的表观等电点（pI）在pH 3.15 - 3.25之间，2条带在pH 3.40 - 3.50之间。
