Welch W J, Feramisco J R
Mol Cell Biol. 1985 Jun;5(6):1229-37. doi: 10.1128/mcb.5.6.1229-1237.1985.
A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.
已开发出一种用于纯化哺乳动物70,000道尔顿（70-kDa）热休克（或应激）蛋白的快速新方法。组成型73-kDa蛋白和应激诱导型72-kDa蛋白均通过两步法进行纯化，该方法采用DE52离子交换色谱，随后在ATP-琼脂糖上进行亲和色谱。在经低渗裂解的热休克处理的HeLa细胞的12,000×g上清液或沉淀中，这两种蛋白的含量大致相等，发现它们以相对均一的形式共纯化。将纯化的蛋白用荧光染料异硫氰酸四甲基罗丹明进行共价标记，并通过显微注射将荧光标记的蛋白重新引入活的大鼠胚胎成纤维细胞中。维持在37℃的显微注射细胞仅显示弥漫性的核和细胞质荧光。对细胞进行热休克处理后，在整个细胞核中观察到荧光，在核仁中更明显。该结果与我们早期的间接免疫荧光研究一致，该研究表明在热休克处理的哺乳动物细胞中内源性72-kDa应激蛋白呈核和核仁分布。该结果还表明，至少对于72-kDa蛋白而言，（i）该蛋白已以明显的“天然”形式纯化，并且（ii）其核仁分布是应激依赖性的。
