Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.
Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.
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哺乳动物 70,000 道尔顿应激蛋白的快速纯化：蛋白质对核苷酸的亲和力。
DOI：
10.1128/mcb.5.6.1229-1237.1985
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发表时间：
1985
影响因子：
5.3
通讯作者：
Feramisco,JR
中科院分区：
文献类型：
--
作者：
Welch,WJ;Feramisco,JR
针对哺乳动物70000道尔顿（70kda）热休克（或应激）蛋白，开发了一种新的快速纯化方法。通过DE52离子交换层析和atp琼脂糖亲和层析两步纯化了73-kDa蛋白和应激诱导的72-kDa蛋白。这两种蛋白质，在12,000个×gsupernatant或低渗裂解的热休克处理的HeLa细胞颗粒中，含量大致相等，被发现以相对均匀的形式共凝。用荧光染料四甲基罗丹明异硫氰酸酯对纯化后的蛋白进行共价标记，并通过显微注射将荧光标记的蛋白导入活大鼠胚胎成纤维细胞。37°C保存的微注射细胞仅显示弥漫性核和细胞质荧光。细胞热休克处理后，荧光在整个细胞核中观察到，核仁内更明显。这一结果与我们早期的间接免疫荧光研究一致，该研究显示内源性72-kDa应激蛋白在热休克处理的哺乳动物细胞中的核和核核分布。结果还表明，至少对于72-kDa蛋白，(i)该蛋白已被纯化为明显的“天然”形式，（ii）其核仁分布依赖于胁迫。
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 ×gsupernatant 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°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.