Feramisco J R, Gross M, Kamata T, Rosenberg M, Sweet R W
Cell. 1984 Aug;38(1):109-17. doi: 10.1016/0092-8674(84)90531-2.
Using an E. coli expression-vector system we have efficiently produced, purified, and characterized the full-length, nonfused, protooncogenic and oncogenic (T-24) forms of the human H-ras gene product. These purified ras proteins have been introduced by microinjection into a variety of somatic cells in an effort to examine their function. Within several hours after injection of the oncogenic form of the human H-ras protein into quiescent cells, we observe dramatic morphological changes followed by transient proliferation of the cells. In contrast, microinjection of the normal, protooncogenic form of the ras protein at the same level appears to have only little effect on the cells. Additional experiments indicate that the effect of the ras protein requires entry into the cells, is temporary, is inhibited by cycloheximide or actinomycin D, and is seen only in established cell lines. This experimental approach demonstrates that the bacterially derived and purified human H-ras proteins retain their ability to function when put back into mammalian cells and furthermore, provides a novel assay for transformation induced in established cells by the human H-ras oncogene protein.
我们利用大肠杆菌表达载体系统高效地生产、纯化并鉴定了人H-ras基因产物的全长、非融合、原癌基因和致癌(T-24)形式。这些纯化的ras蛋白已通过显微注射导入多种体细胞中,以研究其功能。将人H-ras蛋白的致癌形式注射到静止细胞中数小时后,我们观察到显著的形态变化,随后细胞短暂增殖。相比之下,以相同水平显微注射正常的原癌基因形式的ras蛋白似乎对细胞影响很小。额外的实验表明,ras蛋白的作用需要进入细胞,是暂时的,受到环己酰亚胺或放线菌素D的抑制,并且仅在已建立的细胞系中可见。这种实验方法表明,细菌来源并纯化的人H-ras蛋白重新导入哺乳动物细胞时仍保留其功能能力,此外,还为人类H-ras癌基因蛋白在已建立细胞中诱导的转化提供了一种新的检测方法。