Lowe P N, Page M J, Bradley S, Rhodes S, Sydenham M, Paterson H, Skinner R H
Department of Molecular Sciences, Wellcome Research Laboratorie, Langley Court, Beckenham Kent United Kingdom.
J Biol Chem. 1991 Jan 25;266(3):1672-8.
Kirsten-ras is the oncogene most frequently activated in human tumors. Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21. When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease. However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E. coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system. Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation. A C-terminally truncated form (residues 1-166) was also expressed at high levels in E. coli for x-ray crystallographic studies. The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP. Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein.
Kirsten - ras是人类肿瘤中最常被激活的癌基因。对其生物学功能的研究一直受到主要蛋白质产物Kirsten - ras (4B) p21无法大量获取的限制。当在大肠杆菌K12中表达时，重组蛋白在富含赖氨酸的C末端区域细胞裂解时迅速被切割，可能是被ompT蛋白酶切割。然而，当Kirsten - ras基因在缺乏ompT基因的大肠杆菌菌株中表达时，以及在杆状病毒/昆虫细胞表达系统中表达时，可获得可溶性全长蛋白。此外，杆状病毒/昆虫细胞系统产生的约一半Kirsten - ras蛋白以膜相关形式存在，该蛋白经多异戊二烯化和羧甲基化进行翻译后修饰。还在大肠杆菌中高水平表达了一种C末端截短形式（第1 - 166位残基）用于X射线晶体学研究。纯化蛋白的GDP释放动力学和GTP水解动力学与相应的Harvey - ras蛋白相似，尽管它们对GDP和GTP的相对亲和力存在微小差异。通过显微注射到瑞士3T3细胞中证明了全长Kirsten Val - 12 p21的生物学活性，导致形态转化，但其效力低于Harvey Val - 12蛋白。
