STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS P21 PROTEINS

细菌表达rasp 21的结构和功能特性 通过体外和体内分析研究蛋白质。 一系列ras蛋白，包括BALB-MSV、Harvey-MSV和Kirsten-MSV， 在E.大肠杆菌，产品纯度大于95%， 通过用7 M尿素提取细菌沉淀，然后用 SephadexG-100柱层析。 使用相同的程序获得 Harvey-MSV蛋白的缺失突变体，并产生BALB-、Harvey-和 携带正常第12密码子的Kirsten-MSV嵌合蛋白。 小 在氨基和羧基末端都产生缺失。 此外，委员会认为， 跨越几乎整个编码序列的较大缺失产生了 一系列缺少30至115个氨基酸残基的p21衍生物， 羧基末端。 GTP结合的体外分析， 所有表达蛋白的自磷酸化和GT3活性 显示至少需要两个区域来产生所有的 活动 位置6-23和153-165之间的氨基酸序列是 必要但不充分。 此外，单克隆抗体被 产生的抗天然p21 ras-H的表位，并通过 缺失突变体 针对位置1-69和130-152的单克隆抗体 显示GTP结合和相关活性的完全阻断。 两 一组实验表明，至少需要这两个区域 p21 ras蛋白的体外活性。 此外，本发明还提供了一种方法， 显微注射NIH/3 T3细胞的缺失突变体，显示缺乏GTP 结合活性显示GTP结合、GTP酶和 蛋白质的转化活性。 p21的缺失衍生物是 也可以用来表征一个新的功能域的手段， 单克隆抗体识别的表位的定位 Y13-259 这种抗体已被证明能够阻断正常的 p21蛋白的活性并逆转ras-， fms-、fes-和raf-转化的细胞。

The structural and functional properties of bacterially expressed ras p21 proteins were investigated by means of in vitro and in vivo analysis. Aseries of ras proteins, including BALB-MSV, Harvey-MSV and Kirsten-MSV, were expressed in E. coli and the products purified to greater than 95% purity by extraction of bacterial pellets with 7 M urea followed by a sephadex G-100 chromatography. The same procedure was utilized to obtain deleted mutants of Harvey-MSV protein and to generate BALB-, Harvey- and Kirsten-MSV chimeric proteins carrying the normal 12th codon. Small deletions were generated at both amino and carboxy termini. Furthermore, larger deletions spanning almost the whole coding sequence generated a series of p21 derivatives lacking from 30 to 115 amino acid residues from the carboxy terminus. In vitro analysis of GTP binding, autophosphorylation and GTPase activities of all the expressed proteins shown that at least two regions are required to generate all the activities. Amino acid sequences between positions 6-23 and 153-165 are necessary but not sufficient. In addition, monoclonal antibodies were generated against native p21 ras-H and the epitopes localized by means of deleted mutants. Mononclonals directed against positions 1-69 and 130-152 showed a complete blockage of GTP binding and related activities. Both sets of experiments indicate that at least these two regions are required for the in vitro activities of the p21 ras proteins. In addition, microinjection of NIH/3T3 cells by deletion mutants that showed lack of GTP binding activity showed a clear correlation between GTP binding, GTPase and transforming activity of the protein. The deleted derivatives of p21 were utilized as well to characterize a new functional domain by means of the localization of the epitope which is recognized by the monoclonal antibody Y13-259. This antibody has been proved to be able to block the normal activity of p21 proteins and revert the transformed phenotype of ras-, fms-, fes- and raf-transformed cells.