Lydon N B, Adams B, Poschet J F, Gutzwiller A, Matter A
Research Department, Pharmaceutical Division, CIBA-GEIGY Limited, Basel, Switzerland.
Oncogene Res. 1990;5(3):161-73.
A bacterial expression vector containing a segment of the v-abl gene from Abelson murine leukemia virus (A-MuLV) was constructed such that the gag region of v-abl was replaced by a sequence encoding the IgG-binding domain of the S. aureus protein A. pabl HP, a fusion protein encoded by this vector was rapidly purified to near homogeneity by affinity chromatography on IgG-Affigel and Mono Q FPLC. The Km of the pabl HP kinase for ATP varied with [Val5]-angiotensin II concentration and was 21.2 microM at saturating concentrations of [Val5]-angiotensin II. The Km for [Val5]-angiotensin II at saturating concentrations of ATP was 3.8 mM. The turnover number, at 20 degrees C, was 62 mumol min-1 mumol-1. Initial rate studies support a ternary complex kinetic mechanism for phosphoryl transferase. The substrate specificity of the pabl HP kinase was further characterized using synthetic peptides. This expression system, which enables the rapid purification of recombinant v-abl kinase is suitable for the comparative enzymological study of mutant v-abl enzymes generated by site-directed mutagenesis.
构建了一种细菌表达载体，其包含来自艾贝尔森鼠白血病病毒（A-MuLV）的v-abl基因片段，使得v-abl的gag区域被编码金黄色葡萄球菌蛋白A的IgG结合结构域的序列所取代。该载体编码的融合蛋白pabl HP通过IgG-Affigel亲和色谱和Mono Q FPLC快速纯化至接近均一性。pabl HP激酶对ATP的Km随[Val5]-血管紧张素II浓度而变化，在[Val5]-血管紧张素II饱和浓度下为21.2μM。在ATP饱和浓度下，[Val5]-血管紧张素II的Km为3.8 mM。在20℃时，周转数为62μmol min-1μmol-1。初始速率研究支持磷酸转移酶的三元复合物动力学机制。使用合成肽进一步表征了pabl HP激酶的底物特异性。这种能够快速纯化重组v-abl激酶的表达系统适用于对通过定点诱变产生的突变v-abl酶进行比较酶学研究。
