水稻osRACB基因的原核表达及其蛋白质产物的生化特性鉴定  

Prokaryotic Expression and Characterization of Rice Rac Protein osRACB

作　　者：罗敏[1] 唐朝荣[1] 吴乃虎[1] 

机构地区：[1]中国科学院遗传与发育生物学研究所,北京100080

出　　处：《生物化学与生物物理学报》2003年第12期1149-1154,共6页

基　　金：国家科技部转基因植物研究与产业化专项 (No .J0 0 A 0 0 5 );国家重大基础理论研究项目 (No.2 0 0 1CB10 88)资助~~

摘　　要：Rac蛋白作为高等植物中已知的惟一一类分布广泛的信号GTP结合蛋白 ,在植物体众多生命活动调节中起着分子开关的作用。实验将水稻Rac家族新成员osRACB基因克隆于原核表达载体pET 2 8a中 ,转化大肠杆菌BL2 1(DE3)宿主菌 ,经IPTG诱导实现了目标融合蛋白质的高效表达。通过Ni2 + NTA柱纯化 ,获得纯化的目标融合蛋白质 ,经凝血酶作用后得到osRACB非融合蛋白质。该蛋白质经谷胱甘肽氧化还原体系复性和超滤浓缩后 ,用于体外功能鉴定。结果显示 ,osRACB蛋白具有与GTP特异性结合的活性以及水解GTP的功能。与另一Rac蛋白osRACD相比较 ,osRACB具有更强的GTP结合活性和较弱的GTP水解活性。As the sole ubiquitous signal GTP-binding protein family in higher plants, Rac genes act as pivotal molecular switches and participate in many regulations of life activities. In order to study the biochemical characteristics of rice Rac protein osRACB, the complete coding sequence of osRACB was cloned into expression vector pET28a and expressed in E. coli BL21(DE3). After induced by 1 mmol/L IPTG at 37 ℃ for 4 h, the fusion protein His-osRACB was produced in a large amount. The fusion protein was purified by Ni 2+-NTA column and digested by thrombin. After a series of processes including separation and recovery by electrophoresis, renaturation by glutathione and concentration by ultrafiltration, pure osRACB protein in an active form was obtained. The GTP-binding and hydrolyzing assay showed that osRACB has strong GTP special binding and hydrolysis activity regulated by Mg 2+. By comparing it with another rice Rac protein osRACD, it can be concluded that osRACB has stronger GTP-binding activity and weaker hydrolysis activity than osRACD.

关 键 词：水稻 osRACB基因 原核表达 蛋白质 GTP特异结合活性 GTP水解活性 

分 类 号：S511[农业科学—作物学]