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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 only widely distributed signal GTP-binding protein known in higher plants, Rac protein plays a role of molecular switch in the regulation of many living activities in plants. In this study, a new member of rice Rac family, named osRACB, was cloned into the prokaryotic expression vector pET 2 8a and transformed into E. coli BL21 (DE3) host. The target fusion protein was efficiently induced by IPTG. Purified by Ni2 + NTA column to obtain the purified target fusion protein, obtained after thrombin osRACB non-fusion protein. The protein glutathione redox system renaturation and ultrafiltration concentrated for in vitro functional identification. The results show that the osRACB protein has the activity of specifically binding to GTP and the function of hydrolyzing GTP. Compared with another Rac protein osRACD, osRACB has stronger GTP binding activity and weaker GTP hydrolysis activity.