[Prokaryotic expression and characterization of rice Rac protein osRACB]

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 degrees C 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.