Cloning and expression of GSTª²hPBD fusion protein for activated Rac1 protein analysis

    HAN Yaª²Lin£¬ MENG Ziª²Min£¬ KANG Jian£¬ LIU Haiª²Wei£¬ YAN Chengª²Hui£¬ WANG Shiª²Wen

    1Department of Cardiovasology, General Hospital of Chinese PLA, Shenyang Command, Shenyang 110016, China, 2Institute of Cardiovascular Diseases, General Hospital of Chinese PLA, Beijing 100850, China

    ¡¾Abstract¡¿ AIM: To induce and express the GSTª²hPBD fusion protein and to study the expression of Rac1 in endothelial cell line ECV304. METHODS: The gene fragment of hPBD was amplified by RTª²PCR and cloned into the pGEXª²2T prokaryotic expression vector, and the GSTª²hPBD fusion protein was purified. The activated Rac1 protein was detected with GSTª²hPBD by pull down assay. RESULTS: DNA sequencing confirmed that the expression vector of pGEXª²2T/hPBD was constructed successfully and fusion protein was expressed effectively in Escherichia coli, with about 75% of the GSTª²hPBD fusion protein (Mr 36 000). Pull down assay found the activated Rac1 protein in ECV304 cells. CONCLUSION: The pGEXª²2T prokaryotic expression vector is successfully constructed and the GSTª²hPBD fusion protein is purified. Activated Rac1 protein is detected in vascular endothelial cell ECV304. This study lays the basis for the research on the relationship between the Rac1 activity and vascular endothelial cell permeability. ......