[Prokaryotic expression and purification of human GST-Cdc25C fusion protein and preliminary detection of its function].

AIM: To clone prokaryotic expression vector of Cdc25C, purify the fusion protein of GST-Cdc25C, and identify its function preliminarily. METHODS: Human Cdc25C coding region was amplified from human mammary cDNA library by PCR, and cloned into the prokaryotic expression vector pGEX-KG. The fusion protein GST-Cdc25C was expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The function of purified GST-Cdc25C was identified by GST pull-down assay. RESULTS: The GST-Cdc25C recombinant plasmid was successfully obtained by double digestion identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and Western blot analysis showed that the fusion protein was expressed. The fusion protein of about M(r); 80 000 was successfully induced, and identified by SDS-PAGE and Western blot analysis. GST pull-down assay showed that GST-Cdc25C could interact with Chk2 which verified its known function. CONCLUSION: Cdc25C was successfully cloned and purified.