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Published on: October 29, 2013
[Prokaryotic expression and purification of human GST-Cdc25C fusion protein and preliminary detection of its
Zhong-yi Fan1, Xiao-jie Xu, Jia Cao
1Department of Oncology, Chinese PLA Postgraduate Medical School, Beijing, China.
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.
