人源Cdc25C蛋白的融合表达及纯化

目的：表达并纯化有活性的GST—Cdc25C融合蛋白，以用于Cdc25C功能研究。方法：利用RT—PCR克隆MCF-7细胞的cdc25e全长基因；在大肠杆菌中表达GST—Cdc25C融合蛋白；利用GSH交联的琼脂糖珠纯化GST—Cdc25C融合蛋白；通过体外磷酸酶活性分析检测GST—Cdc25C融合蛋白的磷酸酶活性。结果：克隆获得1465bp的人源cdc25c全长基因，并克隆至pGEX-4T-1原核表达载体；在原核系统中可溶性表达了相对分子质量约87x10。的GST—Cdc25C融合蛋白；通过亲和纯化获得的GST—Cdc25C融合蛋白具有较好的磷酸酶活性。结论：得到了有磷酸酶活性的GST—Cdc25C融合蛋白，可用于后续的Cdc25C功能研究。

Prokaryotic Expression and Purification of Human Cdc25C

Objective： To prokaryotic express and purify GST-Cdc25C fusion proteins with bioactivity. Methods： The full-length of human cdc25c gene was cloned from MCF-7 ceils by RT-PCR, then it was inserted into plas- mid pGEX-4T-1 and expressed in E.coli. The GST-Cdc25C fusion protein was purified by method of GSH-aga- rose beads affinity, and its phosphatase activity was measured by using 3-OMFP substrates in vitro. Results： cdc25c gene with 1465 bp was cloned into pGEX-4T-1 vector, and GST-Cdc25C fusion protein about 87 kD was expressed and purified. The GST-Cdc25C fusion protein showed remarkable phosphatase activity. Conclusion： GST-Cdc25C fusion protein with bioactivity was acquired, and it could be used for Cdc25C function research.