关键词: 蛋白磷酸酶2C（PP2C）, 表达, 纯化, 活性

Abstract: Total RNA was isolated from mouse muscle tissue and the gene of protein phosphatase 2C (PP2C) was amplified by RTPCR and inserted into pUCmT vector. After the sequence of the PP2C gene was identified to be correct by DNA sequencing, the fulllength PP2C gene was cloned into expression vector pET28a and the recombined plasmid pET28aPP2C was transformed into E.coli BL21(DE3). The optimum conditions for PP2C expression in E.coli BL21(DE3) were determined to be 0.8 mmol/L IPTG induction for 20 h at 37 ℃. PP2C was eventually expressed under the optimal conditions at a high yield of 18.2% of total E.coliproteins, and the expressed PP2C existed both in the sup ernatant of the cell lysate with 71% of soluble proteins and in the pellet of the cell lysate with 11.1% of insoluble proteins. The expressed PP2C with His tag ha s a molecular weight of about 42 000 by SDSPAGE analysis. The soluble expressed PP2C was further purified on a NiNTA column to homogeneity with a purification yield of 80.5%, and the purified PP2C exhibited a specific activity of 34.5 U/mg.

Key words: protein phosphatase 2C(PP2C), expression, purification, activity