肠球菌肽脱甲酰基酶在大肠杆菌BL21(DE3)中高效表达及其活性检测

The Peptide Deformylase of Enterococcus faecium Overexpressed in E.coli BL21(DE3) and the Enzymatic Activity Assay

肽脱甲酰基酶 (peptidedeformylase ,PDF)存在于所有原核生物中是其生长、代谢、繁殖必不可少的关键酶 ,但不存在于人类与其他哺乳动物细胞内 ,因而被视为新一代广谱抗生素药物筛选的理想靶点。将肠球菌 (Enterococcusfaecium)肽脱甲酰基酶基因连接到高效蛋白表达载体pET 30 A( + )中 ,并转入宿主大肠杆菌BL2 1 (DE3)中进行诱导表达。在该基因的诱导表达中 ,采用不同表达条件进行诱导表达 ,最终获得表达效率极高且可溶的肽脱甲酰基酶。从宿主细胞中提取分离该酶 ,并进行酶活性检测 。

Peptide deformylase, an essential enzyme for growth and multiplication of bacteria, is absent from mammalian cells, therefore it is regarded as one of the most promising targets for discovery of new antibiotics. The def gene of peptide deformylase from Enterococcus Faecium was identified and cloned to protein overexpressed vector pET\|30\|a(+) named pET\|30\|a\| def . The constructed overexpressed vector was used to transform E.coil BL21(DE3) for overexpression. The protein in transformed E.coil BL21(DE3) was induced in different overexpressed condition, and the overexpressed and soluble peptide deformylase was gained. The enzyme that was purified from the overexpressed E.coli cells had strong enzymatic activity.