大肠杆菌N~α-乙酰转移酶RimL^E的表达、纯化及活性测定

Expression,Purifiction and Activity Assay of N~α-Acetyltransferase RimL^E from E.coli

目的:在体外鉴定大肠杆菌Nα-乙酰转移酶RimL(RimLE)的功能。RimL可以通过将原核生物的核糖体蛋白L12的N端氨基乙酰化,使其转化成L7。方法:通过PCR从大肠杆菌基因组中钓取RimLE和L12/L7E的基因,将其插入pET系统,转化大肠杆菌BL21(DE3),培养后提取、纯化得到RimLE和L12E。结果:RimLE在体外可将L12E的N端氨基乙酰化,最大反应速率为25.63/min。结论:为研究原核生物中Nα-乙酰化的分子机制及其功能奠定了基础。

Objective： RimL is responsible for converting the prokaryotic ribosomal protein L12 to L7 by acetylation of its N-terminal amino group. To investigate the function of RimLE from E.coli. Methods： The genes sequence encoding RimL^E and L12^E were amplified from the genome of E.coli by using PCR. These genes were respectively inserted into pET vertors and recombinant RimLE and L12^E were purified from recombinant E.coli BL21（DE3）. Results： RimL^E was capable of acetylating L12^E, exhibiting a Vmax of 25.63/min. Conclusion： This study laid a foundation of exploring the molecular basis for N^α-acetylation and its function in prokaryote.