大肠杆菌对胸腺素α21407;N-末端乙酰化修饰的机理研究

项目名称： 大肠杆菌对胸腺素α21407;N-末端乙酰化修饰的机理研究

项目编号： No.30870050

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 吴军

作者单位： 中国人民解放军军事医学科学院

项目金额： 30万元

中文摘要： 蛋白和多肽的N-乙酰化修饰是真核细胞中普遍存在的一种蛋白修饰，但在原核生物中这种修饰却非常罕见。我们发现大肠杆菌可以同真核生物一样对其表达的胸腺素α21407;（ProTα65289;进行N-乙酰化修饰，从而为研究原核生物乙酰化修饰提供了基础。同时N-乙酰化修饰对包括胸腺素α#22312;内的一些蛋白和多肽的血浆稳定性和生物活性具有重要作用。N-乙酰化修饰的胸腺素α#21450;其前体ProTα22312;病毒性肝炎、肿瘤和艾滋病等疾病的治疗中具有广泛的应用价值。本研究通过大肠杆菌中参与特异性核糖体蛋白乙酰化修饰的蛋白rimL、rimI和rimJ基因的敲除，分析敲除菌表达的ProTα30340;修饰，确定了其对ProTα20057;酰化修饰的影响。发现rimJ基因是负责ProTα-乙酰化修饰的主要基因，RimJ、RimL过表达促进ProTα-乙酰化修饰，而RimI过表达会抑制ProTα-乙酰化修饰，同时发现ProTα30340;N端氨基酸序列的不同会影响ProTα20057;酰化水平，最终基于上述研究理论建立了基因工程方法制备N-乙酰化胸腺素α#30340;技术。本研究为揭示原核生物N-末端乙酰化修饰机理及建立基因工程N-乙酰化修饰多肽表达系统提供了重要的理论和实践基础。

中文关键词： N-末端乙酰化修饰；大肠杆菌；胸腺素α21407;；胸腺素α#65307;多肽药物

英文摘要： N-terminal acetylation is one of the most widespread covalent modifications in eukaryotes, but rare in prokaryotes. Acetylation has been suggested to play an important biological role in modulating enzymatic activity, protein stability, DNA binding, and peptide-receptor recognition N-terminal acetylated thymosin αTα and prothymosin αProTαare widely being developed for treating several diseases, including hepatitis B and C virus (HBV/HCV) infections, non-small cell lung cancer (NSCLC) and AIDS. We have previously reported that a fraction of recombinant human ProTαs N-terminal acetylated when expressed in E. coli. In order to investigate the effect of the three N-terminal acetyltransferases (NATs) of E. coli on human ProTαcetylation, a series of defective E. coli strains were constructed by knocking out three N-terminal acetyltransferase genes (rimJ, rimL and rimI), and expressed proTαn these defective strains. Our study indicated that rimJ gene is responsible for the N-terminal acetylation of ProTαn the wild type DH5αProTαan be completely acetylated when rimJ or rimL were co-expressed, but the acetylation of ProTαas inhibited when rimI was co-expressed. In addition, N-terminal amino acid sequence could affect the level of N-terminal acetylation of ProTαHere we also present a method for production of N-acetylated Tαfrom recombinant ProTαhis study reveals prokaryotic N-terminal acetylation mechanism and provides the theoretical and practical basis for the preparation of recombinant acetylated peptides in E. coli.

英文关键词： N-terminal acetylation;Escherichia coli;Prothymosin αThymosin α peptides