【摘要】 探讨了在大肠杆菌中实现致龋变异链球菌N-乙酰谷氨酸激酶基因(argB)的表达、蛋白纯化和生化特性研究。以变异链球菌基因组DNA为模板,设计特异引物,PCR扩增argB基因。经消化和连接构建重组载体pET28a-argB,测序确认后转化表达菌E.coli BL21(DE3)。SDS-PAGE鉴定argB基因能诱导表达,且表达物可溶。通过镍离子螯合层析和分子筛纯化成功获得N-乙酰谷氨酸激酶(NAGK)重组蛋白。NAGK酶促反应分析表明:精氨酸生物合成的乙酰化环式路径关键酶NAGK活性不受精氨酸反馈抑制,提示可能存在其他调节方式有待进一步研究。此外,分析型分子筛结果显示:具有催化活性NAGK以单体形式存在,显然不同于此前氨基酸激酶家族中的相关报道。更多还原

【Abstract】 Expression,purification and biochemical characterization of N-acetylglutamate kinase gene(argB) from Streptococcus mutans,the leading pathogen of human dental caries,were studied.The argB gene was amplified by polymerase chain reaction(PCR) with S.mutans genomic DNA as template.The PCR fragments were digested and ligated into the cloning/expression vector pET28a to construct the recombinant pET28a-argB.After verified by DNA sequencing,it was transformed into expressing cells E.coli BL21(DE3).The SDS-PAGE assay on the expressing cells E.coli BL21(DE3)/pET28a-argB displayed that the argB gene could be expressed in soluable form after induction with IPTG.The target NAGK was obtained by Ni chelating chromatography and gel size-exclusion chromatography.Enzyme activity assays conformed that the key enzyme S.mutans NAGK isn’t inhibited by arginine,the final product in the acetyl cyclic route of arginine biosynthesis;thus implied that there may be other regulation mechanism,so it was necessary to make further investigation into arginine biosynthesis in S.mutans.The observation of analytical gel chromatography demonstrated that the enzymatic S.mutans NAGK is monomeric,apparently which was inconsistent with the reports about that in the amino acid kinase family.更多还原