| 摘要: |
| 目的:构建含人线粒体亮氨酰tRNA合成酶(mtLeuRS)基因的重组表达载体,表达纯化mtLeuRS并检测其酶促动力学参数.方法:克隆mtLeuRS基因,构建入pET-24a(+)表达载体,转化大肠杆菌BL21-CodonPlus(DE3)-RIL,经诱导产生带6-His标签的mtLeuRS融合蛋白,再经Ni2+亲和层析柱一步法纯化后用酶促反应检测其氨酰化活力.作为mtLeuRS酶促反应底物,人tRNALeu(UUR)由T7 RNA聚合酶体外转录生成.结果:经IPTG诱导,人mtLeuRS蛋白表达量约占细菌总蛋白量的1％～2％,1 L培养液的菌体内可收获约2 mg的纯化酶蛋白.通过体外转录生成的酶底物tRNALeu(UUR)可达转录模板的100倍.经酶促反应,人mtLeuRS可氨酰化人线粒体内tRNALeu(UUR).反应的亲和常数(Km)为16.67 μmol/L,催化常数(Kcat)为0.17 s-1.结论:本实验成功克隆并表达了人mtLeuRS,并成功用于催化tRNALeu(UUR)的氨酰化. |
| 关键词: 亮氨酰tRNA合成酶、线粒体、基因表达、动力学 |
| DOI： |
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| 基金项目: |
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| Expression,purification and kinetics study of human mitochondrial leucyl-tRNA synthetase |
| 汪振诚,王学敏,金由辛,缪明永,焦炳华 |
| () |
| Abstract: |
| Objective: To construct the recombinant expression vector of human mitochondrial leucyl-tRNA synthetase(mtLeuRS),and to express, purify mtLeuRS and determine its kinetic parameters.Methods: The human mtLeuRS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-mtLeuRS,which could direct the synthesis of a mammalian mitochondrial derived protein in E. coli BL21-CodonPlus(DE3)-RIL.The vector allowed production and single-step purification of His6-tagged human mtLeuRS by the facilitation of metal (Ni2+) chelate affinity chromatography.As the substrate of mtLeuRS,the human mitochondrial tRNALeu(UUR) was synthesized and transcribed in vitro with T7 RNA polymerase.The ability of mtLeuRS to aminoacylate mitochondrial tRNALeu(UUR) was assayed by enzymatic reaction when tRNALeu(UUR) between 5-25 μmol/L.Results: The expression level of human mtLeuRS was about 1％-2％ of total cell proteins after isopropyl β-D-thiogalactoside induction.The produced human mtLeuRS-His6 could be purified to homogeneity within 2 h and about 2 mg purified enzyme could be obtained from 1 L cell culture.The procedure of purification was easy and simple.The mitochondrial tRNALeu(UUR) obtained was as high as 100 times of transcription template.The enzymatic reaction showed that the Km of mtLeuRS for tRNALeu(UUR) was 16.67 μmol/L and the turnover (Kcat) was 0.17 s-1. Conclusion: The mature form of human mitochondrial LeuRS has been cloned and expressed in E.coli.It is capable of aminoacylating tRNALeu(UUR) transcripted in vitro. |
| Key words: leucyl-tRNA synthetase mitochondrial gene expression kinetics |
