摘要
目的 克隆表达色氨酸合成酶α亚基编码基因并对其进行功能分析。方法 以结核分枝杆菌( 简称结核杆菌)H37Rv 基因组为模板, 扩增trpA 基因, 构建pET30a-trpA 重组质粒; 转化重组质粒到大肠埃希菌DH5α并在BL21( DE3) 诱导表达, 纯化可溶性的结核杆菌重组色氨酸合成酶α亚基( His-rMtTrpA) 。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳( SDS-PAGE) 和质谱分析测定相对分子质量( Mr) 后, 用圆二色光谱( CD) 分析和同源模建方法检测二级和三级结构。研究不同浓度HisrMtTrpA对β亚基酶活反应的影响。结果 成功克隆了813 bp 的目的基因trpA, 并获得了高纯度的His-rMtTrpA 蛋白。重组蛋白Mr 为33. 151 ×103 ( 含载体蛋白) 。25 ℃时His-rMtTrpA 的二级结构包括31. 8% α 螺旋、31. 8% β 折叠、8. 4% β转角和27. 9%无规则卷曲, 它的三维模型显示为( β/ α) 8 桶状结构。酶学性质研究表明, 在His-rMtTrpA 与MtTrpB 的摩尔比为2. 2时, 色氨酸合成酶α亚基可以最大限度促进β亚基酶活反应。结论 成功得到高纯度的重组目的蛋白His-rMtTrpA, 其功能分析为针对色氨酸合成酶的药物筛选设计提供理论基础。
Abstract
Objective To clone and express the Mycobacterium tuberculosis trptophan synthase αsubunit ( MtTrpA) gene trpAand to study the role of MtTrpA. Methods The trpA gene was amplified by PCR from Mycobacterium tuberculosis H37Rv straingenomic DNA and cloned into a prokaryotic expression vector pET30a. The resulting recombinant expression plasmid pET30a - trpAwas then transformed into the E. coli strain DH5αand a high-level expression E. coli BL21 ( DE3) was established after induction with
IPTG. SDS - PAGE and MALDI - TOF determined the relative molecular weight of this recombinant protein His - rMtTrpA. Itssecondary and 3D structures were determined by circular dichroism and homologous modeling. The enzyme studies tested the functionsof MtTrpA. Results The Mycobacterium tuberculosis trpA gene ( 813 bp) and highly purified recombinant His - rMtTrpA protein wereobtained. The relative molecular weight of recombinant His - rMtTrpA protein was determined to be 33. 151 ×103 ( vector included) .
Secondary structure of His - rMtTrpA had about 31. 8 % α helix, 31. 8% β sheet, 8. 4 % β turn, 27. 9% random coil at 25℃.Homologous modeling shows His - rMtTrpA as ( β/ α) 8 - barrel protein. His - rMtTrpA can most effectively activate βreaction whenmolecular ratio of MtTrpA and MtTrpB was 2. 2. Conclusion This study obtained purified His - rMtTrpA; Functional analysis pavedthe way for further design of inhibitors against this enzyme.
关键词
色氨酸合成酶 /
α亚基 /
同源模建
Key words
Mycobacterium tuberculosis /
Tryptophan synthase /
αsubunit /
Homologous modeling
赵静静; 徐胜凤;王虹军; 沈洪波; 王洪海.
结核分枝杆菌H37Rv 色氨酸合成酶α亚基编码基因的克隆、表达及酶学性质分析[J]. 微生物与感染. 2008, 4(4): 195-199
ZHAO Jing-jing; XU Sheng-feng; WANG Hong-jun; SHEN Hong-bo; WANG Hong-hai.
Cloning, expression and enzymatic analysis of the recombinant tryptophan synthase αsubunit from Mycobacterium tuberculosis H37Rv[J]. Journal of Microbes and Infections. 2008, 4(4): 195-199
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