四氢嘧啶羟化酶的表达纯化及结构解析

发布时间：2018-06-18 21:10

本文选题：四氢嘧啶及5-羟基四氢嘧啶 + 四氢嘧啶羟化酶　；参考：《天津科技大学》2015年硕士论文

【摘要】：四氢嘧啶及5-羟基四氢嘧啶在精细化工、生物医药、生物制造及生产、环保等领域有广泛的用途及应用前景。四氢嘧啶羟化酶能以Fe2+为辅因子、α-酮戊二酸为辅底物,催化四氢嘧啶转化为5-羟基四氢嘧啶,是微生物发酵生产5-羟基四氢嘧啶过程中的关键酶。蛋白质晶体学利用X射线衍射技术对蛋白质的结构进行解析,使我们从原子角度了解蛋白质的结构信息,结合蛋白质的功能特性,确定在实际催化过程中的功能基团及其作用方式,为定向改造提供重要信息。本文以Bacillus pseudofirmus OF4菌株中的四氢嘧啶羟化酶EctD为研究对象,从其表达、纯化以及结晶等方面展开深入研究。利用基因工程技术成功构建了重组表达质粒EctD-pET-22b (+),并在E. coli BL21 (DE3)实现了高效可溶性表达,经Ni-NTA亲和层析、SuperdexTM20010/300 GL分子筛层析、Resource Q离子交换层析纯化过程后,得到纯度大于95%的目的蛋白。利用商业结晶试剂盒通过悬滴和坐滴气象扩散法对目的蛋白的结晶条件进行筛选。通过进一步结晶条件优化,在0.2 mol/L MgCl2,0.1 mol/L BIS-TRIS (pH 6.5),25% (g/mL) PEG 3,350,目的蛋白浓度为6.5 mg/ml结晶条件,运用坐滴气象扩散法下获得了高分辨率的目的蛋白晶体样本。最终收集到分辨率为2.40A的EctD晶体的衍射数据,数据处理结果显示：EctD晶体属于三斜晶系,空间群为P1晶胞参数为a=45.183 A, b=58.871 A, c= 68.812A, a=77.478°, p=86.034°, 7=66.966°。采用原子置换法(MR),成功解析了EctD的三维结构。一个晶胞由两个蛋白质分子组成,每个单体由9个α螺旋和9个β折叠通过转角结构和loop结构链接形成。EctD晶体结构的解析为我们深入理解四氢嘧啶羟化酶的酶结构和生物学功能提供重要的指导信息。另外,针对该蛋白与辅底物α-酮戊二酸及底物四氢嘧啶的复合物结晶工作在进一步进行中,其中EctD与α-酮戊二酸复合物晶体X-射线衍射分辨率达到3.50A,复合物晶体尚需进一步优化。
[Abstract]:Tetrahydropyrimidine and 5-hydroxytetrahydropyrimidine are widely used and applied in fine chemical industry, biomedicine, biological manufacture and production, environmental protection and so on. Tetrahydropyrimidine hydroxylase can catalyze the conversion of tetrahydropyrimidine to 5-hydroxy tetrahydropyrimidine by using Fe _ 2 as cofactor and 伪 -ketoglutaric acid as coagent, which is the key enzyme in the production of 5-hydroxytetrahydropyrimidine by microbial fermentation. Protein crystallography uses X-ray diffraction to analyze the structure of proteins, which enables us to understand the structural information of proteins from an atomic point of view and to combine the functional properties of proteins. The functional groups and their action modes in the actual catalytic process are determined to provide important information for directional transformation. In this paper, the tetrahydropyrimidine hydroxylase EctD from Bacillus pseudofirmus OF4 was used as the research object, and its expression, purification and crystallization were studied. The recombinant expression plasmid EctD-pET-22b was successfully constructed by genetic engineering technique and expressed in E. coli BL21 / DE3. The recombinant plasmid was purified by Ni-NTA affinity chromatography and SuperdexTM20010 / 300GL molecular sieve chromatography with Resource Q ion exchange chromatography. The purity of the target protein was more than 95%. The commercial crystallization kit was used to screen the crystallization conditions of the target protein by the method of droplet and droplet meteorological diffusion. By optimizing the crystallization conditions, the high resolution target protein crystal samples were obtained under the conditions of 0.2 mol / L MgCl _ 2 O _ 1 0.1 mol / L BIS-TRIS pH 6.5% g 路mL ~ (-1) PEG _ (3) O _ (350) and the target protein concentration of 6.5 mg/ml. Finally, the diffraction data of EctD crystal with a resolution of 2.40A were collected. The data processing results show that the crystal belongs to the triclinic system, and the space group is: P1 unit cell parameter (AH45.183 A), BX (58.871 A), c = 68.812A, av 77.478 掳, ptd 86.034 掳, 76.966 掳. The 3D structure of EctD was successfully analyzed by atom replacement method. A unit cell is made up of two protein molecules, The analysis of the crystal structure of .EctD formed by 9 伪 helix and 9 尾 folds of each monomer through angular structure and loop structure provides important guidance information for us to deeply understand the enzyme structure and biological function of tetrahydropyrimidine hydroxylase. In addition, the complex crystallization of the protein with substrates 伪 -ketoglutaric acid and substrate-tetrahydropyrimidine is in progress. The X-ray diffraction resolution of EctD and 伪 -ketoglutaric acid complex crystals is 3.50A, and the complex crystals need to be further optimized.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q814

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