酿酒酵母菌重要蛋白质HMO2的初步晶体学研究

发布时间：2018-06-17 11:47

本文选题：酿酒酵母菌 + HMO2　；参考：《重庆医科大学》2014年硕士论文

【摘要】：背景高速泳动族蛋白（HMGB）是一类广泛存在于真核生物的非组蛋白家族，有着高度保守的三维结构，在转录调控、DNA修复、基因重组、细胞分化和胞外信号转导等过程中扮演着重要的角色。作为HMGB蛋白质超家族的成员之一，HMO2在进化上高度保守且分布广泛提示该蛋白质具有重要的生物学功能，如ATP依赖性染色体重建、DNA损伤修复等。目前，酿酒酵母菌HMO2蛋白的三维结构还没有得到解析，其具体的功能机制也尚不清楚。目的解析酿酒酵母菌重要蛋白质HMO2的三维结构，并基于其结构研究功能，进而了解其具体的功能机制，为进一步的染色体重建和DNA损伤修复研究打下基础。方法将HMO2基因重组到pET22b载体上，构建pET22b-HMO2重组表达质粒，用大肠杆菌原核表达系统表达该蛋白，通过金属离子亲和层析柱（Ni-NTA）、阴离子交换层析（Resource Q）和凝胶排阻层析（分子筛Superdex200）进行纯化，，得到足够纯度的目的蛋白。用分子筛分析蛋白在溶液中的聚集状态。通过气相扩散法，培养蛋白质晶体，用X-射线衍射仪收集晶体衍射数据，再进行数据分析处理以解析晶体三维结构。结果通过分子克隆技术成功构建了重组质粒pET22b-HMO2；通过蛋白表达纯化相关技术成功获得了纯度达98%的目的蛋白质HMO2；凝胶排阻层析（Superdex200）结果显示HMO2蛋白质在溶液中以二聚体形式存在。培养出晶型好、偏光能力强的母体蛋白质单晶。在上海同步辐射光源收到晶体的衍射数据以供结构解析使用。HMO2晶体属于空间群P222或P212121，晶胞参数为a=39.35, b=75.69,c=108.03。根据HMO2的空间群和分子量（约24kDa），以及溶剂含量分析提示，每个晶体学不对称单位含有1个蛋白质分子，表明一个VM值3.193Da-1。但是，多波长反常散射方法仍没能成功解析出蛋白质三维结构。
[Abstract]:Background High Speed Swimming Group protein (HMGB) is a nonhistone family of eukaryotes, with highly conserved three-dimensional structure, DNA repair and gene recombination in transcriptional regulation. Cell differentiation and extracellular signal transduction play an important role. As a member of the HMGB protein superfamily, HMO2 is highly conserved and widely distributed in evolution, which indicates that the HMGB protein has important biological functions, such as ATP-dependent chromosome reconstruction and DNA repair. At present, the three-dimensional structure of HMO2 protein of Saccharomyces cerevisiae has not been elucidated, and its specific functional mechanism is still unclear. Objective to analyze the three-dimensional structure of HMO2, an important protein of Saccharomyces cerevisiae, and to understand its specific functional mechanism based on its structure and function, so as to lay a foundation for further studies on chromosome reconstruction and DNA damage repair. Methods the recombinant expression plasmid pET22b-HMO2 was constructed by recombination of HMO2 gene into pET22b vector and expressed in E. coli prokaryotic expression system. The target protein was purified by metal ion affinity chromatography column (Ni-NTAA), anion exchange chromatography (Resource Q) and gel exclusion chromatography (molecular sieve Superdex200). Molecular sieve was used to analyze the aggregation state of proteins in solution. The protein crystals were cultured by gas phase diffusion method. The crystal diffraction data were collected by X-ray diffractometer, and the data were analyzed and processed to analyze the three-dimensional structure of the crystals. Results the recombinant plasmid pET22b-HMO2 was successfully constructed by molecular cloning, and the target protein HMO2 with 98% purity was obtained by protein expression and purification. The results of gel exclusion chromatography showed that HMO2 protein existed in the form of dimer in solution. The parent protein single crystals with good crystal form and strong polarizing ability were cultured. Diffraction data of crystals were received at Shanghai synchrotron radiation source for structural analysis. HMO2 crystals belong to the space group P222 or P212121. The cell parameters of the crystals are AZ39.35 and BX 75.69 cnc108.03. According to the spatial group and molecular weight of HMO2 (about 24 kDa) and the solvent content analysis, each crystallographic asymmetric unit contains one protein molecule, indicating a VM value of 3.193 Da-1. However, the multi-wavelength anomalous scattering method has not been able to successfully analyze the three-dimensional structure of protein.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378

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