【作者】 高昂；

【导师】 李越中； 吴志红； 刘志杰；

【作者基本信息】 山东大学 ， 微生物学， 2008， 硕士

【摘要】 本研究分为两大内容:一、纤维堆囊菌So9733-1木聚糖酶XynB结构域功能研究;二、四种人源肝脏蛋白的异源表达纯化和晶体生长。重点是XynB结构域功能研究。一、纤维堆囊菌So9733-1木聚糖酶XynB结构域功能研究纤维堆囊菌不但可以产生多种有活性的次级代谢产物,而且还可以有效降解纤维素,但目前关于其纤维素降解机制还未有报道。在本实验室的前期研究中,已经克隆获得了编码纤维堆囊菌So9733-1木聚糖酶XynB的完整基因。该基因全长1197bp,编码398个氨基酸的多肽链。研究表明,该酶为内切木聚糖酶。但进一步的序列分析显示,该酶N端大约100个氨基酸与数据库中的现有序列没有任何同源性,功能未知。在本文中,我们重点研究了纤维堆囊菌木聚糖酶XvnB N端结构域的功能。通过生物信息学方法对木聚糖酶XynB进行了保守结构域预测,结果显示XynB中氨基酸114-398为糖基水解酶家族10保守区,氨基酸1-113功能未知。在此基础上,我们成功构建了木聚糖酶XynB N端结构域、C端结构域和完整XynB的异源表达质粒,并在大肠杆菌中实现了异源表达。通过亲和层析和凝胶过滤层析,分离纯化了XynB C端结构域的重组蛋白GST-C和XynB的重组蛋白15b-XynB。其中15b-XynB表达形成包涵体,随后对其进行了复性,获得了有活性的木聚糖酶。接下来,我们对15b-XynB与GST-C在酶活力、Km值、最适反应温度和最适反应pH值等方面进行了比较分析。结果表明,二者在上述酶学性质方面没有明显差异。因此,我们推测,XynB N端结构域的缺失对XynB与底物的结合能力和降解木聚糖的能力没有显著的影响,XvnB的N端结构域不具有底物结合区等与木聚糖降解有关的功能。随后,我们又比较了15b-C和15b-XynB在其异源表达菌株中与细胞膜的结合能力。结果显示,15b-XynB可以与细胞膜结合,而15b-C不能与细胞膜结合。因此,我们推测,XynB N端结构域的缺失使蛋白丧失了与细胞膜结合的能力,XvnB N端结构域与XynB在细胞中的定位有关。这为更加深入地研究XynB N端结构域功能打下基础。二、四种人源肝脏蛋白的异源表达、纯化和结晶本文的第二部分研究内容是四种人源肝脏蛋白的异源表达、纯化和结晶,是人类肝脏结构基因组计划中的一部分。人类肝脏结构基因组计划旨在通过实验方式(X-射线晶体衍射、核磁共振质谱)或计算方式(Homology Modeling),对肝脏中一些具有重要生物学意义的生物大分子(特别是蛋白质分子)的三维结构进行规模化测定。在参加人类肝脏结构基因组研究工作的一年中,主要参与了80多个基因的克隆、表达和纯化筛选工作,从中获得了一定数量的目的蛋白纯品。对其中GA3、GA8、GA17、GA19四个蛋白进行了结晶条件摸索并获得微晶,为相关工作的深入开展奠定了基础。更多还原

【Abstract】 During the study for my master’s dissertation,my research has been focusing on the functional characterization of the domains of xylanase XynB from Sorangium cellulosum So9733-1.Meanwhile,I have conducted my research concerning the overexpression,purification and crystallization of four human liver proteins.Sorangium cellulosum not only generates a spectrum of secondary metabolism products with enzymatic activity,it also mediates the degradation of cellulose effectively.However,the underlying mechanisms concerning its role in cellulose degradation has not been reported yet.Our preliminary data indicate that the full length of xylanase XynB cloning from Sorangium cellulosum So9733-1 comprise 1197bp nucleotides encoding for a peptide with 398 amino acids.Emerging evidence suggests that this enzyme belongs to endo-xylanase family.Furthermore,sequencing analysis showed that the most N-terminal 100 amino acids have no genetic homology to the reported sequences from data base,and no function about the novel fragment has been reported yet.The undergoing study is designated to explore the function of the domain of xylanase XynB from Sorangium cellulosum So9733-1.After analyzing the conserved sequence of xylanase XynB,we found that the amino acids fragment(114-398)of xylanase XynB belongs to glycosyl hydrolase family concerved sequence,whereas the function of amino acids fragment(1-113)is unknown.Accordingly,we had constructed three plasmids that can heterogenously overexpress the full-length, C-terminal domain or N-terminal structure domain of xylanase XynB,respectively. Resorting to affinity and gel filtration chromatography,we had purified C-terminal domain recombinant protein of xylanase XynB(GST-C)and XynB recombinant protein(15b-XynB).Particularly,XynB recombinant protein(15b-XynB)forms inclusion body,whose xylanase activity can be recovered.Furthermore,we had investigated the differences of 15b-XynB and GST-C recombinant protein in enzyme activity,Km value,and the optimal reaction temperature and pH value.Our data indicate that the loss of XynB N-terminal domain does not exert significant effects on the substrates binding ability and catalytic capability of XynB.Instead,the N-terminal domain of XynB may be involved in the cellular location of XynB within Sorangium cellulosum So9733-1,which sheds lights on the further expatiation of its function within XynB protein.The remaining part of the undergoing study is related to the expression, purification and crystallization of four human liver proteins,which is part of human liver structural genomic project.The human liver structural genomic project is designated to applying experiment modeling(X-ray crystal diffraction analysis and nuclear magnetic resonance mass spectrometry)and Homology Modeling to analyzing three-dimensional structure of the macro-molecules(especially protein molecules).Within this project,I was actively involved in cloning,expressing,and purifying more than eighty genes,and finally obtained certain purified proteins.In particular,after optimize the condition of crystallization,I had got micro-crystals from four proteins,namely GA3、GA8、GA17、GA19,which provides solid platform for following up research.更多还原