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Ⅰ. Functional Characterization of the Domains of Xylanase XynB from Sorangium Cellulosum So9733-1 Ⅱ. Expression, Purification and Crystallization of Four Human Liver Proteins

Author: GaoAng
Tutor: LiYueZhong；WuZhiHong；LiuZhiJie
School: Shandong University
Course: Microbiology
Keywords: Fiber heap sac bacteria Xylanase Expression and purification of Structural genomics of human liver Crystallization
CLC: Q93
Type: Master's thesis
Year: 2008
Downloads: 77
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Abstract

This study is divided into two: one fibrous the heap capsule bacteria So9733-1 xylanase XynB domain function studies; Second, heterologous expression of four human liver protein purification and crystal growth. The focus is on the XynB domain function. A fiber heap capsule bacteria So9733-1 xylanase enzyme XynB structural domain functional fiber heap capsular bacteria not only can produce a variety of activity of secondary metabolites, but can also be effective to degrade cellulose, but on its cellulose degradation The mechanism has not yet been reported. In preliminary studies in our laboratory have been cloned coded fibers heap sac fungi So9733-1 the xylanase XynB the complete gene. This gene is 1197bp, encoding 398 amino acids of the polypeptide chain. The study showed that the enzyme endo-xylanase. However, further sequence analysis showed that the existing sequences in the enzyme N-terminal approximately 100 amino acids with a database does not have any homology with unknown function. In this article, we focus on the fiber heap sac fungus of wood the xylanase XvnB N-terminal domain of the function. Through bioinformatics methods The xylanase XynB conserved domains predicted results showed that amino acids 114-398 glycosyl hydrolase family 10 conserved region XynB amino acids 1-113 of unknown function. On this basis, we have successfully constructed a the xylanase XynB N-terminal domain and the C-terminal domain and complete XynB heterologous expression plasmid and heterologous expression in E. coli. Isolated and purified by affinity chromatography and gel filtration chromatography, XYNB C-terminal domain of the recombinant protein GST-C and XynB recombinant protein 15b-XYNB. Wherein the expression of the 15b-XynB formation of inclusion bodies, and subsequent renaturation to obtain the active xylanase. Next, we on the 15b-XynB GST-C in enzyme activity, Km values, the optimum reaction temperature and the optimum pH value of a comparative analysis. The results show that there is no significant difference in both the enzymatic properties. Therefore, we speculate, XYNB N-terminal domain of the the deletion on XynB substrate binding capacity and ability to no significant degradation of xylan, the XvnB the N-terminal domain does not have a substrate binding region, and the degradation of xylan related functions. Then, we compared the 15b-C and 15b-XynB, in its heterologous expression strain and membrane binding capacity. The results show, 15b-XynB can be combined with the cell membrane, and 15B-C can not be combined with the cell membrane. Therefore, we hypothesized that the lack of XynB N-terminal domain of the protein lost its ability to combine with the cell membrane, XvnB N-terminal domain and XynB cellular localization. This lay the foundation for a more in-depth study of the XynB N-terminal domain of function. Four human liver protein heterologous expression, purification and crystallization of this second part of the study is the four human liver protein heterologous expression, purification and crystallization, is part of the human liver structure Genome Project. Of the liver biologically important biological macromolecules (especially proteins), the three-dimensional human liver structure Genome Project aims to (X-ray crystallography, nuclear magnetic resonance mass spectrometry) experiments or calculations (Homology Modeling) structure determination of scale. Participate in human liver structural genomics research work mainly involved in more than 80 gene cloning, expression and purification screening, derive a certain amount of pure target protein. Which GA3, GA8, GA17, GA19 four protein crystallization conditions groping and get microcrystalline, laid the foundation for the depth development work.

Ⅰ. Functional Characterization of the Domains of Xylanase XynB from Sorangium Cellulosum So9733-1 Ⅱ. Expression, Purification and Crystallization of Four Human Liver Proteins

Abstract

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