Ⅰ. Functional Characterization Of The Domains Of Xylanase XynB From Sorangium Cellulosum So9733-1 Ⅱ. Expression, Purification And Crystallization Of Four Human Liver Proteins

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.