酵母内质网嵌膜gtp酶seylp的表达、纯化及结晶-生物学;微生物学专业论文.docxVIP

优秀毕业论文 精品参考文献资料 AbstractThe Abstract The Endoplasmic Reticulum(ER)is a morphologically and functionally conserved organelle in eukaryotic cells．It consisted by two morphologically different compartments：the tubular ER and sheet ER,which serve different physiological purposes in cells．Studies found that many proteins participate in the morphological maintenance of tubular ER．Seylp is an ER membrane-bound,large GTPase protein in Saccharomyces cereviaiae belongs to the Dynamin super family,and also exclusively localise ER and enriches at ER tubules．Studies also suggested that Seylp and its function orthology Atlastin in mammals play vital roles in the maintenances of tubular ER network．In this study,we employ structural and biochernical assay to investigate the detailed molecular mechanism of Seyl p in maintaining ER morphology and mediating tubular ER fusion／fission． Firstly we successfully cloned the full cytosolic domain at N-termius of Scylp，and then found different protein fragments determined by secondary structure prediction, trysin-digestion,and mass spectrum assay,plus with protein structure domain analysis．The prokaryotic expression system is suitable for the expression of different fragments of Scyl p protein．By using different chromatography systems，the purifications of all proteins Were realized．Then the proteins properties Were further analyzed by gel·filtration and GTPase activities assay,which demonstrate the proteins are stable and have biological activity．Finally,the protein crystals Were obtained by screening various crystallization conditions．However,the crystals have very low diffraction qualities determined by X-my diffraction assay． We applied different protein treatment strategies and optimized the crystallization conditions，and we also emploied post—crystal treatments．Finally the diffzaction qualifies of crystals wore drastically improved．All those data enable US to better understand the Seylp and provide US all insighff试picture of how Sey