The Purification, Crystallization And Crystal Structure Analysis Of Cinnamomin The Expression, Purification, Refolding, And Crystallization Of Lcn6

The thesis consists of two partsPart I The purification, crystallization and crystal structure analysis of cinnamominCinnamomin is a type II ribosome-inactivating protein purified from the seeds of Cinnamonum camphora. Its A-chain is the RNA N-glycosidase(EC3.2.2.22). It could cleave the N-C glycosidic bond of a conserved adenosine in the largest RNA of mammalian ribosome and inhibit the protein synthesis. Just as the ricin, cinnamomin is stored in the seeds. Cinnamomin is lower toxin while ricin is highly toxic, although they share very high amino-acid identity. A rapid and improved method was developed for the purification of cinnamomin based on the fast protein liquid chromatography (FPLC). After crystal screening and optimization, suitable crystals were obtained. A complete data set at 2.78 ? has been collected. Molecular replacement was successfully completed using ricin model as the starting model. The structure was determined at 2.95 ? and the Rfactor is 25.6% while Rfree is 29.7%. The structures of ricin, abrin and cinnamomin have been compared in detail. An adenine base has been built in the Fo-Fc electronic map between the active site tyrosines. And no adenine and its derivatives were added in the crystal solution. The adenine may be the product of enzymatic reaction. Compared to the structures of ricin and abrin, these are more difference on the interaction of Cinnamomin A and B chain in the structure of cinnamomin. And we also identified the possibly binding site for ribosomal RNA. Part II The expression, purification, refolding, and crystallization of Lcn6Lcn6 is a new protein, belonging to the Lipocalin family. It had been proved to exist in Rodent. The amino acid sequences of Lcn6 are very similiar related to Lcn5 and Lcn8, which have been proved to play an important role in the sperm maturation. Immunofluorescent staining shows that Lcn6 was located on the head and tail of spermatozoa. It might bind another protein to form a complex. And the complex may play an important role in the sperm maturation. Lcn6 was expressed in insoluble inclusion bodies in E. coli which has then been purified and refolded successfully. Crystals were obtained with a new method which is designed according to the protein crystalliability. A data-set at 1.90 ? was collected from the native crystal at APS (Advanced Photon Source, USA). And the crystals of seleno-methionyl Lcn6 were obtained in the similar protocol. And a complete data had also been collected at 2.0 ? at APS. The structure model had been built using Single Wavelength Anomalous Diffraction (SAD) to find the phasing. And now the structure analysis is being carried out. .