Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Cloning, Expression, Purification and Preliminary Crystallographic Analysis of the SET/TAF-Iβ and CHMP6

Author XuZhen
Tutor ZuoMaiKun;NiuLiWen
School University of Science and Technology of China
Course Biochemistry and Molecular Biology
Keywords histone chaperone SET/TAF-Iβ acidic domain crystal X-ray diffraction ESCRT CHMP6 expression and purification
Type Master's thesis
Year 2010
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In eukaryotic cells, all genomic DNA with a high degree of evolutionary conservation of basic protein -- histone and histone-related chromatin assembly factors constitute a nucleosome structure; the repetition of this element forms chromatin structure. Chromatin structure would prevent a variety of enzymes and the DNA-associated factor from approaching the DNA molecule. Therefore, the maintenance of chromatin structure plays an important role in the eukaryotic gene regulation. Changes in chromatin structure depend on the histone modification and conformational change. Histone chaperone is a kind of important factors involved in histone regulation.Histone chaperone SET which is also named template activating factor Iβ(TAF-Iβ) is encoded by the set gene. The gene was first identified in acute undifferentiated leukemia (AUL) as a fusion gene with can gene. A series of functional tests show that SET/TAF-Iβis a multi-functional protein, involved in many physiological pathways, such as the histone binding, nucleosome assembly, DNA replication, transcription and apoptosis. Biochemical experiments show that SET/TAF-Iβcontains an acidic domain. In the absence of the acidic domain, SET/TAF-Iβloses the H2A-H2B histone binding activity and inhibition of histone acetylation.Histone chaperones which have been isolated show a high diversity of primary structure. Because of their quaternary structure of the poorly understood, the relationship between structure and function has not been uniform. Recent studies on the structure and function of histone chaperones indicate the similarities and differences between them. Up to date, the structure of full length of SET/TAF-Iβhas not been resolved. To learn more about the SET/TAF-Iβstructure and mechanism, we cloned, expressed and purified the N-terminal truncated 22 amino acids, but with acidic domain of human SET/TAF-Iβprotein. The SET/TAF-Iβprotein was crystallized by the hanging-drop vapor diffusion method using Sodium Acetate as precipitant at 283K. The crystals diffracted to 2.75 ? resolution and belonged to space group P43212. ESCRT(endosomal sorting complex required for transport)is a series of important proteins for transport that mainly regulate the down-regulation of the proteins in the surface of the cell in human. Previous research indicates a core -role this complex plays is transporting the proteins to the lyses or endosal. Additionally, its function is not separated but interacts with some sub-complex factors named as ESCRT-I, II, III which form a complex and accurate system. Molecular evidence demonstrates that ESCRT-III is composed of two subunits, which are homolog of the class E Vps proteins in the Saccharomyces cerevisiae. The important function of this complex drives scholars to research on it and many results and papers have been reported, however, it is still obscure about the concrete mechanism and how it functions and interacts with other factors. So it is significant to study on the structural level, which may provide us more implication and insight into understanding the function of this complex. The work we have done and are doing is mainly focused on the structural research of the CHMP6 from Homo sapiens by approaches of X-ray crystallography. I have completed the former work of this whole project including constructing the artificial fusion protein CHMP6, expressing and purifying this protein to obtain an appropriate candidate for the crystal growth, screening out the optimal conditions for crystallization and going to obtain ideal crystals that can be utilized to collect a set of data of X-ray diffraction for resolving its structure.In this report, a brief introduction of the previous research on the function of ESCRT has been given and some aspects about the homology and consensus viewed from the evolutionary level are discussed, all of which tells the focus interest and the essence of our work. I also discussed the designation, methods and materials and the process of the whole experiment. Finally, some proper speculations have been given according to the careful analysis of the results we have achieved and some ideas for the later experiment are also demonstrated.

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