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Crystallization, Purification and Expression of Proteins Involved in C. Elegans Phagocytosis and Cell-migration Passage

Author: SunDaPeng
Tutor: WangLiAn
School: Hebei Normal
Course: Biochemistry and Molecular Biology
Keywords: programmed cell death,PCD apoptosis cell engulfment
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 89
Quote: 0
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Abstract


Programmed cell death is a process that cells die in a predetermined manner,and is a tightly regulated cell-suicide program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or harmful cells.Defective control of cell apoptosis is associated with various human disorders such as cancer development and neurodegenerative and immunosuppressive and neoplasic,autoimmune diseases.etc.During programmed cell death,cell corpses are rapidly engulfed.This engulfment process involves the recognition and subsequent phagocytosis of cell corpses by engulfing cells.Genetic studies in C.elegans have led to the identification of dozens of genes that are important for the specification of which cells should live or die,the activation of the suicide program,and the dismantling and removal of dying cells.The engulfment process is mediated by two partially redundant pathways.In the CED-1/CED-6/CED-7 pathway,CED-1 and CED-7 act on the surface of the engulfing cell to mediate recognition of engulfment signal(s) on the surface of the dying cell and to transduce the signal through CED-6 to activate the phagocytic machinery of the engulfing cell.CED-7 also acts in dying cells.In the CED-2, CED-5,CED-10 and CED-12 pathway,PSR-1 act in the engulfing cell to mediate the recognition of Phosphatidylserine(PS) externalized by the dying cell and to transduce the signal through the CED-2/CED-5/CED-12 ternary complex to activate CED-10.There are likely other engulfment receptors that act in the CED-2,CED-5,CED-10 and CED-12 pathway.In addition to CED-10,two other Rac-like GTPases,MIG-2 and RAC-2,also contribute to cell corpse phagocytosis.Structural biology research of these proteins and their complex can help us understand the engulfment process of apoptotic cells and try to explain life phenomenon in molecular level.In this paper our works primarily focus on cell corpses engulfment process related protein CED-2.Here,full longth CED-2 CED-2(1-174) fragment and CED-5(1715-1781) fragment have been expressed in Escherichia coli,purified with high purity and large amount by affinity and gel-filtration chromatography.A fter chromatography,the purity of the protein can be obtained up to 95%.In our work we co-expressed CED-2 and CED-5,thus provided an important foundation to the complex structure of CED-2 and CED-5.Here,we co-expressed CED-2 and CED-5 with pET28a and pGEX6p-1 vectors which are thought uncompatible in that both of them are pBR332 origin..Our work show that they are definitely compatible and stable in 3 days with 37℃.or 30 days with 4℃.By means of pull-down assays we ascertained the fragment of CED-5(1759-1775)amino acid that action with CED-2.The crystals of His-tagged CED-2 were got by vapor diffusion method using hanging drop. This work has provided an important foundation to determination of the CED-2 and CED-5 complex structure and the molecular mechanism of cell engulfment.The process of cell-corpse engulfment played a key role in many functions in cell,and involved in many important diseases.Through the research on structure of these proteins can more clarify the molecular and regulative mechanism of related proteins and protein complexes,and then provide the basis of drug design for some related diseases.

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