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Identification and Characterization of Rab-GTPase Involved in Vesicle Traffic in Euplotes

Author: ZhiHui
Tutor: WangWei；LiangAiHua
School: Shanxi University
Course: Biochemistry and Molecular Biology
Keywords: Eight Euplotes Rab proteins Clone Function Immunofluorescence localization
CLC: Q819
Type: Master's thesis
Year: 2005
Downloads: 66
Quote: 2
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Abstract


Endometrial cells are eukaryotic cells separated into many different compartments , the district chamber through a complex microtubule vesicle transport and exchange of materials and information transfer . Vesicle transport is highly selective , Rab-G7Pase vesicle transport as molecular switches , in different stages of vesicle transport regulation specific means of transport vesicles . Ciliates are a class of low single-celled eukaryotes , and its many functions of the cell organelles Nate accomplished between the cells material exchange through vesicle trafficking . Research Rab proteins in which the function of eukaryotic cells help to understand the transport mechanism of vesicle targeting . In this study, eight Euplotes (Euplotes octocarinatus) for the material , its large nuclear DNA cloned 16 kinds of Rab new genes that total length of 767 bp, both ends of the telomere sequence , an open reading frame of 624 bp , encoding 207 amino acids , Eorab gene sequence contains 2-3 universal termination codon TGA, in this encoding cysteine ??. Proteins encoded by these genes to be part of Rab protein family , a family of conserved structural features of Rab named Eorab1b ~ Eorab1u, 16种Rab genes homologous 87.6 ~ 99.5%. From Euplotes small nuclear DNA cloned Rab got one kind of new genes , the gene length of 784 bp, coding frame is also a 624 bp, named miEorab genes. Large and small from the nuclear DNA of the nucleus rab genes for comparison and found miEorab gene contains two IESs (Internal Eliminated Sequences) sequence , two IESs deletion mechanism there are significant differences . The Eorab1f mutation in two TGA TGC. Mutated Eorab1f construct the prokaryotic expression vector pRSETc in . pRSETc-Eorab1f transformed E.coli BL21 (DE3), induced by IPTG , SDS-PAGE and Western blotting analysis identified the target protein expression obtained . Expressed product by IMAC metal chelate affinity chromatography and Resource-Q anion exchange chromatography to obtain pure protein electrophoresis . Brandford assay showed that the purified protein yield of 1.353 mg / L. Western blotting analysis showed that the protein was fused with six

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