Purification and characterization of NCO segment of TWD protein from Trypanosoma brucei
| Autor: | Wen-Shuo Kuo, 郭文碩 |
|---|---|
| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 Trypanosoma brucei is a unicellular eukaryote that parasitizes in mammals and insects to cause African sleeping sickness of human being. A novel gene, TWD, is found in the organism that consists 2154 base of its full length cDNA and encodes a 65.3 kD protein. Similarity search showed that it contains 7 WD repeats protein at its entire C-terminal half and 7 leucine zipper and ERM protein motifs in WD repeat N-terminal extension. TWD obviously is a multi-domain protein which likely has a multi functions in the cell. WD repeat proteins have been found to participate in many important processes of a cell such as cell division, transcription, mRNA modification, proteasome-mediated protein degradation, signal transduction across cell membrane, and cell apoptosis. In database, TWD’s ortholog is PF20, a bridging protein of central tubules of axoneme in a green algae. In this thesis, the focus was set at an NCO I restriction fragment of TWD gene which contains motif of ERM protein, a protein family linking actin and cell membrane. The NCO fragment was expressed as inclusion body inside E. coli with pET system to have His-tag at its C terminus by 0.8mM IPTG induction at 37℃ for 5 hours. The expressed 36kD protein was collected and purified through Ni+2 charged affinity chromatography with two gradient steps to remove urea and later depart the adsorbed protein at 0.52M imidazole. Both Edman degradation and MALDI-TOF methods proved protein sequence as predicted from DNA sequence. This 36kD protein was then used to elicit rabbit polyclonal antiserum, NCO B, which was able to recognize a 65 kD protein in T. brucei cell lysate by Western method. Immuno-coprecipitation experiments showed that NCOB could pull down tubulin but not actin in the cytoplasm. In the cytoskeleton portion, TWD could be detected with tubulin and also with actin. Fractionation experiments showed that TWD resisted Triton X-100 wash and maintained with cytoskeleton. In summary, an antigen corresponding to part of TWD gene was prepared and used to elicit antiserum. Through this antiserum, TWD could be detected in the cell lysate without obvious modification and existed in the cytoskeleton. Most likely, TWD should be a tubulin and actin linker protein. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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