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KMID : 0545120030130060912
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Journal of Microbiology and Biotechnology
2003 Volume.13 No. 6 p.912 ~ p.918
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Detection of Mitotic Centromere-Associated Kinesin (MCAK) During Cell- Cycle Progression of Human Jurkat T Cells Using Polyclonal Antibody Raised Against Its N-Terminal Region Overexpressed in E. coli
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JUN, DO YOUN
RUE, SEOK WOO/KIM, BYUNG WOO/KIM, YOUNG HO
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Abstract
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Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to play a role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region ( 187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli. the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector.
Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE. and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2,4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the GI phase. These results indicate that a polyclonal antibody raised against the N-terminal region ( 187 ad) of HsMCAK. overexpressed in E. coli, specifically detects HsMCAK (81 kDa). and it can analyze the differential expression of HsMCAK protein during the cell cycle.
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