PURIFICATION OF MURINE HEAT SHOCK PROTEIN 90-BETA (84 KDA) FROM MICE TUMOR CELL LINE

	PURIFICATION OF MURINE HEAT SHOCK PROTEIN 90-BETA (84 KDA) FROM MICE TUMOR CELL LINE
Iraqi Journal of Biotechnology
Article 1, Volume 9, Issue 2, 2010, Pages 124-136
Abstract
Murine heat shock protein 90 beta isotype (84 kDa) was purified from the cytoplasm of AMN3 cells (locally established mice mammary adenocarcenoma cell line). This protein does not have an in vitro activity assay, to confirm its presence during purification, detection of protein with molecular weight equal or near 84 kDa was followed in each purification step. The cell cytoplasm fraction of AMN3 cells contained proteins of molecular weight (MW) ranged from 23.4 to 85.1 kDa as determined with SDS-PAGE analysis. The precipitate of 70% ammonium sulfate saturation of cytoplasm fraction exhibited protein bands with molecular weight ranged from 31.6 to 93.3 kDa. Ion exchange chromatography with DEAE-Cellulose column for the ammonium sulfate precipitated cytoplasm proteins produced one prominent peak, eluted between 0.5 and 0.7 M NaCl. This peak contained four proteins with MW ranged from 17.3 to 85.1 kDa. Gel filtration through Sepharose 6B column for the peak eluted from DEAE-Cellulose column was conducted, different peaks were resulted. A peak with estimated molecular weight of 87 kDa was selected. Second round of gel filtration through Sephacryl S-200 was conducted for the 87 kDa selected fractions eluted from the previous gel filtration step. The presence of one peak was emphasized and its estimated MW was 83.176 kDa. The molecular weight and purity of this protein were checked again with SDS-PAGE. The identity of the purified protein as Hsp90β was confirmed with immunofixation assay by gel electrophoresis on 1% agarose using standard monoclonal antibody specific for murine Hsp90β.
Keywords
HSPs; Hsp; cancer cell; Immunotherapy

Statistics Article View: 77 PDF Download: 39