PURIFICATION OFMURINE HEAT SHOCK PROTEIN90-BETA (84 KDA) FROMMICE SPLEEN

Abstract

ABSTRACT
Murine heat shock protein 90 beta isotype (84kDa) was purified from the
cytoplasm of BALB/c mice spleen cells. Since this protein dose not have an in vitro
activity assay and the presence of protein with molecular weight equal or near 84 kDa
was followed in each purification step. Cytoplasm fractions of spleen cells contained
proteins of molecular weight (MW) ranged from 86.0 to 20.9kDa as determined with
SDS-PAGE analysis. The ammonium sulfate 70% saturation precipitate of cytoplasm
fraction exhibited protein bands with molecular weight ranged from 40.7 to 86.1kDa.
Ion exchange chromatography with DEAE-Cellulose column for the ammonium
sulfate precipitated cytoplasm proteins produced one prominent peak, eluted between
0.5 and 0.7M NaCl. This peak contain four proteins with MW ranged from 19 to
83.1kDa. During gel filtration through Sepharose 6B column for the peak eluted from
DEAE-cellulose column, a peak with estimated molecular weight of 87kDa was
selected among others produced. Second round of gel filtration through Sephacryl S-
200 was conducted for the 87kDa 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 was checked again with
SDS-PAGE 10%. The identity of the purified proteins as Hsp90β was confirmed with
immunofixation assay by gel electrophoresis on 1%agarose using standard.
Key words: Heat shock, Protein 90, Spleen cells.