Characterization and Purification

We describe the biochemical characterization and purification of the small 28,000-dalton heat shock protein (28-kDa protein) of mammalian cells. Metabolic pulse labeling of heat shock-treated cells with either [3H]leucine or HaS2P04 and analysis of the labeled proteins by two-dimensional gel electrophoresis revealed increased levels of three 28-kDa proteins differing only in their relative isoelectric point. Using both peptide mapping and immunological analysis, we demonstrate that all three proteins are related isoforms, with two of the isoforms containing phosphate. Cell fractionation studies revealed that the 28-kDa protein localizes predominantly within the nuclear pellet very shortly after the heat shock treatment. With increasing times of recovery of the heat-treated cells back at 37 O C , the majority of the 28-kDa protein was now observed to fractionate within the soluble fraction of the cells. Both gel filtration and velocity sedimentation studies revealed that the 28-kDA protein exists as a higher order structure with an approximate sz0+, palue of 10-18 S, a Stokes radius of about 60-70 A, and an estimated native molecular mass of at least 500,000 daltons. We describe a relatively simple and rapid purification of the proteins employing both ionexchange and gel filtration chromatography.