OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION AND CHARACTERIZATIONOF THE MOLECULAR CHAPERONE HSC70

The 70-kDa heat-shock cognate protein (HSC70), a constitutively expres sed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin -coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a desi gned two-cistron expression vector, and purified to homogeneity in a t wo-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 1l of cell culture. Amino-terminal sequencing of the recombinant protein gives the expect ed sequence, and non-denaturing gel electrophoresis as well as gel fil tration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this ef fect to higher temperatures. The recombinant protein displays a low in trinsic ATPase activity that can be stimulated about threefold by bind ing to apocytochrome c, a permanently unfolded protein, while native c ytochrome c has no effect on the ATPase activity indicating that recom binant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having st ructural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be pr obed.