Expressions of hsc/hsp70 cDNAs in bacteria and comparison with tissue-isolated proteins.

Abstract

Rat hsc70 and human hsp70 have been expressed in bacteria using the T7 polymerase system. The recombinant proteins, which were the major proteins in E.coli, had the same molecular weights as the tissue-isolated proteins and were immunoactive with hsc70/hsp70 antibodies. ATP binding assay by equilibrium dialysis showed a K$\sb{\rm d}$ for ATP of 0.44 $\mu$M. At saturation, 0.4 mole of ATP was bound per mole of hsc70. Both recombinant and tissue-isolated hsc70/hsp70 have ATPase activities. The denatured substrate, reduced carboxyl methylated $\alpha$-lactalbumin (RCMLA), stimulated ATPase rates of bovine tissue-isolated hsc70/hsp70, but the ATPase rates of rat skeletal muscle and recombinant hsc70 were not changed upon the adding of RCMLA. The analysis of two-dimensional gels showed hsc70/hsp70 isolated from different sources had different isoform patterns. It is speculated that each isoform may have its own substrate specificity.