Caenorhabditis elegans Hsp70-1 expresses highly in bacteria, is sufficiently soluble, and has a catalytic constant similar to Hsc70 and BiP

Abstract

Caenorhabditis elegans has been used as a model organism to study the roles of molecular chaperones in cellular processes. C. elegans heat shock protein 70-1 (CeHsp70-1) is the first of the 13-member Hsp70 family genes identified so far in the organism. The protein product of this gene, CeHsp70-1, has been shown to play an important role in conferring thermo-tolerance and longevity on C. elegans. Here, we present the results of the first work to over-express, purify and characterize the ATP hydrolyzing activity of a member of the C. elegans Hsp70s. Recombinant CeHsp70-1 was found to be highly expressed and sufficiently soluble in Escherichia coli. The protein was purified to homogeneity using a combination of nickel affinity, ion exchange and size-exclusion chromatography. Kinetic properties of the basal ATPase activity of the enzyme in a low-salt buffer were determined using a colorimetric assay. The specific activity (Vmax per mg protein), Km and kcat values obtained for CeHsp70-1 were 25nmol/min/mg, 50μM and 0.28min−1, respectively. The catalytic constant (kcat) of the protein was found to be similar to that of heat shock cognate 70 (Hsc70) and binding immunoglobulin protein (BiP). At low concentrations, CeHsp70-1 existed mostly in its monomeric form. This work provides a platform for kinetic studies of other members of the C. elegans Hsp70 molecular chaperones.