Abstract
Heat shock proteins (HSP) perform vital cellular functions and modulate cell response pathways to physical and chemical stressors. A key feature of HSP function is the ability to interact with a broad array of protein binding partners as a means to potentiate downstream response pathways or facilitate protein folding. These binding interactions are driven by ATP-dependent conformational rearrangements in HSP proteins. The HSP70 family is evolutionarily conserved and is associated with diabetes and cancer progression and the etiopathogenesis of hepatic, cardiovascular, and neurological disorders in humans. However, functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Studies of purified human HSP70 proteins are essential for downstream investigations of protein-protein interactions and in the rational design of novel family-specific therapeutics. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5 in either Escherichia coli or Saccharomyces cerevisiae. We also include initial biophysical characterization of HSPA9 and HSPA8. This work provides the basis for future biochemical studies of human HSP70 protein function and structure.