BIOPHYSICAL AND STRUCTURAL ANALYSIS OF A RECOMBINANT HUMAN HSP70 ISOFORM

The 70 kDa heat shock protein family (Hsp70) comprises molecular chaperones involved in critical cellular functions essential for maintaining proteostasis. These proteins assist in protein folding, intracellular transport, and stabilization. While Hsp70s are structurally conserved across prokaryotes and eukaryotes, they differ in expression patterns and subcellular localization. Recent studies suggest that newly identified HSP70 isoforms in humans may be involved in tumorigenesis and cancer progression, either promoting or inhibiting these processes depending on the tumor type. However, their precise roles and mechanisms remain to be elucidated. In this study, we report the heterologous expression, purification, and initial biophysical characterization of a human HSP70 isoform. The gene encoding encoding this isoform was identified, and its cDNA was cloned into an expression vector. The recombinant His-tagged protein was expressed in Escherichia coli and purified through a two-step chromatography process: Ni²⁺ affinity chromatography followed by size-exclusion chromatography yielding a highly pure protein. The structural characterization of the protein was assessed using circular dichroism (CD) spectroscopy, confirming that the protein was properly folded and composed of about 45% alpha-helices. Next steps include size-exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering (SEC-MALS-QELS) to determine absolute molecular mass, oligomeric state, and hydrodynamic radius. This integrative biophysical analysis will enhance the understanding of the structural stability and conformational properties of this chaperone, offering insights into its potential functional roles in cellular proteostasis and stress response pathways.