Poster abstracts

Poster number 62 submitted by Wen-I Luo

Overexpression, purification and characterization of human HscA

Wen-I Luo (Ohio State Biochemistry Program), Eric Dizin (Department of Chemistry), Taejin Yoon (Department of Chemistry), James A. Cowan (Ohio State Biochemistry Program; Department of Chemistry)

Abstract:
Iron-sulfur (Fe-S) clusters are crucial cofactors in biological processes. Proteins containing Fe-S clusters are found in almost all living organisms and are essential in critical biological events, such as photosynthesis, nitrogen fixation, and mitochondrial respiration. The biogenesis of Fe-S clusters in vivo requires a complicated biosynthetic machinery; however, little is known about the regulation and mechanism of this machinery. In eukaryotic systems, Fe-S cluster biogenesis occurs in the mitochondria and is mediated by the iron-sulfur cluster (ISC) machinery. It has been acknowledged that HscA, a member of the Hsp70 family of proteins that is related to cancer and Parkinson’s disease, is engaged in Fe-S cluster biogenesis in the human ISC machinery. Nevertheless, one obstacle toward further investigation of the role of HscA in human Fe-S cluster biosynthesis is the difficulty in obtaining sufficient quantities of pure HscA. Herein, we have successfully overexpressed, purified and characterized human HscA. The protein was overexpressed in Escherichia coli and purified from inclusion bodies by use of an affinity column that resulted in an isolated yield on the milligram scale (30 mg/L cell culture). The purified protein was then refolded with intact ATPase activity. Subsequently, the protein was characterized through spectroscopic methods that included UV-Vis absorbance, fluorescence, and CD spectroscopies. This work provides a basis for understanding the structure and function of this protein, and the underlying mechanisms that link it to human diseases.

Keywords: iron-sulfur cluster, HscA, purification