Expresion and Isolation of a hSfi1 Peptide

The human centrosomal protein, hSfi1, is localized to the centriole.  This protein binds human centrin, a calcium binding EF-hand protein, through 23 centrin binding sites throughout its sequence.  The centrin binding sites are comprised of 33 amino acids (X₉A₁₀X₃LLX₂W/FX₆).  The role of centrin-Sfi1 interactions in the dynamic structure of the centriole is currently unknown.  Recombinant hSfi1 will be overexpressed and purified for future centrin-Sfi1 complex studies.  This construct transformed into BL21(DE3)RIL E. coli cells was grown in a five liter fermentor and induced with IPTG at log phase, for its expression.  Subsequent isolation of hSfi1 was accomplished using GST-Tag chromatography followed by successful removal of the GST-Tag by protease cleavage and size-exclusion chromatography. The isolated protein was then analyzed by SDS-PAGE and UV-Vis spectroscopy.  Electrophoresis was employed in additional studies to determine the protein's solubility and to understand the formation of inclusion bodies. The purified protein sample was sent for MS analysis and partial amino acid sequencing at the Wistar Institute at the University of Pennsylvania.  This work will generate sufficient quantities of purified hSfi1 protein to be biophysically characterized by differential scanning calorimetry (DSC) and FT-IR spectroscopy, essential to understanding its structure and stability.