(116an) 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.