Cloning, overexpression, purification, and spectroscopic characterization of human S100P. |
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Authors: | A Gribenko M M Lopez J M Richardson rd and G I Makhatadze |
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Institution: | A. Gribenko, M. M. Lopez, J. M. Richardson, 3rd, and G. I. Makhatadze |
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Abstract: | The calcium-binding protein S100P has been found to be associated with human prostate cancer. We have overexpressed S100P in Escherichia coli using a T7 expression system. A rapid two-step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of approximately 150 mg per liter of culture. The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques. The far-UV CD shows that secondary structure of recombinant S100P consists predominantly of a-helical structure. Both near-UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation. Ca2+ has a profound effect on S100P structure. Near-UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule. The similarity of far-UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure. |
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Keywords: | bacterial expression Ca2+-binding circular dichroism fluorescence spectroscopy human S100P |
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