Cloning, overexpression, purification, and spectroscopic characterization of human S100P.
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.
Gribenko
A
A
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061, USA.
Lopez
M M
MM
Richardson
J M
JM
Makhatadze
G I
GI
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C103003:S100P protein, human; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine; D002118:Calcium