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Cloning, overexpression, purification, and spectroscopic characterization of human S100P
Author(s) -
Gribenko Alexey,
Lopez Maria M.,
Richardson John M.,
Makhatadze George I.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070123
Subject(s) - recombinant dna , protein tertiary structure , protein secondary structure , fluorescence , chemistry , escherichia coli , tyrosine , circular dichroism , biochemistry , protein structure , microbiology and biotechnology , biophysics , biology , gene , physics , quantum mechanics
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 ∼ 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 α‐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. Ca 2+ 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 Ca 2+ suggests that Ca 2+ binding has only minor effects on secondary structure.