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Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin
Author(s) -
Vorum Henrik,
Madsen Peder,
Rasmussen Hanne H.,
Etzerodt Michael,
Svendsen Ib,
Celis Julio E.,
Honoré Bent
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150171118
Subject(s) - divalent , chemistry , affinity chromatography , nitrilotriacetic acid , biochemistry , fusion protein , recombinant dna , chelation , enzyme , organic chemistry , gene
Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca 2+ ‐binding proteins, and that is highly up‐regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH 2 ‐terminus. The protein was purified by affinity chromatography on Ni 2+ ‐nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion‐exchange chromatography and characterized by two‐dimensional (2‐D) gel electrophoresis and NH 2 ‐terminal sequencing. The ability of rh psoriasin to bind Ca 2+ , Zn 2+ , and Mg 2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca 2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3–1.6 × 10 4 M −1 . This indicates that psoriasin may cooperatively bind several molecules of Ca 2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca 2+ is relatively high. At least eight molecules of Zn 2+ were bound in KCl and four in NaCl, with an affinity just below 1 × 10 4 M −1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn 2+ at physiological concentrations. Mg 2+ and Ca 2+ are bound anti‐cooperatively and binding of each of the ions (Ca 2+ , Zn 2+ , or Mg 2+ ), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.