Abstract
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 NH2-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 NH2-terminal sequencing. The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3-1.6 x 10(4) M-1. This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high. At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.
Udgivelsesdato: 1996-Nov
Udgivelsesdato: 1996-Nov
Original language | English |
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Journal | Electrophoresis |
Volume | 17 |
Issue number | 11 |
Pages (from-to) | 1787-1796 |
Number of pages | 9 |
ISSN | 0173-0835 |
DOIs | |
Publication status | Published - 1996 |
Externally published | Yes |
Keywords
- Amino Acid Sequence
- Base Sequence
- Calcium-Binding Proteins
- Cations, Divalent
- DNA, Complementary
- Electrophoresis, Gel, Two-Dimensional
- Escherichia coli
- Extracellular Space
- Humans
- Molecular Sequence Data
- Multigene Family
- Protein Binding
- Protein Denaturation
- Recombinant Fusion Proteins
- S100 Proteins
- Sequence Alignment
- Sequence Homology, Amino Acid
- Spectrophotometry, Ultraviolet
- Subcellular Fractions