Adipose differentiation related protein: expression, purification of recombinant protein in Escherichia coli and characterization of its fatty acid binding properties

Adipose differentiation related protein (ADRP) is a 53 kDa protein encoded by a cDNA originally cloned by differential hybridization from murine adipo cytes. ADRP is induced during the early onset of the adipose differentiatio n program and is expressed at high level in mature adipocytes. We have demo nstrated that ADRP stimulated the uptake of fatty acids thereby providing e vidence for a functional role of ADRP in lipid metabolism. Tn the present p aper, the murine ADRP has been expressed as a recombinant histidine-tagged protein in Escherichia coli, and purified from expressing cultures in order to examine its biochemical properties. We report here that the purified re combinant ADRP binds fatty acids and exhibits stoichiometric saturable bind ing of NBD-stearic acid with a K-d = 0.145 +/- 0.003 muM and a B-max = 0.99 +/- 0.05. Analysis of fluorescence emission spectra indicates that the pol arity of the ADRP binding site is near epsilon approximate to 23, close to that observed for fatty acid binding sites in other lipid binding proteins such as the liver fatty acid binding protein. The data presented here provi de evidence that isolated ADRP purified in the experimental conditions desc ribed hen call be used for functional studies. (C) 2000 Elsevier Science B. V. All rights reserved.