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 adipocytes. ADRP is induced during the early onset of the adipose differentiation program and is expressed at high level in mature adipocytes. We have demonstrated that ADRP stimulated the uptake of fatty acids thereby providing evidence for a functional role of ADRP in lipid metabolism. In the present paper, 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 recombinant ADRP binds fatty acids and exhibits stoichiometric saturable binding of NBD-stearic acid with a K(d)=0.145+/-0.003 microM and a B(max)=0.99+/-0.05. Analysis of fluorescence emission spectra indicates that the polarity of the ADRP binding site is near epsilon approximately 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 provide evidence that isolated ADRP purified in the experimental conditions described here can be used for functional studies.
Serrero
G
G
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201-1180, USA. gserrero@rx.umaryland.edu
Frolov
A
A
Schroeder
F
F
Tanaka
K
K
Gelhaar
L
L
D009327:4-Chloro-7-nitrobenzofurazan; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D000070780:Perilipin-2; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D014162:Transfection
D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D000070780:Perilipin-2; C000602983:Plin2 protein, mouse; D011994:Recombinant Proteins; D009327:4-Chloro-7-nitrobenzofurazan