Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli.
Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol. In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol. Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation. To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli. The purified E. coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine. Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence. They revealed that E. coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1. Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent. The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment. These studies suggest that E. coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions.
Li
E
E
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Locke
B
B
Yang
N C
NC
Ong
D E
DE
Gordon
J I
JI
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D012177:Retinol-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D054840:Retinol-Binding Proteins, Cellular; D013053:Spectrophotometry
D000596:Amino Acids; C518461:Rbp2 protein, rat; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular