EXPRESSION AND CHARACTERIZATION OF THE 4TH REPEAT OF XENOPUS INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN IN ESCHERICHIA-COLI

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRB P's 1-2 binding sites for retinol is unknown. To begin to understand w hich repeat(s) are responsible for ligand-binding, we expressed the fo urth repeat of Xenopus IRBP in E. coli to determine if it could by its elf bind all-trans retinol. Our expression studies used a polyhistidin e fusion domain to purify the recombinant protein directly from inclus ion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (<3 mu M). The reco mbinant fourth repeat of Xenopus IRBP binds [H-3]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The bi nding is saturable with a K-d = 0.4 mu M. The expression of recombinan t IRBP fragments as fusion proteins in prokayrotes will be useful for defining the structural requirements for ligand binding by this intere sting protein.