Expression and characterization of the fourth repeat of xenopus interphotoreceptor retinoid-binding protein in E. 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 IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 μM). The recombinant fourth repeat of Xenopus IRBP binds [³H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a K_d = 0.4 μM. The expression of recombinant IRBP fragments as fusion proteins in prokayrotes will be useful for defining the structural requirements for ligand binding by this interesting protein.