Zebrafish as a Model to Study Interphotoreceptor Retinoid-Binding Protein (IRBP) Structure & Function

Abstract

Purpose: : Zebrafish IRBP (zIRBP) which is composed of only two modules, each about 300 amino acids (aa), provides a simple system to study IRBP. Zebrafish also offers a model to study trafficking of IRBP in a system where production and turnover of IRBP are highly coordinated in light and dark. However, attempts to express the full-length zIRBP in a soluble form were unsuccessful, and a cryptic enterokinase site prevented removal of the fusion domain. Here, we sought to express zIRBP free of fusion domains allowing ligand-binding analyses, crystal screening, and cellular studies.

Methods: : zIRBP cDNA (Rajendran et al, J.Exp.Biol.1996;199:2775-87) was subcloned into pET-30 Xa/LIC. The fusion domain includes a polyhistidine tag. A tobacco-etch virus (TEV) protease site was inserted upstream of the cDNA to facilitate removal of vector-aa. zIRBP was over-expressed in BL21(DE3)pLysS at 20^oC and purified by Ni²⁺-affinity chromatography. The His-tag was cleaved by TEV protease. zIRBP was further purified by a second Ni²⁺-affinity column, followed by ion-exchange chromatography. The DTT concentration was maintained at 0.5mM throughout the purification. The protein concentration was confirmed by aa analysis. All-trans retinol binding was characterized by fluorescence spectroscopy.

Results: : The new construct yielded soluble protein. TEV protease cleavage was highly efficient allowing purification by resubjection to the Ni²⁺-column. LC-MS/MS confirmed the authenticity of zIRBP. Binding parameters as measured by monitoring retinol-fluorescence enhancement and tryptophan quenching were: N = 1.01 ± 0.06, K_d = 0.12 ± 0.04 µM and N = 0.81 ± 0.22, K_d = 0.23 ± 0.14 µM, respectively.

Conclusions: : We established conditions to express and purify pristine zIRBP in a soluble form free of fusion domains. A single binding site similar to that of Bovine and Xenopus IRBPs was detected. This suggests that the retinol-binding scaffold is formed by both modules, or that only one of the modules binds all-trans retinol.

Keywords: protein structure/function • protein purification and characterization • retina