May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Zebrafish as a Model to Study Interphotoreceptor Retinoid-Binding Protein (IRBP) Structure & Function
Author Affiliations & Notes
  • R. Chandrashekar
    Ophthalmology, Univ of Buffalo SUNY/Ross Eye Institute, Buffalo, New York
    VAMC, Buffalo, New York
  • J. Armenia
    Ophthalmology, Univ of Buffalo SUNY/Ross Eye Institute, Buffalo, New York
    VAMC, Buffalo, New York
  • K. Lee
    Ophthalmology, Univ of Buffalo SUNY/Ross Eye Institute, Buffalo, New York
    VAMC, Buffalo, New York
  • T. Loehfelm
    Ophthalmology, Univ of Buffalo SUNY/Ross Eye Institute, Buffalo, New York
    VAMC, Buffalo, New York
  • J. Griswold
    Hauptman-Woodward Institute, Buffalo, New York
  • F. Gonzalez-Fernandez
    Ophthalmology, Univ of Buffalo SUNY/Ross Eye Institute, Buffalo, New York
    VAMC, Buffalo, New York
  • D. Ghosh
    Hauptman-Woodward Institute, Buffalo, New York
    Roswell Park Cancer Institute, Buffalo, New York
  • Footnotes
    Commercial Relationships  R. Chandrashekar, None; J. Armenia, None; K. Lee, None; T. Loehfelm, None; J. Griswold, None; F. Gonzalez-Fernandez, None; D. Ghosh, None.
  • Footnotes
    Support  NIH grant EY09412 (F.G-F./D.G.); RPB Research Challenge Grant to the Department of Ophthalmology; Veterans Affairs Merit Review Award (F.G.-F.)
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1278. doi:https://doi.org/
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    • Get Citation

      R. Chandrashekar, J. Armenia, K. Lee, T. Loehfelm, J. Griswold, F. Gonzalez-Fernandez, D. Ghosh; Zebrafish as a Model to Study Interphotoreceptor Retinoid-Binding Protein (IRBP) Structure & Function. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1278. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 20oC 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, Kd = 0.12 ± 0.04 µM and N = 0.81 ± 0.22, Kd = 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 
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