Expression and characterization of the fourth repeat of Xenopus interphotoreceptor retinoid-binding protein in E. coli

Curr Eye Res. 1994 Jun;13(6):391-400. doi: 10.3109/02713689408999866.


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 microM). 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 Kd = 0.4 microM. The expression of recombinant IRBP fragments as fusion proteins in prokaryotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Blotting, Western
  • DNA
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Eye Proteins / genetics*
  • Eye Proteins / isolation & purification
  • Eye Proteins / metabolism*
  • Gene Expression
  • Ligands
  • Protein Folding
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Repetitive Sequences, Nucleic Acid*
  • Retinol-Binding Proteins / genetics*
  • Retinol-Binding Proteins / isolation & purification
  • Retinol-Binding Proteins / metabolism*
  • Spectrometry, Fluorescence
  • Xenopus


  • Eye Proteins
  • Ligands
  • Recombinant Proteins
  • Retinol-Binding Proteins
  • interstitial retinol-binding protein
  • DNA