September 1994
Volume 35, Issue 10
Free
Articles  |   September 1994
Biochemical and biophysical properties of recombinant human interphotoreceptor retinoid binding protein.
Author Affiliations
  • Z Y Lin
    Department of Ophthalmology, Emory University, Atlanta, GA 30322.
  • J S Si
    Department of Ophthalmology, Emory University, Atlanta, GA 30322.
  • J M Nickerson
    Department of Ophthalmology, Emory University, Atlanta, GA 30322.
Investigative Ophthalmology & Visual Science September 1994, Vol.35, 3599-3612. doi:
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      Z Y Lin, J S Si, J M Nickerson; Biochemical and biophysical properties of recombinant human interphotoreceptor retinoid binding protein.. Invest. Ophthalmol. Vis. Sci. 1994;35(10):3599-3612.

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Abstract

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) binds and transports retinoids and fatty acids in the interphotoreceptor space (IPS). To understand the relationship between the protein structure and its functions requires bulk quantities of human IRBP. The authors sought to produce recombinant human IRBP (rhIRBP), a perfect duplicate in amino acid sequence of the authentic human protein. This material could serve as a supply of the protein and later could be used to make mutants of the protein. The goals of the present study were to produce human IRBP in an expression system and to examine some of its biochemical properties. METHODS: A cDNA encoding human IRBP was cloned into the transplacement vector, pVL1392, and the plasmid was recombined with linearized baculovirus on cotransfection into Sf9 cells. Viruses containing the human IRBP cDNA were identified by polymerase chain reaction analysis. IRBP was secreted from virus-infected insect cells. rhIRBP was purified from cell medium and was examined by chromatography, N-terminal protein sequencing, immunologic techniques, and fluorometry. Eyecup and retina washes of human donor eyes provided a source of authentic human IRBP (IPS-IRBP). RESULTS: rhIRBP and IPS-IRBP exhibit similar elution profiles on concanavalin A, ion-exchange, and size exclusion chromatography. rhIRBP contains a five-amino-acid propeptide at the N-terminus as deduced from the cDNA sequence. Retinol binding of rhIRBP has been characterized by fluorometric titration. The dissociation constant is approximately 1.04 microM, close to that reported for bovine IRBP. By scanning fluorometry, the emission and excitation maxima are 479 nm and 339 nm, respectively. CONCLUSIONS: The baculovirus system provides an excellent method to produce and secrete human IRBP. The recombinant protein can be readily purified from cell culture medium. Its behavior in chromatography and in binding studies suggests that the recombinant protein is virtually identical to the authentic protein. This validates its use in place of IRBP from human donor eyes. Small, but significant, differences in biochemical properties in comparing human and bovine material highlight the significance of studying the human protein.

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