Detection of NORPEG Expression in Developing Mouse Eye Using an Antibody Raised Against the Ankyrin Repeats Region of This Protein

Abstract

Abstract: : Purpose: NORPEG (novel retinal pigment epithelial cell gene, RAI14) is a gene that we originally characterized from the human retinal pigment epithelial (RPE) cell line ARPE–19 and showed to be regulated by retinoic acid (Kutty et al., J. Biol. Chem. 276:2831, 2001). The developmentally regulated gene encodes a protein consisting of 980 amino acid residues containing an ankyrin repeats region towards the N–terminal and a coiled–coil domain towards the C–terminal. The present study was aimed at expressing the N–terminal 286 amino acid residue region of NORPEG as a HIS tag fusion protein, raising an antibody against it, and using the antibody to analyze the expression of NORPEG in the developing mouse eye. Methods: Part of the open reading frame of NORPEG encoding the N–terminal 286 amino acid residues was amplified by RT–PCR from RNA preparations obtained from ARPE–19 cells, subcloned into an expression vector, and expressed in E. Coli as a HIS tag fusion protein. The protein, purified using Ni–NTA Agarose, was used as an antigen to raise antibodies in rabbits. An affinity purified NORPEG antibody was used for Western blotting, immunoprecipitation and immunolocalization studies of developing mouse eyes. Results: The N–terminal 286 amino acid residues of NORPEG containing the ankyrin repeats region was expressed as a HIS tag fusion protein and used to generate antibodies against the NORPEG protein. The antibody recognized a ∼110 kDa protein band in ARPE–19 cell extracts analyzed by Western blotting and immunoprecipitation. Western blot analysis also showed the presence of the ∼110 kDa immunoreactive band in mouse eye extracts. NORPEG expression was detected in mouse eyes throughout the early postnatal period. Confocal immunolocalization studies detected NORPEG expression in the ganglion cell layer and the outer margin of the outer nuclear layer, in the lens epithelium, ciliary body, iris, cornea and ocular muscles. Conclusions: The expression of the protein encoded by NORPEG, a gene regulated by retinoic acid, in the mouse eye during postnatal development suggests that this protein may play an important role in eye development and function.

Keywords: protein structure/function • immunohistochemistry • retinal development