BIOCHEMICAL AND BIOPHYSICAL PROPERTIES OF RECOMBINANT HUMAN INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN

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 sou ght to produce recombinant human IRBP (rhIRBP), a perfect duplicate in amino acid sequence of the authentic human protein. This material cou ld serve as a supply of the protein and later could be used to make mu tants of the protein. The goals of the present study were to produce h uman IRBP in an expression system and to examine some of its biochemic al properties. Methods. A cDNA encoding human IRBP was cloned into the transplacement vector, pVL1392, and the plasmid was recombined with l inearized baculovirus on cotransfection into Sig cells. Viruses contai ning the human IRBP cDNA were identified by polymerase chain reaction analysis. IRBP was secreted from virus-infected insect cells. rhIRBP w as purified from cell medium and was examined by chromatography, N-ter minal protein sequencing, immunologic techniques, and fluorometry. Eye cup and retina washes of human donor eyes provided a source of authent ic 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 con stant is approximately 1.04 mu M, close to that reported for bovine IR BP. By scanning fluorometry, the emission and excitation maxima are 47 9 nm and 339 nm, respectively. Conclusions. The baculovirus system pro vides an excellent method to produce and secrete human IRBP. The recom binant protein can be readily purified from cell culture medium. Its b ehavior in chromatography and in binding studies suggests that the rec ombinant 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 p rotein.