Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone.
We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.
Redmond
T M
TM
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, MD 20892.
Si
J S
JS
Barrett
D J
DJ
Borst
D E
DE
Rainier
S
S
Kotake
S
S
Gery
I
I
Nickerson
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005123:Eye / Q000276:immunology / Q000473:pathology; D005128:Eye Diseases / Q000209:etiology / Q000276:immunology / Q000473:pathology; D005136:Eye Proteins / Q000235:genetics* / Q000276:immunology; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D012177:Retinol-Binding Proteins / Q000235:genetics* / Q000276:immunology; D000099214:Retinol-Binding Proteins, Interstitial
D004247:DNA; D005136:Eye Proteins; D011993:Recombinant Fusion Proteins; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial