CLONING AND EXPRESSION OF A CDNA-ENCODING BOVINE RETINAL-PIGMENT EPITHELIAL 11-CIS RETINOL DEHYDROGENASE

Purpose. Identification of a 32-kd protein in the bovine retinal pigme nt epithelium. Methods. A bovine retinal pigment epithelium cDNA libra ry was constructed in the bacteriophage lambda ZAP Express. A monoclon al antibody, designated 21-C3/AV, was used to isolate the cDNA encodin g the 21-C3/AV antigen. A positive full-length clone, designated 21-C3 RDH/CD, was sequenced. Northern blot analysis was used to determine th e length of the mRNA and the tissue expression pattern. The entire ope n reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells . Enzymatic activity toward 11-cis retinaldehyde was investigated. Res ults. The complete nucleotide sequence of 21-C3RDH/CD was obtained. Th e deduced amino acid sequence reveals homology with short-chain alcoho l dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also rea cts with other ocular and nonocular tissues, the authors were unable t o demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells syn thesized the 21-C3/AN antigen. This protein showed 11-cis retinol dehy drogenase activity. Conclusions. Homology to the human D-beta-hydroxyb utyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Further more, tissue specificity and molecular weight of the antigen suggest t hat 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis r etinol dehydrogenase. Direct proof came from experiments in which we u sed the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from rec ombinant baculovirus-infected insect cells were found capable of reduc ing 11-cis retinaldehyde.