CLONING OF THERMOPHILIC BILIRUBIN OXIDASE GENES

THERMOGEN, INC., WILL DEVELOP A THERAPEUTIC BIOREACTOR ABLE TO INACTIVATE BILIRUBIN. THE REACTOR WILL BE BASED ON A THERMOSTABLE BILIRUBIN-DEGRADING ENZYME PRODUCED FROM A GENE CLONED FROM A THERMOPHILIC ORGANISM. THE BIOREACTOR WILL BE ABLE TO DETOXIFY BILIRUBIN BY EXTRACORPOREAL OR INTRACORPOREAL TREATMENT OF THE BLOOD OF PATIENTS SUFFERING FROM LIVER DYSFUNCTION. DURING PHASE I, THERMOGEN, INC., WILL SYSTEMATICALLY SCREEN A LARGE VARIETY OF GRAM-NEGATIVE AND GRAM-POSITIVE PROKARYOTIC THERMOPHILIC ORGANISMS FOR THE PRESENCE OF BILIRUBIN-DEGRADING ENZYMES (PREFERABLY OXIDASES). THE SCREENING METHOD IS BASED ON A SIMPLE SPECTROPHOTOMETRIC ASSAY THAT CAN DISTINGUISH BETWEEN THE SUBSTRATE AND ITS DEGRADATION PRODUCTS. THE CLONING OF BILIRUBIN-DEGRADING GENES FROM THE IDENTIFIED ORGANISMS WILL BE ACCOMPLISHED BY SCREENING GENOMIC BANKS IN ESCHERICHIA COLI FOR THE PRESENCE OF BILIRUBIN-MODIFYING ENZYME ACTIVITIES. PHASE II WILL AIM FOR THE COMPLETION OF THE CLONING OF THE IDENTIFIED GENES, THEIR OVEREXPRESSION IN SUITABLE HOSTS, THE PURIFICATION OF THE PROTEIN PRODUCTS, AND THE EVALUATIONOF THEIR PERFORMANCE IN A VARIETY OF PROTOTYPE BIOREACTORS AND AS PIGMENT GALLSTONE-SOLUBILIZING AGENTS. SUBSEQUENT RESEARCH WILL BE DEVOTED TO THE DEVELOPMENT OF U.S. FOOD ANDDRUG ADMINISTRATION-APPROVED BIOREACTORS AND ENZYME PREPARATIONS FOR EXTRACORPOREAL TREATMENT OF JAUNDICE.