PURIFICATION OF HTLV-I RECOMBINANT ENVELOPE ANTIGEN

THE LONG TERM OBJECTIVE OF THE RESEARCH INITIATED BY THIS PROPOSAL IS TO DEVELOP AN IMPROVED DIAGNOSTIC ASSAY FOR DETECTION OF ANTIBODIES TO HTLV-I IN HUMAN SERA USING RECOMBINANT DNA DERIVED ANTIGENS. HTLV-I IS A HUMAN RETROVIRUS THAT HAS BEEN SHOWN TO CAUSE A FORM OF ADULT T-CELL LEUKEMIA, AND HAS BEEN LINKED BY SOME RESEARCHERS TO A NUMBER OF NEUROTROPIC DISEASES. INFECTION IS SPREAD PRIMARILY THROUGH INTIMATE PERSONAL CONTACT OR DIRECT BLOOD CONTACT. AN ASSAY TO DETECT EVIDENCE OF INFECTION BY HTLV-IMUST HAVE EXCELLENT SENSITIVITY AND SPECIFICITY CHARACTERISTICS, AND BE SUITABLE FOR LARGE SCALE AUTOMATED ANALYSIS. KEY COMPONENTS IN THE DEVELOPMENT OF SUCH AN ASSAY ARE PROCEDURES FOR LARGE SCALE FERMENTATION OF THE E. COLI CULTURES PRODUCING THE ANTIGEN, AND PROCEDURES FOR ANTIGEN PURIFICATION. IN GENERAL, RECOMBINANT DNA DERIVED PROTEINS EXPRESSED IN E. COLI HAVE PROVEN TO BE PARTICULARLY DIFFICULT TO PURIFY BECAUSE OF THEIR POOR SOLUBILITY AND TENDENCY TO FORM AGGREGATES IN THE ABSENCE OFSTRONG DENATURANTS. THE RESEARCH PLAN IN THIS PROPOSAL WILLMAKE USE OF CONVENTIONAL PROTEIN PURIFICATION METHODOLOGY, NOVEL PROTEIN MODIFICATION METHODS THAT ALLOW SOLUBILITY IN NON-DENATURING BUFFERS, AND HIGH PERFORMANCE CHROMATOGRAPHY TO PURIFY HTLV-I RECOMBINANT ENVELOPE ANTIGENS.