"BIOSYNTHESIS OF A PROTEIN WHICH BINDS TO MURINE IGM IMMUNOGLOBIN"

THE OBJECTIVE OF THIS PROPOSED PROGRAM IS TO DEVELOP AN AFFINITY PROTEIN THAT IS SPECIFIC FOR MURINE IGM. THE APPROACH FOR ACHIEVING THIS OBJECTIVE IS TO PRODUCE A SINGLE-CHAIN ANTIBODY (SCA) PROTEIN WITH SPECIFICITY FOR MOUSE IGM. THE SCA PROTEIN WILL BE DESIGNED FROM THE VARIABLE REGIONS OF THE ANTI-MOUSE IGM MONOCLONAL ANTIBODY, BET-2, (ATCC HB88). A GENE ENCODING THIS PROTEIN WILL BE CONSTRUCTED AND THE PROTEIN WILL BE EXPRESSED IN E. COLI. THE PROTEIN WILL BE RENATURED, PURIFIED, AND ASSAYED FOR ITSBINDING TO MOUSE IGM. THIS NEW AFFINITY PROTEIN, WHEN IMMOBILIZED, WILL HAVE UTILITY AS A REAGENT FOR THE PURIFICATION OF MURINE IGM ANTIBODIES AND MURINE IGM MONOCLONAL ANTIBODIES. AVAILABLE AFFINITY CHROMATOGRAPHIC METHODS USING PROTEIN G OR PROTEIN A ARE NOT SPECIFIC FOR IGM NOR ARE CLASSIC METHODS SUCH AS ION EXCHANGE. THIS PRECLUDES THE DEVELOPMENT OF IGM MONOCLONAL-BASED PRODUCTS. AN IGM SPECIFIC SCA PROTEIN WOULD OVERCOME THIS OBSTACLE. TECHNIQUES FOR SPECIFIC IMMOBILIZATION THE SCA PROTEIN TO SUITABLE SEPARATIONS MEDIA AND VARIOUS PROTOTYPE MURINE IGM SEPARATION PRODUCTS WILL BE DEVELOPED DURING PHASE II.