CRYSTALLIZATION OF IL-3\AN ANTIBODY MEDIATED APPROACH

INTERLEUKIN 3 WILL BE CRYSTALLIZED USING A NEW APPROACH THAT MAY PROVIDE A GENERAL METHOD FOR GENERATING CRYSTALS OF RECOMBINANT PROTEINS. HUMAN IL-3 WILL BE PRODUCED IN E. COLI AS A FUSION PROTEIN CONTAINING A SHORT HYDROPHILIC MARKER PEPTIDE SEQUENCE (FLAG PEPTIDE), FUSED TO ITS AMINO TERMINUS. THIS EIGHT AMINO ACID FLAG SEQUENCE, ASP- TYR-LYS-ASP-ASP-ASP-ASP-LYS, HAS BEEN SHOWN TO ALLOW FLAG-CONTAINING FUSION PROTEINS TO BE PURIFIED TO HOMOGENEITY ON AN AFFINITY COLUMN OF AN ANTI-FLAG MONOCLONAL ANTIBODY (MAB) UNDER EXTREMELY MILD CONDITIONS (PHOSPHATE BUFFERED SALINE, PH 7.2, + OR - CALCIUM). THIS UNIQUE AND MILD ONE-STEP PURIFICATION PROCEDURE YIELDS FLAG FUSION PROTEIN PRODUCTS OF HIGH PURITY AND ACTIVITY, BOTH OF WHICH ARE CRITICALLY IMPORTANT FOR CRYSTALLIZATION. ONCE THE FLAG IL-3 FUSION PROTEIN HAS BEEN PURIFIED, CRYSTALLIZATION TRIALS WILL BE CARRIED OUT, BOTH ON THE FREE FLAG- IL-3 PRODUCT AND ALSO ON ITS COMPLEX WITH THE FAB FRAGMENT OF THE SAME ANTI-FLAG MAB. BECAUSE FAB FRAGMENTS AND FAB/ANTIGEN COMPLEXES ARE RELATIVELY EASY TO CRYSTALLIZE, IT IS LIKELY THAT FORMING THE COMPLEX WILL ALTER THE CRYSTALLIZABILITY OF THE FLAG-IL-3 MOLECULE. SUCCESSFUL CRYSTALLIZATION OF FLAG IL-3 WILL SERVE THE DUAL PURPOSES OF (L) PROVIDING THE STARTING MATERIAL FOR SOLVING THE THREE DIMENSIONAL STRUCTURE OF IL-3 AND (2) TESTING THE UTILITY OF THE FLAG ANTIBODY MEDIATED CRYSTALLIZATION CONCEPT. THE CRYSTALS OBTAINED WILL BE USED TO DETERMINE A THREE DIMENSIONAL STRUCTURE OF IL-3 IN PHASE II STUDY. THIS STRUCTURE, IN TURN, CAN BE USED TO CARRY OUT RATIONAL DRUG DESIGN PROCEDURES TO FIND NEW AND IMPROVED MIMICS OR ANTAGONISTS FOR IL-3, WHICH WILL BE OF CLINICAL AND COMMERCIAL VALUE IN THE TREATMENT OF DISEASES THAT ARE RELATED TO THE ROLE OF IL-3 IN EARLY HEMATOPOIESIS AND IMMUNITY.