PURIFICATION OF A PROHORMONE - PROCESSING PROTEASE

THE SPECIFIC AIM OF THIS RESEARCH IS TO ELABORATE A METHOD TO PURIFY AND DEFINE THE SPECIFICITY OF A YEAST PROTEOLYTIC ENZYME KNOWN AS KEX2 PROTEASE. THIS ENZYME CLEAVES PEPTIDE BONDS AT THE C-TERMINAL SIDE OF A PAIR OF BASIC RESIDUES, A PROTEOLYTIC CLEAVAGE THAT IS A CHARACTERISTIC OF THE MATURATION OF NUMEROUS HORMONES AND NEUROPEPTIDES SECRETED BY EUKARYOTIC CELLS. AS A SOURCE OF THE ENZYME, THESE STUDIES WILL USE A YEAST STRAIN TRANSFORMED WITH A PLASMID THAT OVERPRODUCES SEVERAL HUNDREDFOLD THE KEX2 PROTEASE-ENCODED GENE. IT IS ANTICIPATED THAT THE ISOLATED ENZYME WILL HAVE POTENTIAL USES FOR BOTH TECHNOLOGICAL INNOVATION AND COMMERCIAL APPLICATIONS IN: (1) IN VITRO PRODUCTION OF HORMONES FROM PROHORMONES CONTAINING A PAIR OF BASIC RESIDUES AT THE PROCESSING SITE, (2) IN VITRO PROCESSING OF FUSION PROTEINS CONTAINING A GENETICALLY INTRODUCED ARG-ARG OR LYS-ARG AT THE JUNCTION SITE, AND (3) THE SELECTIVE CLEAVAGE OF PROTEINS TO PRODUCE ONLY A FEW FRAGMENTS.