SPECIFIC CLEAVAGE OF HYBRID PROTEINS BY PROTEINASE ENCODED BY THE KEX2 GENE

A method for isolation of the KEX2-gene-encoded membrane-bound protein ase from alpha-cells of Saccharomyces cervisiae yeast has been modifie d. The isolated enzyme hydrolyzes peptides and proteins with basic ami no acid pairs which are cleaved at the C-ends of their peptide bonds. Because KEX2 proteinase is located within the Golgi compartment, it ma p be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min. By extracting the fraction that contains the active enzyme by a detergent solution, a protein has been obtained with specific activity 30 times higher than that of the memb rane extract prepared according to the standard technique. This protoc ol decreases the number of steps required to isolate the enzyme. The e ffects of pH and inhibitors on KEX2 proteinase-catalyzed hydrolysis of Ac-Leu-Lys-Arg-pNA were studied. KEX2 proteinase can participate in p eptide hormone processing because it cleaves human proinsulin at the p eptide bond between Arg32 and Glu33. The KEX2 proteinase can specifica lly cleave large recombinant proteins, for example, a protein consisti ng of a gamma-interferon fragment linked to HIV1-proteinase via a Lys- Arg-containing peptide.