SCREENING METHOD FOR UBIQUITIN AND UBIQUITIN-LIKE PROTEIN SPECIFIC PROTEASES USING CI GENE OF LAMDA PHAGE, IN WHICH THE ENZYMES ARE USEFUL FOR DEVELOPMENT OF DRUGS FOR TREATING BRAIN NEURONAL DISEASES

摘要

PURPOSE: A screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage is provided, thereby easily screening ubiquitin and ubiquitin-like protein specific proteases and simultaneously measuring activity of enzymes, so that the enzymes are useful for development of drugs for treating brain neuronal diseases. CONSTITUTION: The screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage comprises the steps of: (1) cloning Ub, UbI and cI genes, preparing a mutant of cI gene, preparing an expression plasmid of Ub-cI* or UbI-cI* fusion protein, and preparing transformed E. coli using the expression plasmid; (2) introducing cDNA library into the transformed E. coli using recombinant lambda-phage; (3) selecting E. coli expressing desired protein by heat shock at 42 deg. C for 2 hours; and analyzing enzyme activity of the expressed protein, wherein the Ub-cI* or UbI-cI* fusion protein is produced by fusing ubiquitin-like protein Nedd8 or SUMO-1 with cI protein mutant(cI*); and the fusion protein expression plasmid is pACYC184.