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The library number and peptide structures are the most important aspects to find out the substrates of a protease. We introduced mRNA-display to screening for protease substrates and immobilized cDNA/mRNA-peptide library on acylated hexa-lysine bead by transglutaminase (TG) activity.1,2 TG generates the covalent bond by its specific peptide sequence containing glutamine. We identified the reacted substrates for 1×10-5 unit of thrombin, V8 protease, and caspase3. Screening of protease substrates supplied critical information for its specificity. We confirmed the proteolytic cleavage site of thrombin and V8 protease and founded novel peptides reacted by caspase3.