Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

The peptide sequence (DKTHC)-D-N was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a hum anized gamma 1 Fab' as a model protein. Here we show that conservative muta tions to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved, They were cleaved more eff iciently by CU2+, such that cleavage reactions could be shorter, of lower p H or at a lower temperature. Some were even found to be measurably cleaved by Ni2+, Use of these new cleavage sequences along with cupric ions may pro vide a more rapid and less harsh method for cost-effective, large-scale pro teolytic cleavage of fusion proteins and peptides.