Highly active and selective endopeptidases with programmed substrate specificities

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (K-cat/K-M > 10(4) M-1 s(-1)). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu down arrow Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.