Highly active and selective endopeptidases with programmed substrate specificities

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Abstract
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 (kcat/KM > 104 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↓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.