Protease screening methods and proteases identified thereby

A method for identifying or selecting a protease or catalytically active portion thereof that cleaves a cleavage sequence in a protein target substrate, comprising:

• a) contacting a collection comprising a plurality of different mutant proteases and/or catalytically active portions thereof that comprise the mutations(s) with a protease trap polypeptide, wherein:
• the protease trap polypeptide is a serpin or modified serpin, an alpha macroglobulin or a modified member of the alpha macroglobulin family, or a p35 protein or a modified member of the p35 family;
• the protease trap polypeptide comprises a reactive site containing a cleavage sequence for the protein target substrate, or is modified in its reactive site by amino acid replacement(s), deletion(s), or substitution(s) to include the cleavage sequence of the protein target substrate;
• contacting is effected under conditions for cleavage of amino acids in the cleavage sequence in the protease trap polypeptide by a protease or a catalytically active portion thereof in the collection to form stable complexes containing the protease trap polypeptide covalently linked to a protease or catalytically active portion thereof;
• upon cleavage of amino acids in the cleavage sequence in the protease trap polypeptide by a protease or catalytically active portion thereof in the collection, the protease trap polypeptide forms a stable complex with a protease or catalytically active portion thereof in the collection;
• b) separating the complexed proteases from uncomplexed members of the collection;
• and
• c) identifying or selecting the protease or catalytically active portion thereof in the complex, thereby identifying or selecting a protease or catalytically active portion thereof that cleaves the protein target substrate.