PROTEASE SCREENING METHODS AND PROTEASES IDENTIFIED THEREBY

摘要

Provided is 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 mutant proteases and/or catalytically active portions of mutant proteases with a protease trap polypeptide, wherein: the protease trap polypeptide comprises a reactive site containing a cleavage sequence for the protein target substrate; whereby upon cleavage of amino acids in the reactive site by a protease or catalytically active portion thereof, the protease trap polypeptide forms a covalent bond with the protease or catalytically active portion thereof to form a complex; contacting is effected under conditions for cleavage of amino acids in the cleavage sequence included in the reactive site in the protease trap polypeptide by a protease or a catalytically active portion thereof in the collection; and upon cleavage of amino acids included in the reactive site of the protease trap polypeptide by a protease in the collection, the protease trap polypeptide forms stable complexes containing the protease trap polypeptide covalently linked to the protease or catalytically active portion thereof; and b) separating the complexed proteases from uncomplexed members of the collection; and c) identifying or selecting a protease or catalytically active portion thereof in a complex, to identify or select a protease or catalytically active portion thereof that cleaves the cleavage sequence in the protease trap polypeptide, thereby identifying or selecting a protease or catalytically active portion thereof that cleaves the protein target substrate. Further provided are combinations of mutant proteases and protease trap polypeptides for practising the invention.