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 of 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 selected from plasminogen activator inhibitor-1 (PAI-1), antithrombin III (AT3), and modified forms thereof, wherein the modified form contains one or more amino acid replacements, deletions, or substitutions in the reactive site in the protease trap polypeptide to include a cleavage sequence of the protein target substrate;
• the protease trap polypeptide comprises a cleavage sequence for the protein target substrate;
• upon cleavage of amino acids in the cleavage sequence included in the reactive site in the protease trap polypeptide by a protease or catalytically active portion thereof in the collection, the protease trap polypeptide forms a covalent bond with a protease or catalytically active portion thereof in the collection to form a stable complex containing the protease or catalytically active portion thereof with a protease trap polypeptide;
• 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.

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 of 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 modified serpin and the one or more amino acid replacements is/are in the reactive site loop of the serpin polypeptide;
• the protease trap polypeptide comprises a cleavage sequence for the protein target substrate;
• upon cleavage of amino acids in the cleavage sequence included in the reactive site in the protease trap polypeptide by a protease or catalytically active portion thereof in the collection, the protease trap polypeptide forms a covalent bond with a protease or catalytically active portion thereof in the collection to form a stable complex containing the protease or catalytically active portion thereof with a protease trap polypeptide;
• 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.

An iterative method for selecting or identifying a mutant protease or catalytically active portion thereof with specificity/selectivity and/or activity for at least two different cleavage sequences in a protein target substrate, comprising:

• a) contacting a collection comprising a plurality of mutant proteases and/or catalytically active portions thereof that comprise the mutation(s) with a first protease trap polypeptide, wherein:
• the first protease trap polypeptide comprises a first cleavage sequence;
• contacting is effected in the presence of a competitor to thereby enhance selectivity of identified protease(s) or catalytically active portion(s);
• and
• upon cleavage of amino acids in the cleavage sequence in the protease trap polypeptide by the protease or catalytically active portion thereof, the protease trap polypeptide forms a covalent bond with the protease to form stable complexes containing the first protease trap polypeptide 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 proteases or catalytically active portions thereof that form complexes with the first protease trap polypeptide;
• d) contacting the identified or selected proteases or catalytically active portion thereof with a second protease trap polypeptide in the presence of a competitor, wherein:
• the second protease trap polypeptide comprises a second cleavage sequence;
• and
• e) identifying or selecting the proteases or catalytically active portions thereof that form complexes with the second protease trap polypeptide,
• whereby the identified or selected protease or catalytically active portion thereof has substrate specificity and/or cleavage activity for at least two cleavage sequences, wherein:
• the first and second protease trap polypeptides are the same or different;
• each of the protease trap polypeptides is a serpin, alpha macroglobulin, p35 family protein or a modified form thereof that comprises in the reactive site amino acid residues containing a cleavage sequence of a protein target substrate;
• and
• upon cleavage of the amino acid residues in the protease trap polypeptide by a protease or catalytically active portion thereof in the collection, covalent complexes of the protease trap polypeptide with the protease or catalytically active portion thereof in the collection are produced.

A method of selecting a protease, comprising:

• a) contacting a collection comprising a plurality of different mutant proteases or catalytically active portions thereof that comprise the mutation(s) with a first protease trap polypeptide, wherein:
• the first protease trap polypeptide is a serpin, alpha macroglobulin, p35 family protein or a modified form thereof that comprises in the reactive site amino acid residues containing a cleavage sequence in a protein target substrate;
• and
• upon cleavage of the amino acid residues in the protease trap polypeptide by a protease or catalytically active portion thereof in the collection, covalent complexes of the protease trap polypeptide with the protease or catalytically active portion thereof in the collection are produced;
• b) separating the complexed proteases from uncomplexed proteases;
• c) isolating or selecting or identifying the proteases in the complex;
• d) generating a second collection of proteases or catalytically active portions of proteases based on the selected proteases;
• e) repeating steps a) - c) by contacting the second collection of proteases or catalytically active portions thereof with a second protease trap polypeptide, wherein:
• the second protease trap polypeptide is a serpin, alpha macroglobulin, p35 family protein or a modified form thereof that comprises in the reactive site amino acid residues containing a cleavage sequence of a protein target substrate, but is different from the first protease trap polypeptide to form complexes with proteases or catalytically active portions of proteases;
• f) separating the complexed proteases from uncomplexed proteases;
• and
• g) isolating, selecting or identifying the proteases in the complex.