# US 8,211,428 B2 · Protease screening methods and proteases identified thereby

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- Number: US 8,211,428 B2
- Document ID: US8211428B2
- Status: Granted
- Kind: B2
- Application number: 11/825,627
- Filed: 2007-07-05
- Published: 2009-05-14
- Granted: 2012-07-03
- Earliest claimed: 2006-07-05
- Assignees: Torrey Pines Institute for Molecular Studies; Catalyst Biosciences, Inc.
- Inventors: Edwin L. Madison
- Web page: https://patentgrep.com/patent/US8211428B2

## CPC

- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 7/08: Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock (artificial tears A61P27/04)
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/08: Vasodilators for multiple indications
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/14: Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
- A61P 11/06: Antiasthmatics
- A61P 13/12: of the kidneys
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/02: Muscle relaxants, e.g. for tetanus or cramps
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/64: derived from animal tissue
- C12N 9/6408: {Serine endopeptidases (3.4.21)}
- C12N 9/6462: {u-Plasminogen activator (3.4.21.73), i.e. urokinase}
- C12Q 1/37: involving peptidase or proteinase
- C12Y 304/21073: u-Plasminogen activator (3.4.21.73), i.e. urokinase
- C12Y 304/21109: Matriptase (3.4.21.109)
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/21: containing a His-tag
- C07K 2319/23: containing a GST-tag
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/41: containing a Myc-tag
- C07K 2319/42: containing a HA(hemagglutinin)-tag
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Methods for identifying modified proteases with modified substrate specificity or other properties are provided. The methods screen candidate and modified proteases by contacting them with a substrate, such as a serpin, an alpha macroglobulins or a p35 family protein or modified serpins and modified p35 family members or modified alpha macroglobulins, that, upon cleavage of the substrate, traps the protease by forming a stable complex. Also provided are modified proteases.

## Claims

### Claim 1

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 replacement of one or more of amino acids in the P4-P2′ positions of the protease trap polypeptide 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 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mutant proteases that are members of the collection comprise one or more of amino acid replacements, deletions or insertions in the primary sequence of an unmodified protease.

### Claim 3 (depends on 1)

The method of claim 1, wherein:
the protease trap polypeptide is labeled for detection or separation; and
separation is effected by capture of complexes containing the detectable protease trap polypeptide with a protease or catalytically active portion thereof.

### Claim 4 (depends on 3)

The method of claim 3, wherein the protease trap polypeptide is labeled with biotin.

### Claim 5 (depends on 3)

The method of claim 3, wherein:
the protease trap is labeled with a His tag; and
separation is effected by capture with a metal chelating agent.

### Claim 6 (depends on 5)

The method of claim 5, wherein the metal chelating agent is selected from nickel sulphate (NiSO4), cobalt chloride (CoCl2), copper sulphate (CuSO4) or zinc chloride (ZnCl2).

### Claim 7 (depends on 1)

The method of claim 1, wherein the method is multiplexed, and wherein:
a plurality of different protease trap polypeptides are contacted with the collection; and at least two protease trap polypeptides can be identifiably detected, whereby more than one protease or catalytically active portion thereof are identified.

### Claim 8 (depends on 1)

The method of claim 1, wherein the proteases or catalytically active portions thereof in the complexes are displayed on a microorganism, and wherein following identification or selection of a protease or catalytically active portion thereof in the collection, the microorganism displaying the protease or catalytically active portion is grown to thereby amplify the nucleic acid molecules that encode the identified or selected protease or catalytically active portion thereof to produce a second collection of proteases and/or catalytically active portions thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein the collection contains at least 5, 10, 50, 100, 500, 103, 104, 105, 106 or more different members.

### Claim 10 (depends on 1)

The method of claim 1, where the proteases or catalytically active portions thereof are serine and/or cysteine proteases.

### Claim 11 (depends on 1)

The method of claim 1, wherein the proteases or catalytically active portions thereof are displayed on a microorganism.

### Claim 12 (depends on 11)

The method of claim 11, wherein the proteases or catalytically active portions thereof are displayed in a phage display library.

### Claim 13 (depends on 1)

The method of claim 1, wherein the protease or catalytically active portions thereof are displayed on a solid support, on a cell surface, or on the surface of a microorganism.

### Claim 14 (depends on 1)

The method of claim 1, wherein the proteases or catalytically active portions thereof are displayed on the surface of a yeast, bacterium, a virus, a phage, a nucleic acid, an mRNA molecule or on ribosomes.

### Claim 15 (depends on 1)

The method of claim 1, wherein the proteases are provided as catalytically active portions of a protease.

### Claim 16 (depends on 1)

The method of claim 1, wherein:
at least two different protease trap polypeptides are contacted with the collection; and
at least one of the protease trap polypeptides is detectably labeled to effect capture of stable complexes containing the detectable protease trap polypeptide and a protease or catalytically active portion thereof.

### Claim 17 (depends on 16)

The method of claim 16, wherein:
only one protease trap polypeptide is detectably labeled; and
the other or others are each present in excesses compared to the detectably labeled protease trap polypeptide.

### Claim 18 (depends on 16)

The method of claim 16, wherein the detectable label is a fluorescent label or a His tag.

### Claim 19 (depends on 16)

The method of claim 16, wherein the detectable label is biotin.

### Claim 20 (depends on 1)

The method of claim 1, wherein the proteases or catalytically active portions thereof are selected from the chymotrypsin, subtilisin, caspases or the papain family of proteases.

### Claim 21 (depends on 1)

The method of claim 1, wherein:
the protease is a serine protease, cysteine protease, or catalytically active portion thereof; and
the protease or catalytically active portion thereof is selected from granzyme B, testisin, trypstase beta 1, kallikrein hk5, corin, kallikrein 12, DESC1 oritesase, trypstase gamma 1, kallikrein hK14, hyaluronan-binding serine protease, tryptase, kallikrein hK15, trypsin, neutrophil elastase, mannan-binding lectin-associated serine protease-3, cathepsin G, myeloblastin, granzyme A, granzyme M, chymase, granzyme K, granzyme H, chymotrypsin B, pancreatic elastase, pancreatic endopeptidase E, pancreatic elastaste II, enteropeptidase, chymotrypsin C, prostasin, kallikrein 1, kallikrein hK2, kallikrein 3, mesotrypsin, Factor XII, plasma kallikrein KLK3, factor XI, factor IX, factor VII, factor Xa, thrombin, protein C, acrosin, hepsin, hepatocyte growth factor activator, urinary plasminogen activator (uPA), tissue plasminogen activator (tPA), plasmin, neurosin, neurotrypsin, neuropsin, kallikrein hK10, epitheliasin, prostase, chymopasin, kallikrein 11, membrane type 1 matrix metalloprotease (MT-SP1), spinesin, cathepsin L, cathepsin V, cathepsin K, cathepsin S, cathepsin F, cathepsin B, papain, cruzain, subtilisin, thermitase, C5a peptidase, fervidolysin, lactocepin, furin, kexin, caspase-1, caspase-3, caspase-7, caspase-6, caspase-2, caspase-4, caspase-5, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-1, caspase-13, or caspase-14.

### Claim 22 (depends on 21)

The method of claim 21, wherein the proteases or catalytically active portion thereof are selected from urokinase plasminogen activator (u-PA), tissue plasminogen activator (t-PA) or membrane type 1 matrix metalloprotease (MT-SP1).

### Claim 23 (depends on 21)

The method of claim 21, wherein the protease trap polypeptide is a serpin or a p35 family member that comprises a cleavage sequence from the target protein substrate.

### Claim 24 (depends on 1)

The method of claim 1, wherein the protease trap polypeptide is alpha2- macroglobulin, or a modified form thereof, wherein the modified form contains one or more amino acid replacements, in the reactive site in the protease trap polypeptide to include a cleavage sequence of the protein target substrate.

### Claim 25 (depends on 1)

The method of claim 1, wherein the target substrate is a protein involved in the etiology of a disease or disorder.

### Claim 26 (depends on 1)

The method of claim 1, wherein the target substrate is a vascular endothelial growth factor receptor (VEGFR), a complement protein, or a substrate of tissue plasminogen activator (tPA).

### Claim 27 (depends on 26)

The method of claim 26, wherein the VEGFR is VEGFR2.

### Claim 28 (depends on 26)

The method of claim 26, wherein the complement protein is C2.

### Claim 29 (depends on 1)

The method of claim 1, wherein the protease trap polypeptide is modified in the reactive site by one or more amino acid replacements corresponding to a cleavage sequence that has a sequence of amino acids set forth in any of SEQ ID NOS: 389, 479 and 498.

### Claim 30 (depends on 1)

The method of claim 1, wherein the protease trap polypeptide is alpha 2 macroglobulin, and the one or more amino acid replacements are in the bait region of the polypeptide.

### Claim 31 (depends on 1)

The method of claim 1, wherein the method of identifying proteases is performed a plurality of times, whereby the process is iterative.

### Claim 32 (depends on 31)

The method of claim 31, wherein the method is performed until a mutant protease is identified that exhibits increased substrate specificity and/or activity for a cleavage sequence in a target substrate compared to a non-target substrate that is a substrate of the corresponding protease not containing the mutation(s).

### Claim 33 (depends on 31)

The method of claim 31, wherein a plurality of different proteases are identified at least in the first iteration of the method.

### Claim 34 (depends on 31)

The method of claim 31, further comprising comparing the amino acid sequences of the identified proteases to identify hot spots, wherein:
a hot spot is a modified locus that occurs in at least two, three, four, five or more of the identified mutant proteases compared to the protease not containing the mutation(s).

### Claim 35 (depends on 1)

The method of claim 1, further comprising:
after identifying a first protease or proteases, preparing a second collection of proteases, wherein an identified first protease is used as a template to make further mutations in the protease sequence or catalytically active portion thereof such that members of the second collection contain polypeptides having mutations of the identified first proteases and additional mutations;
then contacting the second collection with a second protease trap polypeptide that is either identical or different from the first protease trap polypeptide used to isolate the first protease or proteases, wherein:
the second 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 the protein target substrate; and
upon cleavage of the amino acid residues in the second 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; and
identifying a second protease(s) or catalytically active portion(s) thereof from the collection in the complex, whereby the second identified protease(s) has greater activity or specificity towards the target substrate than the first identified protease.

### Claim 36 (depends on 1)

The method of claim 1, wherein in step a) the method further comprises adding a competitor to the reaction between a protease trap polypeptide and a protease or catalytically active portion thereof to thereby enhance selectivity of identified protease(s) or catalytically active portion(s).

### Claim 37 (depends on 36)

The method of claim 36, wherein the competitor is human serum or plasma.

### Claim 38 (depends on 36)

The method of claim 36, wherein the competitor is a cell or tissue extract or a biological fluid.

### Claim 39 (depends on 36)

The method of claim 36, wherein the competitor is a wild-type form of a protease trap polypeptide or a variant of a protease trap polypeptide.

### Claim 40 (depends on 1)

The method of claim 1, further comprising screening proteases or catalytically active portions thereof that formed complexes to assess the substrate specificity of these proteases or catalytically active portions thereof for the cleavage sequence of the target substrate compared to a non-target substrate.

### Claim 41 (depends on 1)

The method of claim 1, wherein the protease is human kallikrein 8.

### Claim 42 (depends on 1)

The method of claim 1, wherein the collection contains 2 different members.

### Claim 43 (depends on 1)

The method of claim 1, wherein the target substrate is selected from Interleukin-5 (IL-5), IL-5 receptor, IL-1, IL-1 receptor, IL-13, IL-13receptor, IL-12, IL-12 receptor, IL-4, IL-4 receptor, tumor necrosis factor (TNF), TNF receptor, C-C chemokine receptor type 5 (CCRS), CXC chemokine receptor 4 (CXCR4), gp120, gp141, CD4, RSV fusion protein, hemagglutinin, B7, CD28, IgE; IgE receptor, CD2, CD3, CD40, IL-2, IL-2 receptor, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), HER2, C-C chemokine receptor type 1 (CCR1), CXC chemokine receptor 3 (CXCR3), C-C chemokine receptor type 3(CCR3), Src, Akt, Bc1-2, BCR-Abl, GSK-3, Cdk-2, Cdk-4, EGFR, VEGFR-1, VEGFR-2 or a complement protein.

### Claim 44 (depends on 1)

The method of claim 1, wherein the protease trap polypeptide is modified in the reactive site by one or more amino acid replacements corresponding to a cleavage sequence that has a sequence of amino acids set forth in any of SEQ ID NOS: 406-432 and 480-489.

### Claim 45 (depends on 1)

The method of claim 1, wherein the protease is a serine protease, cysteine protease, or catalytically active portion thereof.

### Claim 46

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.

### Claim 47 (depends on 46)

The method of claim 46, wherein the protease is a serine protease, cysteine protease, or catalytically active portion thereof.

### Claim 48

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.

### Claim 49 (depends on 48)

The method of claim 48, wherein the one or more amino acid replacements are in any one or more of the P4-P2′ positions.

### Claim 50 (depends on 49)

The method of claim 49, wherein the serpin has a sequence of amino acids set forth in any of SEQ ID NOS: 497, 499, 610 and 611.

### Claim 51

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.

### Claim 52 (depends on 51)

The method of claim 51, wherein the at least two cleavage sequences are in one target substrate.

### Claim 53 (depends on 51)

The method of claim 51, wherein the at least two cleavage sequences are in two different target substrates.

### Claim 54 (depends on 51)

The method of claim 51, wherein the first and second protease trap polypeptide are different and each is modified in the reactive site of the protease trap polypeptide by one or more amino acid replacements containing a cleavage sequence of a different target substrate.

### Claim 55 (depends on 51)

The method of claim 51, further comprising repeating steps a)- c) or a)- e) at least once more until an identified or selected protease with a predetermined substrate specificity and/or cleavage activity to at least two cleavage sequences is isolated.

### Claim 56 (depends on 51)

The method of claim 51, wherein the first and second protease trap polypeptides are selected from a serpin, a p35 family member, an alpha-macroglobulin family member, a modified serpin, a modified alpha macroglobulin family member, or a modified p35 family member, wherein the modified form thereof is modified in the reactive site by one or more amino acid replacements, deletions or substitutions to include amino acids of the cleavage sequence of the protein target substrate.

### Claim 57

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.

### Claim 58 (depends on 57)

The method of claim 57, wherein the first and second protease trap polypeptides are modified to contain two different target substrate cleavage sequences, whereby the identified or selected protease has specificity and high cleavage activity to at least two cleavage sequences.

### Claim 59 (depends on 57)

The method of claim 57, wherein the first and second protease trap polypeptides are selected from a serpin, a p35 family member, an alpha-macroglobulin family member, a modified serpin, a modified alpha macroglobulin family member, or a modified p35 family member, wherein the modified form thereof is modified in the reactive site by one or more amino acid replacements, deletions or substitutions to include amino acids of the cleavage sequence of the protein target substrate.