WO2012135902A1

WO2012135902A1 - Protease activity assay

Info

Publication number: WO2012135902A1
Application number: PCT/AU2012/000343
Authority: WO
Inventors: Patrick Schaeffer
Original assignee: James Cook University
Priority date: 2011-04-08
Filing date: 2012-04-04
Publication date: 2012-10-11

Abstract

The present invention discloses methods for assessing protease activity and fusion protein substrates suitable for use in such methods. The fusion protein substrates comprise a stable fluorescent protein, a linker and an unstable non- fluorescent protein, wherein the linker comprises a protease recognition sequence recognised by the protease to be quantified. More particularly, this invention relates to improved assays for quantifying protease activity.

Description

TITLE

PROTEASE ACTIVITY ASSAY

FIELD OF THE INVENTION THIS INVENTION relates to protease activity. More particularly, this invention relates to improved assays for quantifying protease activity.

BACKGROUND TO THE INVENTION

Proteases, also know as peptidases, proteinases, peptide hydrolases, or proteolytic enzymes, are critical protein-cleaving enzymes classified according to the presence of catalytic residues including Ser, Cys, Thr, or Glu (Turk, Nat. Rev. Drug Discov. 5:785-99, 2006). Proteases include exopeptidases (proteases that catalyse the hydrolysis of amino acids from the end of a polypeptide) and endopeptidases (proteases that catalyse the hydrolysis of non-terminal peptide bonds in a polypeptide). Protease substrate specificity is described by residue position relative to a scissile bond between PI and ΡΓ (Turk, Nat. Rev. Drug Discov. 5:785-99, 2006; Schechter and Berger, Biochem. Biophys. Res. Commun. 27: 157-62, 1967).

Serine and cysteine proteases are important to many biological processes such as regulatory pathways, through activation or clearance of their protein substrates (Heutinck et al, Mol. Immunol. 47: 1943-55, 2010; Turk and Stoka, FEBS Lett. 581 :2761-67, 2007). The serine protease known as neutrophil elastase (NE) targets a wide range of biological substrates arising from a low recognition specificity (Hedstrom, Chem. Rev. 102:4501 -23, 2002). It is implicated in respiratory inflammatory conditions, including Cystic Fibrosis, predominantly through its erratic degradation of lung epithelial lining tissue (Kelly et al. , Expert Opin. Ther. Targets 12:145-57, 2008).

Serine proteases are also involved in physiological processes such as blood coagulation {i.e. , coagulation factors), complement activation (i.e. , complement components/factors), phagocytosis, and turnover of damaged tissue. Many serine proteases function to degrade proteins during inflammatory responses, and inadequate control by their natural inhibitors can cause these enzymes to degrade healthy constituents of the extracellular matrix, and thereby contribute to inflammatory disorders such as asthma, emphysema, bronchitis, psoriasis, allergic rhinitis, viral rhinitis, ischemia, arthritis, and reperfusion injury.

Caspases, which are cysteine proteases, by contrast are heavily involved in intracellular apoptotic processes. They are implicated in the pathogenesis of numerous cancers (Kolenko et al, Apoptosis 5: 17-20, 2000) and neurodegenerative conditions such as Alzheimer' s Disease (Cotman et al. , J. Ne ropathol. Exp. Neurol. 64:104-12, 2005). Malfunction, or a reduction in expression of caspase 3 (C3) specifically plays a role in the elevated proliferation rate of tumour cells, and reduced sensitivity to chemotherapy and cytotoxic drugs (Kolenko et al. , Apoptosis 5:17-20, 2000).

Additional cysteine proteases involved in the aetiology of disease include the calpains, a group of calcium-dependent, non-lysosomal cysteine proteases that are involved in a variety of diseases, including cancer, and the cathepsins, which play a vital role in the turnover of cellular proteins. Cathepsin L, for example, is a major lysosomal protease that is synthesized as a proenzyme and is secreted from cells where it is activated and promotes the degradation of the extracellular matrix and basement membrane required for tumour metastasis. Increased cathepsin L activity is linked to invasive and metastatic cancers, including prostate, colorectal and melanoma cancers. This cysteine protease also plays a role in the pathology of degenerative cartilage and bone disorders, such as rheumatoid arthritis and osteoporosis.

Another group of proteases involved in the aetiology of disease are the metalloproteases, a large family of secreted, membrane-bound and cytosolic proteases, including ADAMs (A Disintegrin and Metalloproteases) and MMPs (Matrix Metalloproteases). The metalloproteases play a fundamental role in diverse processes such as asthma, angiogenesis and cancer through their activities in cell adhesion/ usion, membrane protein shedding and breaking down components of the extracellular matrix (ECM).

In view of the important role of proteases in mediating a variety of diseases, there is an urgent need to develop inexpensive, rapid assays for accurately quantifying protease activity and screening protease inhibitors. SUMMARY OF THE INVENTION .

The present invention is directed to methods for assessing protease activity and/or a fusion protein substrates suitable for use in such methods.

In one aspect, the invention provides a method for quantifying protease activity, the method including the steps of: a) combining a fusion protein and a protease to form a mixture, the fusion protein comprising a stable fluorescent protein, a linker and an unstable non-fluorescent protein, wherein the linker comprises a protease recognition sequence recognised by the protease; b) exposing the mixture to a denaturant; c) separating the mixture into soluble and insoluble fractions, wherein the soluble fraction comprises soluble fluorescent protein and the insoluble fraction comprises aggregates of the fusion protein; and d) measuring fluorescence of the fluorescent protein in the soluble fraction as an indicator of protease activity.

In one embodiment, the method further comprises the step of producing and/or purifying the fusion protein prior to combining the fusion protein and the protease.

In another embodiment, the fusion protein is produced by expressing the fusion protein in an expression system, wherein the expression system comprises a nucleic acid molecule encoding the fusion protein and a promoter active in the expression system operably linked to the nucleic acid molecule; and extracting a protein sample from the expression system, wherein the protein sample comprises the fusion protein.

The expression system can comprise an expression construct, wherein the nucleic acid molecule is operably linked to one or more regulatory sequences in the expression construct and the promoter is active in a host cell, and the fusion protein is expressed in the host cell.

The host cell can be a bacterial cell, an insect cell, a yeast cell, a nematode cell, or a mammalian cell.

Furthermore, the expression system can comprise an in vitro transcription/translation system.

In a further embodiment, producing a fusion protein comprises joining the stable fluorescent protein via the linker to the unstable non-fluorescent protein. joining the stable fluorescent protein via the linker to the unstable non- fluorescent protein can comprise ligating the stable fluorescent protein via the linker to the unstable non-fluorescent protein or the self-assembly of the stable fluorescent protein, the linker and the unstable non-fluorescent protein.

In a yet a further embodiment, the stable fluorescent protein is C-terminal to the unstable non-fluorescent protein.

In an alternative embodiment, the stable fluorescent protein is N-terminal to the unstable non-fluorescent protein.

This aspect includes linkers of five to fifty or more amino acids connecting the stable fluorescent protein and the unstable non-fluorescent protein.

Furthermore, this aspect extends to all proteases, including, for example, aspartic proteases, cysteine proteases, metalloproteases, and serine proteases.

In some embodiments, combining the fusion protein and the protease to form a mixture occurs in a well of a microtiter plate. For example, a plurality of the same or different fusion proteins can be combined with the same or different proteases in separate wells of a microtiter plate.

In one embodiment, exposing the combined fusion protein and protease mixture to a denaturant comprises heating the mixture to a pre-determined temperature.

In another embodiment, centrifugation is used to separate the mixture into soluble and insoluble fractions following exposure to a denaturant.

In yet another embodiment, an aliquot of the mixture is spotted onto a selectively permeable matrix (e.g., a gel surface, such as an agarose gel surface or a polyacrylamide gel surface), following exposure to a denaturant to separate the mixture into soluble and insoluble fractions.

In still another embodiment, filtration is used to separate the mixture into soluble and insoluble fractions following exposure to a denaturant.

In a further embodiment, the protease is a mutant protease comprising one or more amino acid substitutions, insertions or deletions, and the method for quantifying protease activity comprises a method for quantifying the activity of the mutant protease. This aspect extends to screening potential inhibitors of a protease, wherein combining the fusion protein and a protease to form a mixture includes combining the fusion protein and a potential inhibitor of the protease, and wherein combining the potential inhibitor with the fusion protein can occur before, after or simultaneously with combining the protease. Thus, in certain embodiments, the method for quantifying protease activity comprises a method for screening potential inhibitors of a protease.

In another aspect, the invention provides a method for quantifying protease activity, the method including the steps of: a) combining a fusion protein and a protease to form a mixture, the fusion protein comprising a stable fluorescent protein, a linker and an unstable non-fluorescent protein, wherein the linker comprises a protease recognition sequence recognised by the protease; b) separating the mixture by gel electrophoresis; and c) measuring fluorescence of the fluorescent protein as an indicator of protease activity.

In one, embodiment, the gel electrophoresis is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

In a further aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding a stable fluorescent protein in-frame with a polynucleotide encoding an unstable non-fluorescent protein, and an internal cloning site between the stable fluorescent protein and the unstable non-fluorescent protein coding sequences into which a heterologous polynucleotide encoding a protease recognition sequence can be inserted in-frame with the stable fluorescent protein and the unstable non-fluorescent protein coding sequences.

In still a further aspect, the invention provides a genetic construct comprising the isolated nucleic acid molecule of the above aspect.

The genetic construct can be an expression construct, wherein the isolated nucleic acid molecule is operably linked or connected to one or more regulatory sequences in an expression vector.

In yet a further aspect, the invention provides an isolated fusion protein comprising an amino acid sequence of a stable fluorescent protein, a peptide linker amino acid sequence and an amino acid sequence of an unstable non- fluorescent protein, wherein said linker comprises a protease recognition sequence.

In a further embodiment, the stable fluorescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, red fluorescent protein, and orange fluorescent protein.

In yet another aspect, the invention provides a method for quantifying protease activity in a biological sample, the method including the steps of: a) isolating the biological sample from a subject; b) combining the isolated fusion protein of the above aspect with the biological sample to form a mixture; c) exposing the mixture to a denaturant; d) separating the mixture into soluble and insoluble fractions, wherein the soluble fraction comprises soluble fluorescent protein and the insoluble fraction comprises aggregates of the fusion protein; and e) measuring fluorescence of the fluorescent protein in the soluble fraction as an indicator of protease activity in the biological sample.

This aspect extends to diagnosis of a protease-related disease.

In still a further aspect, the invention provides a kit for quantifying protease activity, the kit comprising an expression vector comprising a polynucleotide encoding a stable fluorescent protein in-frame with a polynucleotide encoding an unstable non-fluorescent protein, and an internal cloning site between the stable fluorescent protein and the unstable non-fluorescent protein coding sequences into which a heterologous polynucleotide encoding a protease recognition sequence can be inserted in-frame with the stable fluorescent protein and the unstable non- fluorescent protein coding sequences.

In one embodiment, the kit comprises one or more oligonucleotide primer pairs for introducing a promoter, a ribosomal binding site, and a linker tor generating a fusion gene comprising a gene coding for a stable fluorescent protein joined in- frame with a gene coding for an unstable non-fluorescent protein, wherein the linker comprises a protease recognition sequence.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Principle of Tus-GFP-based protease activity assay (TG-PA).

(A) Tus-GFP is cleaved by the protease at the interdomain linker during incubation. (B) Thermal denaturation results in precipitation of uncleaved Tus-GFP, leaving GFP in solution.

(C) After centrifugation, the fluorescence of the GFP-containing supernatant, corresponding to protease activity, is quantified.

Figure 2. Interdomain linker sequences of Tus-GFP substrates and Tus-GFP substrate susceptibility to trypsin, NE and C3.

(A) Details of interdomain linker sequences of Tus-GFP substrates. Substrate sequences are enclosed an outline, flanked by the Kpnl and Spel translated restriction sites (underlined). Vertical double lines indicate end and start of structured regions of Tus-7¾r complex and GFP respectively (Mulcair et ai, Cell 125: 1309-19, 2006; Ormo et ai, Science 273:1392-95, 1996). Single Arg enclosed under asterisk represents potential trypsin cleavage site within C-terminal region of unbound Tus.

(B) In silico proteolysis maps of Tus-GFP substrate susceptibility to trypsin, NE and C3. Arrowheads represent theoretical cleavage sites.

Figure 3. Proteolysis and stability of Tus-GFP substrates obtained with increasing concentrations of trypsin, NE and C3.

TerB-bound or free Tus-GFP substrates (10 μΜ) were incubated with increasing concentrations of protease for 30 min at 37 °C. Half of the reactions were electrophoretically separated by SDS-PAGE without further treatment. The remaining reactions were stopped by heating at 72 °C for 1 minute and centrifuged at 18,000 g for 10 minutes to remove uncleaved Tus-GFP, prior to electrophoresis. For reactions in the presence of TerB, Tus-GFP was incubated with TerB (1 : 1) for 15 minutes at room temperature prior to incubation with protease.

(A) Proteolysis of Tus-G-GFP with trypsin (100 - 500 nM).

(B) Proteolysis of Terfl-bound Tus-G-GFP with trypsin (100 - 500 nM).

(C) Proteolysis of Tus-NE-GFP with NE (2.5 - 25 nM).

(D) Proteolysis of TerB-bovaad Tus-NE-GFP with NE (2.5 - 25 nM).

(E) Proteolysis of free and TerB-bound Tus-C3-GFP with C3 (50 - 250 nM), neither denatured nor centrifuged. (F) Comparison of the thermal stability of TerS-bound (37 °C ) and free Tus- G-GFP, TUS-C3-GFP, Tus-Ml 3-GFP, and Tus-NE-GFP at 25 and 37 °C using GFP- Basta. All Reactions were incubated for 30 minutes. Fluorescence was normalized against free Tus-GFP substrates kept on ice for the duration of incubation.

(G) pH stability of free and TerB-bound Tus-G-GFP in 1% glycerol using

GFP-Basta. Reactions were incubated for 30 minutes at 37 °C prior to centrifugation for 10 minutes at 18,000 g. Fluorescence was normalized against free Tus-G-GFP (TerB-bound or free) kept on ice for the duration following background subtraction.

(H) pH stability of free and 7¾r5-bound Tus-G-GFP in 10% glycerol. D: denatured at 72 °C for 1 minute; ND: neither denatured nor centrifuged.

Figure 4. Fluorometric protease activity assays.

Reactions were incubated with increasing concentrations of protease and TerB-bound or free Tus-GFP substrates (5 μΜ) as described in Figure 3. All reactions were stopped by heating at 72 °C for 1 minute and centrifuged for 10 minutes at 18,000 g. Supernatants (5 μΐ) were diluted in buffer A (60 μΐ) and fluorescence analysed by a fluorescence plate reader.

(A) Tus-G-GFP with trypsin (5 nM - 1 μΜ).

(B) Tus-C3-GFP with C3 (1 nM - 1 μΜ).

(C) Tus-NE-GFP with NE (0.5 nM - 1 μΜ).

(D) Effect of extended incubation on NE activity assay (50 pM - 1 μΜ) with TerB bound. Incubation time was extended to 4 hours to improve the detection limit.

(E) Effect of non-specific Tus-Ml 3-GFP on NE activity.

(F) Effect of non-specific Tus-NE-GFP on C3 activity.

Figure s. Fluorometric NE inhibition assay.

MSACK (25 - 300 μΜ) was pre-incubated with NE (50 nM) for 30 minutes at room temperature. Tus-NE-GFP bound to TerB (5 μΜ) was then incubated with inhibited NE for 30 minutes at 37 °C, and denatured at 72 °C for 1 minute before centrifugation at 18,000 g to remove uncleaved Tus-NE-GFP. Supernatants were diluted in buffer A (60 μΐ) and fluorescence recorded. IC_>o value was determined using Graphpad Prism 5 software. Standard error of the IC50 value is represented by a fine dashed line.

Figure 6. Detection of caspase 3 activity by gel-TG-PA.

(A) Human serum (10%) was spiked with increasing concentrations of caspase 3 and incubated with Tus-C3-GFP for 1 hour at 37 °C, then subjected to SDS-PAGE and photographed under UV exposure.

(B) Fluorescence intensity of GFP bands following incubation for 1 hour, integrated using ImageJ.

(C) The same protocol was performed as for (A), however the reactions were incubated for 4 hours at 37 °C.

(D) Fluorescence intensity of GFP bands following 4 hours incubation were integrated as above.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:l Nucleotide sequence of a PGR primer.

SEQ ID NO:2 Nucleotide sequence of a PCR primer.

SEQ ID NO:3 Amino acid sequence of a linker peptide.

SEQ ID NO:4 Nucleotide sequence of a cloning oligonucleotide that encodes a protease substrate linker.

SEQ ID NO:5 Nucleotide sequence of a cloning oligonucleotide that encodes a protease substrate linker.

SEQ ID O:6 Amino acid sequence of a linker peptide.

SEQ lD NO:7 Nucleotide sequence of a cloning oligonucleotide that encodes a protease substrate linker.

SEQ ID NO:8 Nucleotide sequence of a cloning oligonucleotide that encodes a protease substrate linker.

SEQ ID NO:9 Amino acid sequence of a linker peptide.

SEQ ID NO: 10 Nucleotide sequence of a cloning oligonucleotide that encodes a protease substrate linker.

SEQ ID NO: 1 1 Nucleotide sequence of a cloning oligonucleotide that encodes a protease substrate linker.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved methods for quantifying protease activity.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Preferably, the method further comprises the step of producing and/or purifying the fusion protein prior to combining the fusion protein and the protease.

In one embodiment, producing a fusion protein comprises expressing a fusion protein in an expression system, wherein the expression system comprises a nucleic acid molecule encoding the fusion protein and a promoter active in the expression system operably linked to the nucleic acid molecule; and extracting a protein sample from the expression system, wherein the protein sample comprises the fusion protein.

By "protein" is meant an amino acid polymer. The amino acids can be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art.

The term "protein" includes and encompasses "peptide", which is typically used to describe a protein having no more than fifty (50) amino acids and "polypeptide", which is typically used to describe a protein having more than fifty (50) amino acids.

As used herein, "fusion protein" describes a protein formed by the joining of two or more individual proteins to produce a contiguous or fused protein in which the two or more individual proteins retain their individual activities. This term includes a protein formed by way of ligation or self-assembly of two or more individual proteins, as well as an expressed protein resulting from the joining of two or more genes or gene fragments.

Fusion proteins can be produced using any number of ligation and/or self- assembly methodologies, as are well known to one of skill in the art. Exemplary protein ligation techniques include reductive amination, diazo coupling, thioether bond, disulfide bond, amidation, thiocarbamoyl chemistries, sortase-mediated ligation (Mao et al , J. Am. Chem. Soc. 126:2670-71, 2004), expressed protein ligation utilizing intein domains (Pickin etal, J. Am. Chem. Soc. 130:5667-69, 2008; Seyedsayamdost et al, Nat Protoc. 5 : 1225-35, 2007), and ligation reactions between thioester peptides and bis-cysteinyl linkers (Ziaco et al, Org. Lett., 10: 1955-58, 2008).

Self-assembly techniques for the production of fusion proteins are likewise well known and include, for example, the assembly of proteins individually labelled with avidin/streptavidin and biotin.

Fusion proteins can also be produced by linking at least a first nucleic acid molecule encoding at least a first amino acid sequence to at least a second nucleic acid molecule encoding at least a second amino acid sequence, so that the encoded sequences are translated as a contiguous amino acid sequence either in vitro or in vivo. Fusion protein design and expression are well known in the art, and methods of fusion protein expression are described, for example, in U.S. Pat. No. 5,935,824.

Fusion proteins of the invention comprise a stable fluorescent protein, a linker and an unstable non-fluorescent protein, wherein the linker includes a protease recognition sequence recognised by a protease.

The term "stable", as in a "stable" fluorescent protein that is part of a fusion protein of the invention, indicates that the fluorescent protein is less susceptible to denaturation following exposure to a denaturant than the unstable non- fluorescent protein that is also part of the fusion protein.

By "denaturant" is meant any condition or substance that causes denaturation and/or serves as a denaturing agent, and which causes the loss of tertiary and secondary protein structure. Denaturants include, but are not limited to, changes in temperature (i. e. , heat) changes in pH (e.g. , an alkaline pH or a basic pH), acids (e.g. , acetic acid, trichloroacetic acid and sulfosalicyclic acid), solvents (e.g., methanol, ethanol and acetone), cross-linking agents (e.g., formaldehyde and glutaraldehyde), chaotropic agents (e.g., urea, guanidinium chloride and lithium perchlorate), and disulfide bond reducers (e.g., 2-mercaptoethanol and dithiothreitol). Denatured proteins can exhibit a wide range of characteristics, including loss of function, loss of solubility (leading to precipitation) and aggregation.

In some embodiments, the stable fluorescent protein that is part of a fusion protein of the invention has a higher melting temperature than the unstable non- fluorescent protein that is also part of the fusion protein. Thus, in these instances, "thermostable^'" or "heat stable" can be used interchangeably with "stable" and with each other herein.

"Melting temperature", as the term relates to proteins, is used herein as it is in the art. Namely, the melting temperature is the temperature at which the populations of folded and unfolded protein in a sample are equal. There are numerous methods for determining the melting temperature of a protein, as will be well known to one of ordinary skill in the art. Provided that the method for determining the melting temperature of the fluorescent and non-fluorescent proteins are the same, then a "stable" protein, for the purposes of the present invention, is a protein that has a higher melting temperature than the unstable protein.

One example of a method of measuring the melting temperature of a protein includes, but is not limited to, circular dichroism, which is a spectrophotometric method that differentially measures the absorption of right-handed and left-handed circularly-polarized light to monitor the three dimensional configuration of a protein. Methods of Circular Dichroism are discussed by Miles and Wallace (Chem. Soc. Rev. 35:39-51 , 2006).

The term "fluorescent protein" includes a protein that, in response to incident radiation in the visible or ultraviolet spectra, emits radiation at a wavelength longer than the incident radiation. The term "fluorescent domain" is used to indicate the portion of a fluorescent protein having a structure distinct from an adjacent portion(s) of the protein and which is responsible for the fluorescence. In practice, fluorescent proteins and fluorescent protein domains generally emit in the visible portion of the spectrum.

Fluorescent proteins are well know in the art. These include fluorescent proteins derived from the jellyfish Aequorea victoria, for example, green fluorescent protein (GFP) and its variants, such as yellow fluorescent protein ( YFP) and blue (or cyan) fluorescent protein (BFP) (see, e.g. , Waldo et al. , Nat. Biotechnol. 17:691 -95, 1999; Tsien, R.Y. Annu. Rev. Biochem. 67:509-44, 1998; Griesbeck et al., J. Biol. Chem. 276:29188-94, 2001 ; Zacharias et al. , Science 296:913-16, 2002; Nagai et al. , Nat. Biotechnol. 20:87-90, 2002; Nguyen et al., Nat. Biotechnol. 23:355-60, 2005; Pvizzo el al., Nat. Biotechnol. 22:445-49, 2004).

Also included are fluorescent proteins derived from Discosoma sp., for example, red fluorescent protein (RFP) and orange fluorescent protein (OFP) (Wang et al. , Proc. Natl. Acad. Sci. USA 101 : 16745-49, 2004; Shaner et al. , Nat. Biotechnol. 22: 1567-72, 2004; U.S. Patent No. 7,193,052), as well as an OFP derived from Fungia concinna (Karasawa et al., Biochem. J. 381 :307-12, 2004).

Additionally, fluorescent proteins include thermostable fluorescent proteins as described, for example, in published U.S. Pat. App. No. 20100222551. Also included are fusion proteins that require a co-factor to fluoresce, such as, for example, luciferase.

By "linker" is meant a segment that functionally joins two amino acid sequences in a fusion protein. The term "functionally joins" denotes a connection between the two amino acid sequences in the fusion protein that maintains and/or facilitates proper folding (and hence function) of each of the sequences. Linkers can include amino acids, including amino acids capable of forming disulfide bonds, but can also include other molecules such as, for example, polysaccharides or fragments thereof For example, as described herein, a linker joins a stable fluorescent protein to an unstable non-fluorescent protein in a fusion protein. The linker can be C- terminal to the stable fluorescent protein and N-terminal to the unstable non- fluorescent protein in the fusion protein. Alternatively, the linker can be C-terminal to the unstable non-fluorescent protein and N-terminal to the stable fluorescent protein in the fusion protein.

The linker used in the fusion protein comprises a protease recognition sequence. As used herein, the terms "protease recognition sequence", "protease target site", "substrate recognition site", and "cleavage sequence" are interchangeably used to refer to the peptide sequence recognized by the active site of a protease that is cleaved by that protease. Typically, for example, for a serine protease, a protease recognition sequence is made up of the P 1 -P4 and P 1 '-Ρ4' amino acids in a peptide substrate, where cleavage occurs between the PI and ΡΓ positions.

The terms "P1-P4" and "Ρ -Ρ4'" refer to the residues in a substrate peptide that specifically interact with the S 1 -S4 residues and S 1 '-S4' residues, respectively, in a protease, and are cleaved by the protease. P1-P4 refer to the residue positions on the N-terminal side of the cleavage site, while P 1 '-P4' refer to the residue positions on the C-terminal side of the cleavage site. Peptide numbering nomenclature for proteases can be found in Schechter and Berger (Biochem. Biophys. Res. Commun. 27:157-62, 1967).

Such a linker can be any length, so long as it contains a protease recognition sequence. For example, the linker can comprise 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In one embodiment, the linker includes a protease recognition sequence recognized by neutrophil elastase, caspase 3, caspase 6, matrix metalloproteinase 9, or matrix metalloproteinase 13.

In certain embodiments, the stable fluorescent protein is joined C-terminal to the unstable non-fluorescent protein via the linker in the fusion protein. In other embodiments, the stable fluorescent protein is joined N-terminal to the unstable non- fluorescent protein via the linker in the fusion protein.

As discussed herein, by "unstable", as in an "unstable" non- fluorescent protein that is part of a fusion protein of the invention, is meant that the non- _ fluorescent protein is more susceptible to denaturation following exposure to a denaturant than the stable fluorescent protein that is also part of the fusion protein. Provided that the method for determining the melting temperature of the non- fluorescent and fluorescent proteins are the same, then an "unstable" protein, for the purposes of the present invention, is a protein that has a lower melting temperature than the stable protein.

Fusion proteins of the invention are exposed or subjected to a protease by combining the fusion proteins and the protease to form a mixture (i.e. , an incubation period or step). As will be understood by one of ordinary skill in the art, such exposure will be for a period of time sufficient for the activity of the protease to be assessed, and this period of time may be determined empirically. Examples of periods of time that may be appropriate, include minutes (e.g. , 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes) and hours (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, and 24 hours).

As used herein, the terms "protease", "proteinase" and "peptidase" are interchangeably used to refer to enzymes (i.e., proteins that act as catalysts, increasing the rate at which chemical reactions occur) that catalyse the hydrolysis of covalent peptide bonds. These designations include zymogen forms and activated single-, two- and multiple-chain forms thereof. For clarity, reference to a protease refers to all types of proteases, and includes, but is not limited to, aspartic proteases, cysteine proteases, metalloproteases, and serine proteases. The term also encompasses a mutant protease comprising one or more amino acid substitutions, insertions and/or deletions compared to its wild-type counterpart.

By "zymogen" is meant a protease that is activated by proteolytic cleavage, including maturation cleavage, such as activation cleavage, and/or complex formation with other protein(s) and/or cofactor(s). A zymogen is an inactive precursor of a proteolytic enzyme that is converted to a proteolytic enzyme by the action of an activator.

Mutant proteins, for example mutant proteases, can be produced by a variety of standard, mutagenic procedures known to one of skill in the art. A mutation can involve the modification of the nucleotide sequence of a single gene, blocks of genes or a whole chromosome, with the subsequent production of one or more mutant proteins. Changes in single genes may be the consequence of point mutations which involve the removal, addition or substitution of a single nucleotide base within a DNA sequence, or they may be the consequence of changes involving the insertion or deletion of large numbers of nucleotides.

Mutations occur following exposure to chemical or physical mutagens. Such mutation-inducing agents include ionizing radiation, ultraviolet light and a diverse array of chemical agents, such as alkylating agents and polycyclic aromatic hydrocarbons, all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids. The DNA lesions induced by such erivironmental agents may lead to modifications of base sequence when the affected DNA is replicated or repaired and thus to a mutation, which can subsequently be reflected at the protein level. Mutation also can be site-directed through the use of particular targeting methods.

Mutagenic procedures of use in producing mutant proteases for study according to the methods disclosed herein include, but are not limited to, random mutagenesis {e.g. , insertional mutagenesis based on the inactivation of a gene via insertion of a known DNA fragment, chemical mutagenesis, radiation mutagenesis, error prone PCR (Cadwell and Joyce, PCR^' Methods Appl. 2:28-33, 1992)) and site- directed mutagenesis {e.g. , using specific oligonucleotide primer sequences that encode the DNA sequence of the desired mutation). Additional methods of site- directed mutagenesis are disclosed in U.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377; and 5,789,166.

Following production of a fusion protein, a purification step can be performed to separate he ^"fusion protein from the two or more individual proteins that were joined to produce the fusion protein. One method for purification, involving ultrafiltration in the presence of ammonium sulfate, is described in U.S. Pat. No. 6,146,902. Alternatively, fusion proteins can be purified away from unreacted individual proteins by any number of standard techniques including, for example, size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, or ammonium sulfate fractionation. See, for example, Anderson et al. {J. Immunol. 137: 1 181 -86, 1986) and Jennings & Lugowski (J Immunol. 127:1011-18, 1981). The composition and purity of the fusion protein can be determined by GLC-MS and MALDI-TOF spectrometry.

As described herein, a fusion protein of the invention can be expressed in an expression system, wherein the expression system comprises a nucleic acid molecule encoding the fusion protein and a promoter active in the expression system operably linked to the nucleic acid molecule.

The term "expression system" as used herein designates a system that comprises a nucleic acid molecule encoding a fusion protein of the invention, a promoter active in the expression system operably linked to the nucleic acid molecule and the necessary biological and/or chemical elements to allow for transcription and translation of the nucleic acid molecule.

By "nucleic acid molecule" is meant single- or double-stranded mRNA, RNA, cRNA, and DNA inclusive of cDNA and genomic DNA.

In one embodiment, the expression system comprises an expression construct, wherein the nucleic acid molecule is operably linked to one or more regulatory sequences in the expression construct and the promoter is active in a host cell, and the fusion protein is expressed in the host cell.

By "expression construct" is meant a genetic construct wherein the nucleic acid molecule to be expressed is operably linked or operably connected to one or more regulatory sequences in an expression vector.

An "expression vector" can be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.

In one aspect of the invention, the expression vector is a plasmid vector.

By "operably linked" or "operably connected" is meant that the regulatory sequence(s) is/are positioned relative to the nucleic acid molecule to be expressed to initiate, regulate or otherwise control expression of the nucleic acid molecule.

Regulatory sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

One or more regulatory sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences, and enhancer or activator sequences.

Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lacl promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes, such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Exemplary eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter.

Constitutive or inducible promoters as known in the art can be used and include, for example, tetracycline-repressible, IPTG-inducible, alcohol-inducible, acid-inducible and/or metal-inducible promoters.

In one aspect, the expression vector comprises a selectable marker gene. Selectable markers are useful whether for the purposes of selection of transformed bacteria (such as bla, kanR and tetR) or transformed mammalian cells (such as hygromycin, G418 and puromycin).

Suitable host cells for expression can be prokaryotic or eukaryotic, such as E. coli (DH5a for example), yeast cells, SF9 cells utilized with a baculovirus expression system, nematode cells, or any of various mammalian or other animal host cells, without limitation thereto.

Introduction of expression constructs into suitable host cells can be by way of techniques including, but not limited to, electroporation, heat shock, calcium phosphate precipitation. DEAE dextran-mediated transfection, liposome-based transfection (e.g., lipofectin, lipofectamine), protoplast fusion, microinjection or microparticle bombardment, as are well known in the art.

Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract comprising the fusion protein is exposed to one or more test conditions, or retained for further purification. Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycl ing, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed fusion proteins can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, nickel affinity chromatography (Ni-NTA), anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, lectin chromatography, and gel filtration. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.

In another embodiment, the expression system comprises the in vitro production of a fusion gene comprising a gene coding for the stable fluorescent protein fused in-frame with the linker and a gene coding for the unstable non- fluorescent protein. Methods of in vitro production of a fusion gene are well known in the art, and include, for example, overlap extension PCR, which utilizes one or more oligonucleotide primer pairs to introduce a promoter, a ribosomal binding site and a linker sequence for generating the fusion gene. Expression of the fusion protein from the fusion gene can be accomplished using a cell-free transcription/translation system. Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding a fusion protein of the invention. In some aspects, the DNA construct can be linearised prior to conducting an in vitro transcription reaction. The transcribed mRN A is then incubated with an appropriate cell-free translation extract, such as an E. coli extract, a rabbit reticulocyte extract, or a wheat germ reticulocyte extract to produce the desired fusion protein.

As discussed herein, fusion proteins of the invention are exposed or subjected to one or more proteases by combining the fusion proteins and the one or more proteases to form a mixture. Optionally, fusion proteins of the invention can also be exposed or subjected to a test condition in conjunction with exposure to one or more proteases. The term "'test condition" refers to a substance, compound, molecule, mixture, or treatment with which the fusion protein can be contacted or treated, for purposes of evaluating the effect thereof on the cleavage of the protease recognition sequence as found in the linker of the fusion protein.

Test conditions include, but are not limited to, physical and chemical treatments, such as pH (e.g. , between 0 and 14, inclusive, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, and 13), ionic strength, salt (e.g., NaCl, KC1, CaCl₂, (NH₄)₂S0₄, MgCl₂, and MgSC^) concentration (e.g. , between 50 mM and 500 mM, such as 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, and 450 mM), an oxidizing agent (e.g.., nitric acid, peroxides, sulfoxides, permanganate salts, hypochlorite, chlorite, chlorate, perchlorate, and halogens), a reducing agent (e.g. , hydrogen, metals, and hydrocarbons), and a detergent (e.g., N-Lauroylsarcosine, sodium dodecyl sulfate, sodium deoxycholate, T WEEN® 20, TWEEN* 80, Triton® X-100, saponin, CHAPS, Nonidet™ P 40, Polyethylene glycol), Polysorbate 20, Polysorbate 60, and Polysorbate 80).

To evaluate the effect of multiple proteases and/or multiple test conditions on one or more fusion proteins, any number of high-throughput assays can be utilized. The assays are designed to screen large combinations of fusion proteins and proteases/test conditions by automating the assay steps and providing the necessary components from any convenient source to assay, which are typically run in parallel (e.g., in microtiter formats using robotic assays). Thus, by using high-throughput assays it is possible to screen several thousand different protease/test condition/fusion protein combinations in a short period of time, for example, 24 hours. In particular, each well of a microtiter plate can be used to run a separate assay against a selected protease or test condition to evaluate the effect thereof on the same fusion protein, or, alternatively, each well of the microtiter plate can be used to run a separate assay against a selected fusion protein to evaluate the effect thereof on of the same protease or test condition. As will be understood by one of skill in the art, various groupings (including multiple wells of the same fusion protein/protease/test condition to provide duplicates) and arrangements on the microtiter plate are useful in high- throughput assays.

For example, robotic high-throughput systems for screening multiple proteases/test conditions on one or more fusion proteins typically include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a detector, a data storage unit, and an assay component such as a microtiter dish comprising a well that includes a fusion protein. A number of robotic fluid transfer systems are available, or can easily be made from existing components. For example, commercially-available robotics systems (e.g. , TekCel Corporation, Hopkinton, MD, USA) can be used to set up several parallel simultaneous high-throughput systems.

Thus, in some embodiments, exposing the fusion protein to a protease and, optionally, a test condition occurs in a well of a microtiter plate. For example, a plurality of the same or different fusion proteins can be exposed to the same or different proteases and, optionally, test conditions in separate wells of a microtiter plate.

Following exposure of a fusion protein (including a mixture containing a fusion protein) of the invention to a protease and, optionally, a test condition, the fusion protein/mixture containing a fusion protein is exposed or subjected to a denaturant. For example, following exposure of a fusion protein (including a mixture containing a fusion protein) of the invention to a protease and, optionally, a test condition, the fusion protein/mixture containing a fusion protein is heated to a predetermined temperature. As will be understood by one of ordinary skill in the art, the temperature to which the fusion protein (including a mixture containing a fusion protein) is to be heated will depend on the melting temperatures of the stable fluorescent protein and the unstable non-fluorescent protein. A temperature will be selected such that the unstable non-fluorescent protein unfolds and precipitates, while the stable fluorescent protein remains folded and in solution. Exemplary temperatures include, for example, 25 °C to 100 °C, such as 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C. 90 °C, or 95 °C.

In some embodiments, following exposure of a fusion protein (including a mixture containing a fusion protein) of the invention to protease and, optionally, a test condition, centrifugation is used to separate the resulting protein components into soluble and insoluble fractions, wherein the soluble fraction comprises soluble fluorescent protein and the insoluble fraction comprises aggregates of the fusion protein and/or aggregates of the unstable non- fluorescent protein. Diffusion through a selectively permeable matrix can be used to separate the protein components into soluble and insoluble fractions following exposure to a protease and, optionally, a test condition, wherein the soluble fraction comprises soluble fluorescent protein and the insoluble fraction comprises aggregates of the fusion protein and/or aggregates of the unstable non-fluorescent protein. Specifically, one or more aliquots of the protein components can be spotted onto the surface of a selectively permeable matrix following exposure to a protease and, optionally, a test condition to separate them into soluble and insoluble fractions.

Well known selectively permeable matrices can comprise aqueous gels or various types of sediment or fibrous substances. The most conventional matrix is one in which a gel is used, in particular an agar or an agarose gel, suitably comprising a buffered 1 %-solution of agar or agarose which is permitted to solidify prior to the application of a fusion protein. Additional polymers useful in forming selectively permeable matrices include, for example, polyacrylamide, poly(a-hydroxy acids) such as polylactic acid (PLA), polyglycolic acid (PGA) and copolymers thereof (PLGA), poly(orthoesters), polyurethanes, and hydrogels, such as polyhydroxyethyl methacrylate (poly-HEMA) or polyethylene oxide-polypropylene oxide copolymer (PEO-PPO).

By "selectively permeable" is meant that soluble proteins are able to enter the matrix, while protein aggregates are unable to. For example, in 1% agarose gel, aggregates larger than about 0.4 μηι are unable to enter the gel. Methods of controlling the permeability of a matrix (e.g., by varying the amount of matrix material and/or including various cross- linking reagents) are well know in the art.

Filtration can be used to separate the protein components into soluble and insoluble fractions following exposure to a protease and, optionally, a test condition, wherein the soluble fraction comprises soluble fluorescent protein and the insoluble fraction comprises aggregates of the fusion protein and/or aggregates of the unstable non-fluorescent protein. Filtration includes gel filtration, gel permeation, diafiltration, and ultrafiltration, although without limitation thereto, as are well understood in the art.

In certain embodiments, -the method for quantifying protease activity described herein encompasses a method for screening potential inhibitors of a protease, wherein combining the fusion protein and a protease to form a mixture includes combining the fusion protein and a potential inhibitor of the protease, and wherein combining the potential inhibitor with the fusion protein can occur before, after or simultaneously with combining the protease.

By "potential inhibitors of a protease" is intended molecules to be tested for their ability to inhibit protease activity using the methods described herein. Potential inhibitors of a protease may be mutant versions of known protease inhibitors. Examples of molecules that can be tested for their ability to inhibit protease activity using the methods described herein include, but are not limited to, peptides, nucleic acids, carbohydrates, and small molecules. The term is meant to encompass both natural compounds (e.g., purified from a biological source) as well as synthetic compounds.

In particular embodiments, mutant versions of proteases can be screened for their abilities to recognize and/or cleave a protease recognition sequence as found in the linker of the fusion protein using this aspect of the invention. For example, libraries of mutant proteases can be screened for variants with increased (or decreased) affinity for a protease recognition sequence, as compared to a wild-type ^' protease. Alternatively, libraries of mutant proteases can be screened for variants with improved stability or function generally (or in the presence of one or more test conditions), relative to a wild-type protease.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding a stable fluorescent protein in-frame with a polynucleotide encoding an unstable non-fluorescent protein, and an internal cloning site between the stable fluorescent protein and the unstable non-fluorescent protein coding sequences into which a heterologous polynucleotide encoding a protease recognition sequence can be inserted in-frame with the stable fluorescent protein and the unstable non-fluorescent protein coding sequences.

In a further aspect, the invention provides a genetic construct comprising the isolated nucleic acid molecule of the above aspect.

The genetic construct can be an expression construct, wherein the isolated nucleic acid molecule is operably linked or connected to one or more regulatory sequences in an expression vector as described herein.

In yet a further aspect, the invention provides an isolated fusion protein comprising an amino acid sequence of a stable fluorescent protein, a peptide linker amino acid sequence and an amino acid sequence of an unstable non-fluorescent protein, wherein said linker comprises a protease recognition sequence, as described herein.

By "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.

In yet another aspeet, the invention provides a method for quantifying protease activity in a biological sample, the method including the steps of: a) isolating the biological sample from a subject; b) combining the isolated fusion protein of the invention with the biological sample to form a mixture; c) exposing the mixture to a denaturant; d) separating the mixture into soluble and insoluble fractions, wherein the soluble fraction comprises soluble fluorescent protein and the insoluble fraction comprises aggregates of the fusion protein; and e) measuring fluorescence of the fluorescent protein in the soluble fraction as an indicator of protease activity in the biological sample.

The term "biological sample" refers to a sample obtained from a subject. As used herein, biological samples include all clinical samples useful for quantifying protease activity in subjects, including, but not limited to, cells; tissues; bodily fluids, such as blood, derivatives and fractions of blood, such as serum; and biopsied or surgically removed tissue, including tissues that are, for example, unfixed or frozen.

By "subject" is meant a human or non-human animal.

In one embodiment, the method for quantifying protease activity in a biological sample includes a method for diagnosing a protease-related disease in the subject, by detecting altered protease levels and/or activity in the subject, particularly increased protease levels and/or activity, relative to a disease-free subject. In still a further aspect, the invention provides a kit for quantifying protease activity, as well as for screening potential inhibitors of proteases, for use in the methods of the aforementioned aspects. In one embodiment, the kit includes an expression vector comprising a polynucleotide encoding a stable fluorescent protein in-frame with a polynucleotide encoding an unstable non-fluorescent protein, and an internal cloning site between the stable fluorescent protein and the unstable non- fluorescent protein coding sequences into which a heterologous polynucleotide encoding a protease recognition sequence can be inserted in-frame with the stable fluorescent protein and the unstable non-fluorescent protein coding sequences. The expression vector can include one or more regulatory sequences as described herein.

In a further embodiment, the kit comprises one or more oligonucleotide primer pairs for introducing a promoter, a ribosomal binding site, and a linker for generating a fusion gene comprising a gene coding for a stable fluorescent protein joined in-frame with a gene coding for an unstable non-fluorescent protein, wherein the linker comprises a protease recognition sequence; the kit further comprising one or more reagents necessary to carry out in vitro amplification reactions, including DNA sample preparation reagents, appropriate buffers (for example, polymerase buffer), salts (for example, magnesium chloride), and deoxyribonucleotides (dNTPs).

In such a kit, an appropriate amount of the aforementioned one or more oligonucleotide primer pairs is provided in one or more containers, or held on a substrate. An oligonucleotide primer can be provided in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers are provided in pre-measured single use amounts in individual (typically disposable) tubes or equivalent containers.

The amount of each oligonucleotide primer pair supplied in the kit can be any appropriate amount, and can depend on the market to which the product is directed. General guidelines for determining appropriate amounts can be found, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ; Ausubel et al. (eds.), Short Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1999; and Innis et al. , PCR Applications, Protocols for Functional Genomics, Academic Press, Inc., San Diego, CA, 1999.

So that the invention may be readily understood and put into practical effect, the following non-limiting Examples are provided.

EXAMPLES

Example 1 - Fluorescent Proteins as a Reporter of Protease Activity

Experimental Procedures

Buffers

Lysis Buffer: 50 mM Na₂P0₄ [pH 7.8], 300 mM NaCl, 2mM β-mercaptoethanol.

Buffer A: 45 mM Na₂HP0₄, 5 mM NaH₂P0₄, 10% glycerol, 10 mM imidazole, 2 mM β-mercaptoethanol (pH 7.8).

Buffer B: 20 mM Tris, 150 mM NaCl (pH 8).

Buffer NE: 51 mM sodium acetate, 150 mM NaCl (pH 5,5).

Buffer C3A: 50 mM HEPES, 100 mM NaCl, 10 mM β-mercaptoethanol, 10% glycerol (pH 7.4).

Buffer C3B: 37.5 mM HEPES, 350 mM NaCl, 10 mM β-mercaptoethanol (pH 7.4).

Cloning

The plasmid pET-GFP (Moreau et al., Mol. Biosyst. 6:1285-92, 2010) was engineered to contain the Tus-G-GFP protease linker cloning cassette (N-terminal,

Hisg-tagged). The Tus sequence was amplified from the plasmid pMMOOl (Dahdah et al. , Chem. Commun. 21 :3050-52, 2009) with the following primers, introducing a

5' Aflll site, and 3 ' Kpnl and Spel sites: '

psJCU123 (forward) 5'-AAAAAAC L4 lGGCGCGTTACGATCTCG TAGACCG-3' (SEQ ID NO:l);

psJCU124 (reverse) 5'-ΑΑΑΑΑΑ CTAGTAAAGGTA CTATATG ACCG CCGGATCCCAAATT-3' (SEQ ID NO:2).

The PCR product and pET-GFP were digested with Aflll and Spel. This digestion resulted in the removal of a 52 bp fragment from pET-GFP. The PCR fragment was then ligated into pET-GFP by T4 DNA ligase to create the Tus-G-GFP encoding plasmid pSA036.

Protease substrate linker coding sequences were inserted into the Tus-G-GFP cloning cassette by the following method. The plasmid pSA036 was digested with Kpnl and Spel between the Tus and GFP coding sequences. Complementary oligonucleotides (final concentration 1 μΜ) coding for protease substrate sequences were mixed and annealed by heating to 80 °C and cooling slowly to room temperature . The annealed double strand oligonucleotides containing Kpnl and Spel overhangs were then ligated into pSA036 by T4 DNA ligase to yield the different Tus-GFP substrate coding plasmids. The oligonucleotides and resulting plasmids include:

Tus-C3-GFP [C3 linker DEVDGDEVDG (SEQ ID NO:3)] coding plasmid pSA038:

psJCU125 (forward) 5 '-C G ATG AGGTGG ATGG AG ATG AGGTGG ATGGAA.3' (SEQ ID NO:4);

psJCU126 (reverse) 5'-CTAGTTCCATCCACCTCATCTCCATCCACO TCATCGGT-AC-3' (SEQ ID NO:5).

Tus-NE-GFP [NE linker GEPVSGEPVS (SEQ ID NO :6)] coding plasmid pSA042:

psJCU 133 (forward) 5'-CGGTGAACCTGTATCCGGTGAACCTG

TATCCA-3^* (SEQ ID NO:7);

psJCU 134 (reverse) 5'-CTAGTGGATACAGGTTCACCGGATACAGGTT CACCGG-TAC-3' (SEQ ID NO:S).

Tus-MB-GFP [MM 13 linker GPQGLGPQGL (SEQ ID NO: 9)] coding plasmid pSA041 :

psJCUBl (forward) 5'-CGGACCTCAAGGGTTGGGACCTCAA GGGTTGA-3^* (SEQ ID NO: 10);

psJCU132 (reverse) 5'-CTAGTCAACCCTTGAGGTCCCAACCCTTGA GGTCCGG-TAC-3' (SEQ ID NO: 1 1 ).

Protein expression and purification

E. coli strain BL21-(DE3)-RIPL was used to express all proteins. In this strain, the expression of the T7 RNA polymerase required to initiate the transcription of the inserts is controlled by the Lac promoter .that is repressed in the presence of glucose. This strain is deficient in the Lon and OmpT proteases and contains extra copies of genes coding for tRNAs (RIPL) that may limit translation of heterologous proteins. Protein expression was not induced with IPTG.

An auto-inducible media was prepared according to the Studier protocol (ZY, MgS0₄, lOOOx Trace Metal Mix, 20xNPS, 50x 5052) (Studier Prot. Exp. Pur. 41 :207-234, 2005) with the following modifications: sodium molybdate was replaced by ammonium molybdate, ZnS0₄ by ZnCl₂, cobalt chloride by cobalt sulphate, copper chloride by copper sulphate, Na₂SeO₃(₃H₂0) by Na₂Se0₄, and N-Z Amine by peptone. Proteins were also expressed in commercial Overnight Express Instant Medium (Novagen, San Diego, CA, USA).

Bacteria cultures were started from single colonies of overnight transformants plated on LB agar supplemented with 100 μg/ml ampicillin and 50 ng/ml chloramphenicol. Cell cultures (250 mis) where first incubated at 37 °C in 1L flasks. Bacteria expressing GFP fusion proteins were transferred at 16 °C when they entered the stationary phase of growth at OD = 6.6 (Overnight Express Instant Medium) and OD = 1 1 (Studier medium) to allow the proper folding of GFP. Cells were grown at 16 °C for 2 to 3 days until bacterial pellet (from centrifuged culture aliquots) showed bright fluorescence. Cells were harvested 48 hours after cells entered the stationary phase at OD = 6.6 (Studier Media).

Cells were centrifuged at 8,000 rpm for 10 minutes at 4 °C in a Beckman Coulter (Fullerton, CA, USA) Avanti J-20XP centrifuge and re-suspended in ice-cold lysis buffer at 7^' ml/g of cells. E. coli cells were lysed by two to three passes at 12,000 p.s.i. in a cooled French Pressure cell press. The lysate was centrifuged at 18,000 rpm for 40 minutes at 4 °C in a JA-20 rotor in a Beckman Coulter Avanti J- 20XP centrifuge to eliminate cells debris. Cleared lysate was frozen in liquid nitrogen and stored at -80 °C until purification.

Proteins were purified using the Ni-charged resin Profinity IMAC (Bio-Rad, Hercules, CA, USA). Briefly, 500 μΐ of resin was pre-equilibrated in lysis buffer prior to being added to the cleared lysate. FIis₆-Tagged proteins were allowed to bind nickel beads for 1 hour at 4 °C with rocking. The beads-containing lysate was next transferred into a standard filtered column and beads were allowed to settle to the bottom. The flow through (i.e., lysate minus beads) was passed twice through the column. Ni-charged beads were then washed 3 times with 1 ml and one time with 15 ml of lysis buffer supplemented with 10 mM imidazole. Retained proteins were eluted from the beads in lysis buffer supplemented with 200 mM imidazole. Elution fractions containing the proteins were pooled and proteins were precipitated by the addition of 0.5 g/ml (NH₄)₂S0₄ followed by one hour incubation at 4 °C under gentle shaking. The solution was then centrifuged at 18,000 rpm for 40 minutes at 4 °C. The pellet obtained was resuspended in 1 ml of buffer A and was frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined by standard Bradford assay. Protein purity was assessed by NEXT-GEL SDS-PAGE (Amresco, Solon, OH, USA) and band quantification using the image analysis software ImageJ (see the website at rsbweb.nih.gov/ij/). Proteolysis profiles

Tus-GFP substrates (10 μΐ, 20 μΜ in buffer A) were mixed with 10 μΐ of protease solutions at increasing concentrations. Trypsin (Sigma) was at concentrations ranging from 0.2 - 1 μΜ in buffer A; NE (Enzo Life Sciences) at 5 - 50 nM in buffer NE:A mix (1 :4); and C3 (Prospec) at 0.1 - 0.5 μΜ in buffer C3A:C3B mix (1 :4). Reactions (20 μΐ) were incubated at 37 °C for 30 minutes, then half of the reactions were stopped by heating at 72 °C for 1 minute and centrifuged at 18,000 g for 10 minutes at 4 °C. In the case of C3, no denaturation or centrifugation steps were performed following the initial 30 minute incubation.

To evaluate the effect of TerB on proteolysis, Tus-GFP substrates (5 μΐ, 40 μΜ in buffer A) were incubated with stoichiometric amounts of TerB (5 μΐ, 40 μΜ in buffer B) for 15 minutes at room temperature prior to mixing with protease solutions as described above. Following incubation or denaturation, all mixtures were placed on ice for 5 minutes to prevent further proteolysis from occurring. All mixtures (10 μΐ), or supernatants of those centrifuged, were then subjected to separation by SDS- PAGE (NEXT-GEL Amresco). Gels were stained with Coomassie blue and destained in 40% isopropanol/10% acetic acid. Theoretical trypsin and C3 cleavage site maps on their specific Tus-GFP substrates were generated using PeptideCutter software (Gasteiger et al. in John M. Walker (ed) The Proteomics Protocols Handbook, Humana Press, 2005). Theoretical cleavage sites for NE were defined as He or Val residues not following Tip, Cys, Asn, Asp or Tyr residues.

Fluorometric protease activity assay

Tus-GFP substrates (5 μΐ, 10 μΜ in buffer A) were mixed with 5 μΐ of protease solutions at increasing concentrations. Trypsin concentrations ranged from 0.01 - 2 μΜ in buffer A; NE from 0.001 - 2 μΜ in buffer NE:A mix (1 :4); and C3 from 0.002 - 2 μΜ in buffer C3A:C3B mix (1 :4). Reactions (10 μΐ) were incubated at 37 °C for 30 minutes.

Reactions were also performed in the presence of TerB. Here, Tus-GFP substrates (2.5 μΐ, 20 μΜ in buffer A) were mixed with stoichiometric amounts of TerB (2.5 μΐ, 20 μΜ in buffer B) and incubated for 15 minutes at room temperature prior to the addition of protease as described above.

In addition, Tus-NE-GFP in presence of TerB was also incubated for an extended period of 4 hours with NE ranging in concentration from 0.1 - 100 nM in buffer NE:A mix (1 :4).

Following incubation, reactions were stopped by heating at 72 °C for 1 minute then placed on ice for 5 minutes to prevent further proteolysis. All reactions were centrifuged for 10 minutes at 18,000 g and 4 °C. The supernatants (5 μΐ) were diluted in buffer A (60 μΐ) into separate wells of a black 96-we microtitre plate and fluorescence analysed with a fluorescence plate reader (Victor V Wallac 1420, Perkin-Elmer) set at 460 nm excitation/535 nm emission (lamp energy 6192, shaking for 2 second at 0.1 mm prior to first measurement). Fluorescence values were normalised by subtracting the baseline fluorescence (Tus-GFP without protease, denatured at 72 °C) from the proportion of GFP fluorescence compared to a Tus-GFP control that was not denatured and did not contain protease. This represented the detectable proportion of original Tus-GFP fluorescence given by the liberated GFP after assay incubation, and was indicative of the level of protease activity. Normalisation was calculated as follows:

Normalised fluorescence = (GFP fluorescence after incubation / Tus-GFP residual fluorescence) - baseline residual fluorescence. Fluorometric NE inhibition assay

MSACK (2.5 μΐ, 0.1 - 1.2 mM in DMSO) was mixed with NE (2.5 μΐ, 0.2 μΜ in buffer NE) and incubated for 30 minutes at room temperature. Tus-NE-GFP (2.5 μΐ, 20 μ in buffer A) was mixed with TerB (2.5 μΐ, 20 μΜ in buffer B) and incubated for 15 minutes at room temperature. The inhibited NE (5 μΐ) was then mixed with an equal volume of TerB-bound Tus-NE-GFP, incubated and processed according to the fluorometric, end-point assay protocol. The IC50 value was determined by fitting the data to a (log)inhibitor vs. response - Variable slope curve using Graphpad Prism 5 software. Thermal and pH stability of free and TerB-bound

Tus-GFP substrates using GFP-Basta

Thermal and pH stability data for Tus-GFP substrates were obtained using GFP-Basta (Moreau ei al, Mol. Biosyst. 6: 1285-92, 2010). Tus-G-GFP, Tus-Ml 3- GFP, Tus-NE-GFP and Tus-C3-GFP (10 μΐ, 5 μ in buffer A) were incubated separately at 4, 25 or 37 °C for 30 minutes. All reactions were then placed on ice for 5 minutes to prevent further aggregation and centrifuged for 10 minutes at 18,000 g and 4 °C to remove aggregates. The supernatants (5μ1) were diluted in buffer A (60 μΐ) in separate wells of a black.96-.well microtitreplate and the. fluorescence analysed with a fluorescence plate reader (Victor³ V Wallac 1420, Perkin-Elmer) set at 460 nm excitation/535 nm emission (lamp energy 6192, shaking linearly for 2 seconds at 0.1 mm prior to first measurement).

Reactions were also performed in the presence of stoichiometric amounts of TerB for comparison (37 °C only). Substrates (5 μΐ, 10 μΜ in buffer A) were premixed with TerB (5 μΐ, 10 μΜ in buffer B) for 15 minutes at room temperature prior to incubation at 37 °C as described above.

To evaluate the effect of pH, Tus-G-GFP (1 μΐ, 50 μΜ in buffer A) was diluted in sodium phosphate buffers of various pH (9 μΐ, 50 mM) to produce reaction conditions with their final pH ranging from 2.3 to 1 1.9. The effect of glycerol was also evaluated by adjusting the final concentrations to 1 and 10% (v/v), respectively, with the same range of pH.

To evaluate the effect of TerB on Tus-G-GFP substrate stability under varying pH conditions (i.e., 2.1 - 1 1.9) and glycerol concentrations, reactions were performed and processed as described above with the exception that Tus-G-GFP (0.5 μΐ, 100 μΜ in buffer A) was incubated with stoichiometric amounts of TerB (0.5 μΐ, 100 μΜ in buffer B) for 15 minutes at room temperature prior to dilution and incubation in sodium phosphate buffers, followed by centrifugation.

The residual fluorescence was normalized against the fluorescence of a control sample (on ice in buffer A) following background subtraction, representing 00% fluorescence.

Results

Fluorescent proteins as a reporter of protease activity

Taking advantage of the biophysical properties of Tus (Moreau el al. , Mol. Biosyst. 6:1285-92, 2010; Coskun-Ari et al. . Biol. Chem. 269:4027-34, 1994) and GFP (Sniegowski et al, Biochem. Biophys. Res. Commun. 332:657-63, 2005; Tsien, Anna. Rev. Biochem. 67:509-44, 1998; Moreau et al. , Mol. Biosyst. 6:1285- 92, 2010) we aimed to develop a Tus-GFP-based protease activity assay (TG-PA). To do this, several Tus-GFP fusion protein reporters were engineered containing protease substrates within their interdomain linkers. TG-PA exploits the fact that when the linker between the GFP and Tus domains is proteolysed. the

fluorescence of liberated GFP, reflecting protease activity, can be measured after elimination of the uncleaved fusion protein (e.g. , through heat denaturation and centrifugation) (Figure 1). The assay is adapted to function uniquely with various proteases to detect their activity and screen for inhibitors. Here we describe the validation of TG-PA with three proteases having different substrate specificities - trypsin, NE and C3.

First, we engineered a protease linker cloning cassette derived from pMMOOland pET-GFP (Dahdah et al., Chem. Commun. 21 :3050-52, 2009; Morin et al, Mol Biosyst. 6: 1 173-75, 2010) that was used for the production of a generic trypsin substrate (Tus-G-GFP). From Tus-G-GFP the specific NE (Tus-NE-GFP) and C3 (Tus-C3-GFP) substrates, as well as an additional substrate for MMP13 (Tus-M13-GFP) were subsequently produced. Tus-G-GFP contains a single Lys recognised by trypsin in its interdomain linker. Tus-NE-GFP contains a double NE substrate (GEPVSGEPVS) (SEQ ID NO:6), Tus-C3-GFP a double C3 substrate (DEVDGDEVDG) (SEQ ID NO:3) and Tus-M13-GFP a double MMP13 substrate (GPQGLGPQGL) (SEQ ID NO:9). The specific substrate scissile bonds are located between the Val and Ser, Asp and Gly, and Gly and Leu of the linkers, respectively. Specific sequences and flanking sequences are illustrated in Figure 2A. For trypsin and NE, several other potential protease sites were identified (Figure 2B) in both the Tus and GFP domains, although for GFP it has been shown that its structure is highly resistant to proteolysis (Sniegowski et al, Biochem. Biophys. Res. Comm n. 332:657-63, 2005). In contrast, the same analysis showed that for Tus-C3-GFP, only the linker is susceptible to C3.

The E. coli replication terminator protein Tus binds to 21 bp Ter DNA consensus sequences, termed TerA-J, that arrest DNA replication forks. Binding of Ter to Tus greatly increases its thermal stability and resistance to proteolysis, which is advantageous for long incubation times at elevated temperatures (Moreau et al, Mol Biosyst. 6: 1285-92, 2010; Coskun-Ari et al., J. Biol Chem. 269:4027- 34, 1994). We compared the proteolytic profiles of trypsin, NE and C3 on their respective free and fer-bound substrates by SDS-PAGE followed by Coomassie blue staining (Figure 3 A-E). Briefly, each protease was incubated at increasing concentrations with its free or 7¾r-bound substrate at 37 °C for 30 minutes. Half of the reactions were then denatured at 72 °C for 1 minute and centrifuged. For the non-denatured reactions, the upper band (-66 kDa) represents undigested substrate. Cleavage at the interdomain linker generates a ~27 kDa GFP and -37 kDa Tus fragment.

The Tus domain of Tus-G-GFP was highly susceptible to trypsinolysis. This can be seen as a decrease in intensity of the 37 kDa band with increased trypsin concentration (Figure 3 A). The addition of Te B protected the Tus domain from non-specific trypsinolysis, but also from cleavage at the interdomain linker Lys residue (Figure 3B). An explanation for this is that the location of the Lys residue in the interdomain linker is too close to the structured GFP domain to be efficiently cleaved, meaning that hydrolysis occurs at another site in a loosely structured C-terminal region of unbound Tus, such as the C-terminal Arg (Figure 2A). This is supported by the highly structured C-terminal region seen in the Tus- Ter complex (Mulcair et ai, Cell 125: 1309-19, 2006) as well as the high proteolytic susceptibility of unbound Tus (Coskun-Ari et al., J. Biol. Chem.

269:4027-34, 1994). An alternative explanation would be that TerB is preventing trypsin access to the target interdomain Lys (Coskun-Ari et al. , J. Biol Chem. 269:4027-34, 1994). It is important to note that the GFP band intensity is not altered after heat denaturation and centrifugation although all other bands including the substrate were totally removed.

When free Tus-NE-GFP was incubated with NE, proteolysis occurred at the linker region, but also in the Tus domain at concentrations above 1 nM NE (Figure 3C). The addition of TerB protected the Tus domain from proteolysis but NE was still able to access the Tus-NE-GFP linker, shown by the presence and increase in intensity of the 27 and 37 kDa bands when TerB was bound (Figure 3D). The ability to ensure resistance of the Tus-NE-GFP substrate to non-specific proteolysis is important to the assay. The promiscuous activity of proteases such as NE may result in cleavage at numerous sites in the Tus domain. Limiting the cleavage to a single specific site is desirable for applications such as inhibitor screening in drug discovery research. The binding of TerB to the Tus domain allows for the use of TG-PA in such applications.

In contrast, when free Tus-C3-GFP was incubated with C3, proteolysis occurred exclusively at the interdomain linker (Figure 3E). C3 has a very stringent substrate specificity compared to trypsin and NE (Figure 2B; Figure 3A and C). Indeed, based on the specificity of C3 for the P4 - PI sequence of DEVD (Fang et al., J. Mol. Biol. 360:654-66, 2006), these results were expected as Tus does not contain this sequence. The presence of TerB did not have a significant effect on C3 activity, suggesting that the interdomain linker was readily accessible. Furthermore, in the case of Tus-NE-GFP and Tus-C3-GFP, the interdomain linkers are much larger than in Tus-G-GFP and their first scissile bond is located seven residues away from the structured GFP domain (Figure 2A).

The thermal instability and potential for spontaneous aggregation of Tus- GFP proteins (Moreau et al. , Mol. Biosyst. 6: 1285-92, 2010) presents a potential complication with TG-PA. We compared the aggregation susceptibility of free Tus-GFP substrates at 25 and 37 °C to TerB-hound substrates during 30 minutes using GFP-Basta (Moreau et al, Mol. Biosyst. 6:1285-92, 2010). The different free substrates showed comparable stabilities and had aggregated to the extent of approximately 5 - 22% at 25° C, and 36 - 48% at 37 °C under these conditions (Figure 3F). When bound to TerB however, the stability of the substrates were greatly increased and no more than 5% aggregation was observed (Figure 3F). Aggregation could potentially affect the assay as the substrate level is continually changing during the assay period and the possibility exists that the enzyme becomes trapped in the aggregates, resulting in reduced enzyme levels. This is particularly important if longer incubation periods are required. With the exception of Tus-G-GFP, we did not see any significant changes in activity when we compared free or TerB-bourd Tus-GFP substrates, suggesting that the linkers are still fully accessible to the proteases within the aggregates and that aggregation of substrates is not an issue in TG-PA. Ter can be used to ensure specificity and stability during longer incubation times, with the exception of the trypsin substrate for the reasons mentioned herein.

Proteases require specific pH conditions to optimally cleave substrates. Therefore, it is useful to know the stability of the Tus-GFP substrates to pH to define the limits of our assay. The stability of Tus-G-GFP in phosphate buffers ranging from pH 2.1 - 1 1.9 containing either 1 or 10% glycerol was investigated using GFP-Basta (Moreau et al., Mol. Biosyst. 6: 1285-92, 2010) (Figure 3G and H). Glycerol has previously been shown to stabilize Tus by reducing its aggregation rate (Moreau et αΙ., Μοί Biosyst. 6: 1285-92, 2010). In the presence of 1% glycerol, -70 and 20% aggregation had occurred in free and TerS-bound Tus-G-GFP, respectively, after 30 minutes at 37 °C over a large pH range (Figure 3G). Aggregation was reduced to 50 and 6%, respectively, in the presence of 10% glycerol (Figure 3H). As expected, increasing the concentration of glycerol leads to an overall stabilizing effect on Tus-G-GFP. It is interesting to note that the rate of aggregation of Tus-G-GFP is not affected by increasing the pH above 7 (Figure 3G). This is also true for 7¾r5-bound Tus-G-GFP from a slightly more acidic pH value of 6.1, which is probably due to the additional ligand induced stabilization (Figure 3H). A very large destabilizing effect leading to substrate aggregation is observed below these values. At pH 1 1.9 in 10% glycerol, a uniform drop in fluorescence of -10% could be seen both with and without TerB (Figure 3H). This was not observed in 1% glycerol, possibly due to the already high level of aggregation. Consequently, the Tus-GFP protease substrates can be used at a pH ranging from 7 - 1 1, and 6 - 11 when bound to TerB in the presence of 10% glycerol.

All proteolytic studies performed using the three different Tus-GFP substrates demonstrated that protease activity could be monitored by measuring the residual GFP fluorescence after the heat denaturation step (Figure 3). TG-PA was validated in a fluorometric, end-point 96-well format using a fluorescence plate reader. A range of concentrations of Trypsin, HE and C3 were incubated with their 7er#-bound or free substrates (triplicate) at 37 °C for 30 minutes followed by a denaturation step at 72 °C for 1 minute and centrifugation to remove aggregates. The residual GFP fluorescence was measured following dilution of the supernatants in buffer A with a fluorescence plate reader (Victor³ V Wallac 1420, Perkin-Elmer) set at 460 nm excitation and 535 nm emission.

Trypsin, C3 and NE activities were detectable at concentrations as low as 25, 10 and 2 nM respectively after 30 minutes incubation at 37 °C in absence of TerB (Figure 4A-C). Here again, addition of TerB prevented the detection of trypsin activity at concentrations lower than 100 nM (Figure 4A). This result supported our initial data obtained by SDS-PAGE (Figure 3 A and B).

Fluorometric detection of C3 and NE activity with and without TerB (Figure 4B "and C) were comparable, and were in agreement with the SDS-PAGE analyses (Figure 3C-E). The detection limit of the NE assay was improved over ten-fold (200 pM) by extending the incubation time to 4 hours in the presence of stoichiometric amounts of TerB (Figure 4D).

Previously published protease activity assays using luminescence or fluorescent proteins as reporter systems have detection limits ranging from the pM to the nM range (Fan et al. , ACS Chem. Biol. 3 :346-51 , 2008; Aoki et al. , Anal. Biochem. 378:132-37, 2008; Zhang, Biochem. Biophys. Res. Commun. 323:674- 78, 2004; Boeneman et al., J. Am. Chem. Soc. 131 :3828-29, 2009). Generally, such sensitivity has been achieved through incubation of the assay in excess of 1 hour. TG-PA is able to produce sensitive results in the low nM (after 30 minutes of incubation) to mid pM range with incubation up to 4 hours (Figure 4D). The high thermal stability of the Tus-GFP substrates when coupled with TerB indicate that this sensitivity can also be improved further by increasing the incubation beyond 4 hours (e.g., 5 hours, 6 hours, 7 hours, 8 hours,. 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours).

Using TG-PA, we then compared the substrate specificity of NE to C3.

Here we incubated NE with Tus-M13-GFP, and C3 with Tus-NE-GFP, both in the presence of TerB. The NE activity was reduced five- fold (limit of detection 10 nM) when incubated with Tus-M13-GFP, compared to the optimal Tus-NE-GFP substrate (Figure 4E). Cleavage of Tus-M13-GFP still occurred due to the low substrate specificity of NE (Hedstrom, Chem. Rev. 102:4501-23, 2002). NE hydrolyzes preferentially at the C-terminus of small hydrophobic residues such as Ala, Ser, Val and Cys preceded by medium sized hydrophobic residues such as Pro (Blow et al., Biochem. J. 161 : 17-19, 1977; Korkmaz et al., Biochimie 90:227- 42, 2008). The presence of a Ser preceded by a Gly (Figure 2A) in the interdomain linker of the Ml 3 substrate probably resulted in the limited activity observed with Tus-M 13-GFP.

When C3 was incubated with Tus-NE-GFP, no protease activity was detectable after 30 minutes, demonstrating the high substrate specificity of C3 (Figure 4F). As expected, a non-specific linker such as Tus-NE-GFP resulted in markedly less, if any, proteolysis by C3. Indeed, the substrate linker of Tus-NE- GFP does not contain any of the aforementioned characteristics required for hydrolysis by C3 (Fang et al , J. Mol Biol. 360:654-66, 2006).

Overall, our results demonstrated that a fusion protein comprised of a highly stable fluorescent protein and a comparatively unstable protein such as Tus was very useful for the production of a protease substrate providing a practical means for measuring protease activity based on an increase in fluorescence after heat inactivation and centrifugation. In addition, we showed that for NE and C3 our interdomain linkers were optimally designed with regards to length and specificity, and appropriately positioned between the Tus and GFP domains to allow protease access even in the presence of TerB. Previous studies have shown that altering the substrate residues between P5 and P5 ', or substrate length, can significantly affect protease binding (Fang et al. , J. Mol. Biol. 360:654-66, 2006). Our protease assay design addresses both of these issues. The protease linker cloning cassette can be engineered to express substrates containing any desired protease site in excess of 10 residues, ensuring protease recognition is maximised at each position between P5 and P5' by simple subcloning of a synthetic oligonucleotide.

Finally, TG-PA was validated for use in protease inhibitor screening, a major application for protease activity assays. Screening for inhibitors is essential for identifying lead compounds in drug discovery (Zhang, Biochem. Biophys. Res. Commun. 323:674-78, 2004). Inhibition of NE activity by the commercial NE inhibitor MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) was examined to determine the suitability of our assay for inhibitor screening. MSACK irreversibly inhibits NE activity through cross-linking of the NE His57 and Serl95 residues via covalent bonds formed with the inhibitor methylene and ketone carbonyl carbons respectively (Navia et al, Proc. Natl. Acad. Sci. USA 86:7-11, 1989). For this, NE (50 nM) was pre-incubated with increasing concentrations of MSACK for 30 minutes at room temperature, prior to incubation in the presence of TerB-bound Tus-NE-GFP (5 μΜ) for 30 minutes at 37 °C to determine the IC_SQ value (Figure 5). Inhibition of NE was first detectable and increased sharply above ~-50 μΜ MSACK until it reached complete inhibition at -250 μΜ MSACK. An IC₅₀ value of 121.9 ± 2.95 μΜ MSACK was determined using the mathematical software Graphpad Prism 5 (Figure 5). This result was generally in agreement with results obtained from other NE inhibition studies using MSACK where, for example, 77.4% inhibition of NE was achieved with 100 μΜ MSACK (Oltmanns et al, Am. J. Respir. Cell. Mol. Biol. 32:334-41, 2005). Therefore, TG-PA can be used for the screening of inhibitors of protease drug targets.

In conclusion, we have developed a new, sensitive and highly adaptable protease assay using a reporter consisting of a Tus (i.e. , an unstable non- fluorescent protein) and a GFP domain (i.e. , a stable fluorescent protein) separated by a linker containing a protease site. The various fusion constructs are ideal substrates for use as protease activity reporters. Using Tus-NE-GFP and Tus-C3- GFP we were able to detect protease activity in the low nM range after 30 minutes incubation, and sensitivity could be improved to the pM range by increasing the incubation time in the presence of the stabilising TerB.

TG-PA is also well suited to the screening of protease inhibitors, particularly implemented in a high-throughput format as a single shot comparative inhibitor screening system to analyse compound efficacy and potency. Indeed, the denaturation step at 72 °C, corresponding to quenching, is streamlined with the incubation step using a thermal cycler, meaning that there is no handling nor pipetting needed for quenching. The centrifugation step could optionally be avoided by introducing a filtration step under vacuum using adequate commercial multi-well membrane-bottom filter plates.

The spectrum of applicability of the assay also ex tends to protease-related disease diagnostics. The sensitivity of the assay has the ability to detect protease activity in serum. The concentrations of proteases used in this study are known to fluctuate significantly from normal levels during disease pathogenesis (Donnelly et al., Am. J. Respir. Crit. Care Med. 151 :1428-33, 1995; Artigas et al.. Postgrad. Med J. 57:219-22, 1981; Louneva et al.. Am. J. Pathol. 173:1488-95, 2008). Analysis of protease activity kinetics is also possible using the Tus-GFP reporter. Because the basis of the assay relies on the separation of two small domains from their 66 kDa fusion product, techniques such as fluorescence polarization can be used to monitor this separation as a real-time indication of proteolysis. Example 2 - Protease Detection by TG-PA Using Fluorescent Band Integration Following SDS-PAGE

The TG-PA assay is a rapid and sensitive protease detection method amenable to both highly specific and generic proteases for the purposes of activity and mutant screening as well as drug discovery and library compound profiling. Here, TG-PA has been adapted to an SDS-PAGE gel-based format (gel-TG-PA), where the activity of specific proteases can be detected in complex media such as human serum. For validation of gel-TG-PA, human male serum (10%) was spiked with human recombinant caspase 3 (C3) at increasing concentrations (0.5 nM-1 μΜ) and incubated with Tus-C3-GFP (5 μΜ), which contains the duplicated C3 substrate linker DEVDG between the Tus and GFP domains. Reactions were incubated for either 1 hour or 4 hours at 37 °C, migrated on a 15% SDS-PAGE gel, and photographed under UV exposure. Fluorescent bands were inetgrated using ImageJ software.

Fluorescent bands corresponding to the molecular weights of both GFP and Tus-GFP were visible under UV exposure after 1 hour incubation (Figure 6A). Digestion of the substrate resulted in the highly specific liberation of the GFP domain, which appeared as a double-band at approximately 27 kDa, which reflected digestion at both of the C3 proteolysis sites in the substrate linker of

Tus-C3-GFP. Protease digestion was detectable as low as 3.3 nM after 1 hour by band integration (Figure 6B), reaching 100% digestion by 333.3 nM. At 1 μΜ, only a^"single band was visible, which was likely due to complete digestion of both substrate linker C3 sites due to the high protease concentration.

When incubated for 4 hours at 37 °C, the same double-band of GFP. was present in the linear concentration range of C3 (Figure 6C), which also again returned to a single band at the maximum concentration. The sensitivity of the assay was improved approximately three-fold to 1 nM with the extended incubation, and complete digestion of the substrate linker was determined to be 100 nM (Figure 6D).

During incubation for both time periods, no non-specific digestion of the Tus domain of the Tus-C3-GFP substrate was detected by promiscuous proteases such as trypsin and neutrophil elastase. The specificity of the assay for C3 provides an accurate and quantifiable adaptation of TG-PA in complex media, and is adaptable for other proteases having stringent substrate specificity. The ability to utilise gel-TG-PA in 10% human serum demonstrates utility for application of the assay in detecting changes in activity or increases in secretion/expression of proteases in disease diagnostics with rapid turnaround and simple analysis.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Claims

1. A method for quantifying protease activity, the method including the steps of:

a) combining a fusion protein and a protease to form a mixture, said fusion protein comprising a stable fluorescent protein, a linker and an unstable non-fluorescent protein, wherein said linker comprises a protease recognition sequence recognised by said protease;

b) exposing said mixture to a denaturant;

c) separating said mixture into soluble and insoluble fractions, wherein said soluble fraction comprises soluble fluorescent protein and said insoluble fraction comprises aggregates of said fusion protein; and

d) measuring fluorescence of said fluorescent protein in said soluble fraction as an indicator of protease activity.

2. The method of claim 1 , further comprising the step of producing and/or purifying said fusion protein prior to combining said fusion protein and said protease.

3. The method of claim 2, wherein the fusion protein is produced by:

expressing said fusion protein in an expression system, wherein said expression system comprises a nucleic acid molecule encoding said fusion protein and a promoter active in said expression system operably linked to said nucleic acid molecule; and

extracting a protein sample from said expression system, wherein said protein sample comprises said fusion protein.

4. ^" The method of claim 3, wherein said expression system comprises an expression construct, wherein said nucleic acid molecule is operably linked to one or more regulatory sequences in said expression construct and said promoter is active in a host cell, and said fusion protein is expressed in said host cell.

5. The method of claim 3 , wherein said expression system comprises an in vitro transcription/translation system.

6. The method of claim 2, wherein producing said fusion protein comprises joining said stable fluorescent protein via said linker to said unstable non- fluorescent protein.

7. The method of any one of claims 1 -6, wherein said stable fluorescent protein is C-terminal to said unstable non-fluorescent protein.

8. The method of any one of claims 1 -6, wherein said stable fluorescent protein is N-terminal to said unstable non-fluorescent protein.

9. The method of any one of claims 1 -8, wherein said linker comprises 5 to 50 amino acids.

10. The method of any one of claims 1 -9, wherein said protease is selected from the group consisting of neutrophil elastase, caspase 3, caspase 6, matrix metalloproteinase 9, and matrix metalloproteinase 13.

1 1. The method of any one of claims 1-10, wherein combining said fusion protein and said protease occurs in a well of a microtiter plate.

12. The method of any one of claims 1 -1 1 , wherein exposing said mixture to a denaturant comprises heating said mixture to a pre-determined temperature.

13. The method of any one of claims 1-12, wherein separating said mixture into soluble and insoluble fractions comprises centrifugation of said mixture following exposure to said denaturant.

14. The method of any one of claims 1-12, wherein separating said mixture into soluble and insoluble fractions comprises spotting an aliquot of said mixture onto a selectively permeable matrix following exposure to said denaturant.

15. The method of claim 14, wherein said selectively permeable matrix comprises , an agarose gel or a polyacrylamide gel.

16. The method of any one of claims 1-12, wherein separating said mixture into soluble and insoluble fractions comprises filtration of said mixture following exposure to said denaturant.

17. A method for quantifying protease activity, the method including the steps of:

a) combining a fusion protein and a protease to form a mixture, said fusion protein comprising a stable fluorescent protein, a linker and an unstable non-fluorescent protein, wherein said linker comprises a protease recognition sequence recognised by said protease; b) separating said mixture by gel electrophoresis; and

c) measuring fluorescence of said fluorescent protein as an indicator of protease activity.

18. A method for quantifying protease activity, the method including the steps of:

a) expressing a fusion protein in an expression system, wherein said expression system comprises

(i) a nucleic acid molecule encoding said fusion protein, said fusion protein comprising a stable fluorescent protein, a linker and an unstable non-fluorescent protein, wherein said linker comprises a protease recognition sequence recognised by said protease, and

(ii) a promoter active in said expression system operably linked to said nucleic acid molecule;

b) extracting a protein sample from said expression system, wherein said protein sample comprises said fusion protein;

c) combining said protein sample and a protease to form a mixture; d) exposing said mixture to a denaturant;

e) separating said mixture into soluble and insoluble fractions, wherein said soluble fraction comprises soluble fluorescent protein and said insoluble fraction comprises aggregates of said fusion protein; and

f) measuring fluorescence of said fluorescent protein in said soluble fraction as an indicator of protease activity.

19. The me thod of any one of claims 1 - 18, further comprising screening potential inhibitors of said protease, wherein combining said fusion protein and said protease to form a mixture indludes combining said fusion protein and a potential inhibitor of said protease.

20. An isolated nucleic acid molecule, comprising a polynucleotide encoding a stable fluorescent protein in-frame with a polynucleotide encoding an unstable non- , fluorescent protein, and an internal cloning site between said stable fluorescent protein and said unstable non-fluorescent protein coding sequences into which a heterologous polynucleotide encoding a protease recognition sequence can be inserted in-frame with said stable fluorescent protein and said unstable non- fluorescent protein coding sequences.

21. An isolated fusion protein, comprising an amino acid sequence of a stable fluorescent protein, a peptide linker amino acid sequence and an amino acid sequence of an unstable non-fluorescent protein, wherein said linker comprises a protease recognition sequence.

22. The fusion protein of claim 21 , wherein said protease recognition sequence is recognised by neutrophil elastase, caspase 3, caspase 6, matrix metalloproteinase 9, or matrix metalloproteinase 13.

23. The fusion protein of claim 21 , wherein said stable fluorescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, red fluorescent protein, and orange fluorescent protein.

24. The fusion protein of claim 23, wherein said fluorescent marker protein is green fluorescent protein.

25. The fusion protein of any one of claims 21-24, for use according to the method of any one of claims 1 -6.

26. A method for quantifying protease activity in a biological sample, the method including the steps of:

a) isolating said biological sample from a subject;

b) combining said isolated fusion protein of claim 21 with said biological sample to form a mixture; —

c) exposing said mixture to a denaturant;

d) separating said mixture into soluble and insoluble fractions, wherein said soluble fraction comprises soluble fluorescent protein and said insoluble fraction comprises aggregates of said fusion protein; and

e) measuring fluorescence of said fluorescent protein in said soluble fraction as an indicator of protease activity in said biological sample.

27. The method of claim 26, wherein said method for quantifying protease activity in a biological sample comprises a method for diagnosing a protease-related disease in said subject.

28. A kit for .quantifying protease activity, said kit comprising an expression vector comprising a polynucleotide encoding a stable fluorescent protein in-frame with a polynucleotide encoding an unstable non-fluorescent protein, and an internal cloning site between said stable fluorescent protein and said unstable non-fluorescent protein coding sequences into which a heterologous polynucleotide encoding a protease recognition sequence can be inserted in-frame with said stable fluorescent protein and said unstable non-fluorescent protein coding sequences.

29. A kit for quantifying protease activity, said kit comprising one or more oligonucleotide primer pairs for introducing a promoter, a ribosomal binding site, and a linker for generating a fusion gene comprising a gene coding for a stable fluorescent protein joined in-frame with a gene coding for an unstable non- fluorescent protein, wherein said linker comprises a protease recognition sequence.

30. A kit for quantifying protease activity, said kit comprising the fusion protein of any one of claims 21 -24. for use according to the method of any one of claims 1 -6.