System for detectng protease

Abstract

Disclosed is a system for detecting a protease inside a cell. In one embodiment, the system includes a chimeric protein that comprises as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. The invention has a wide spectrum of applications including use in the detection of novel protease inhibitors inside cells and tissue.

1 . A chimeric protein comprising at least one signal protein that has a trafficking signal targeting to a subcellular organelle and at least one proteolytic cleavage site for a protease, which is constructed such that
 (a) the trafficking signals of all the signal proteins are inactivated by linking the proteolytic site or a signal masking protein through the proteolytic site to the N- or C-terminus of the signal proteins, and thus the chimeric protein is present in cytosol;   (b) the trafficking signal of at least one signal protein is activated when the proteolytic cleavage site is cleaved by the protease, and as a result at least one fragment protein that includes the activated signal protein is transported to a subcellular organelle; and   (c) the chimeric protein is labeled with at least one fluorescent protein and the position and intensity distribution of the fluorescent label signal in the cell is altered depending on the cleavage by the protease.   
     
     
         2 . A chimeric protein comprising at least two signal proteins that have trafficking signals targeting to subcellular organelles and at least one proteolytic cleavage site for a protease, which is constructed such that
 (a) the trafficking signal of one signal protein remains active, and those of the rest of the signal proteins are inactivated by linking the proteolytic site or a signal masking protein through the proteolytic site to the N- or C-terminus of the signal proteins, and thus the chimeric protein is transported to a specific subcellular organelle targeted by the trafficking signal of the active signal protein;   (b) at least one proteolytic site and at least one inactivated signal protein are exposed to cytosol after the chimeric protein is transported to the subcellular organelle;   (c) the trafficking signal of the at least one inactivated signal protein exposed to cytosol is activated when the proteolytic cleavage site is cleaved by the protease, and as a result the fragment protein that includes the activated signal protein is transported to a subcellular organelle that is different from the subcellular organelle to which the chimeric protein was transported; and   (d) the chimeric protein is labeled with at least one fluorescent protein and the position and intensity distribution of the fluorescent signal in the cell is altered depending on the cleavage by the protease.   
     
     
         3 . The chimeric protein according to any of  claims 1  and  2 , wherein
 among the fragment proteins produced by the proteolytic cleavage, at least two fragment proteins with different cellular localization characteristics includes a fluorescent protein in each and the fluorescent proteins used consist of at least two fluorescent proteins with different wavelengths. 
 
     
     
         4 . The chimeric protein according to any of  claims 1  and  2 , wherein
 among the fragment proteins including a signal protein whose inactivated trafficking signal is activated by the proteolytic cleavage, at least one fragment protein includes a fluorescent protein. 
 
     
     
         5 . A chimeric protein comprising a signal protein that has a trafficking signal targeting to a subcellular organelle and a fluorescent protein linked to the signal protein through a proteolytic cleavage site of a protease, which is constructed such that,
 (a) the trafficking signal of the signal protein remains active in the chimeric protein and thus the chimeric protein is transported to the subcellular organelle;   (b) the proteolytic cleavage site and the fluorescent protein are exposed to cytosol after the chimeric protein is transported to the subcellular organelle; and   (c) therefore, the fluorescent protein exposed to cytosol is released to cytosol when the proteolytic cleavage site exposed to cytosol is cleaved by the protease.   
     
     
         6 . The chimeric protein of  claim 5 , wherein the signal protein remaining active is further labeled with a fluorescent protein that has a fluorescence wavelength different from that of the fluorescent protein exposed to cytosol, and this additional fluorescence protein remains attached to the active signal protein after the proteolytic cleavage occurs. 
     
     
         7 . The chimeric protein according to any of  claims 1  and  2 , wherein the trafficking signal of the inactivated signal protein is a signal targeting to a subcellular organelle selected from the group consisting of mitochondria, chloroplast, and peroxisome. 
     
     
         8 . The chimeric protein according to any of  claims 1  and  2 , wherein the signal protein that is inactivated is a full length protein selected from the group consisting of  Arabidopsis  outer envelope membrane protein 7 (AtOEP7), Rubisco small subunit (RbcS), Chlorophyll a/b binding protein (Cab), Rubisco activase (RA), F1-ATPase, and Peroxisome-targeting motif (SKL), or a portion thereof that includes the trafficking signal. 
     
     
         9 . The chimeric protein according to any of  claims 2  and  5 , wherein the trafficking signal of the signal protein remaining active is a signal targeting to one selected from the group consisting of outer membranes of mitochondria, chloroplast, and nucleus, peroxisome membrane, and plasma membrane. 
     
     
         10 . The chimeric protein according to any of  claims 2  and  5 , wherein the signal protein remaining active is a protein that binds specifically to a specific phospholipid. 
     
     
         11 . The chimeric protein according to any of  claims 2  and  5 , wherein the signal protein remaining active is a full length protein selected from the group consisting of  Arabidopsis  outer envelope membrane protein 7 (AtOEP7), H + -ATPase, Pleckstrin homology domain (PH), and pleckstrin homology domain of FAPP (family A (phosphoinositide binding specific) member 3), or a portion thereof that includes the trafficking signal. 
     
     
         12 . The chimeric protein according to any of  claims 1  and  2 , wherein the signal masking protein is selected from the group consisting of amino acids, peptides, and proteins. 
     
     
         13 . The chimeric protein according to any of  claims 1 ,  2 , and  5 , wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), mutants thereof, and derivatives thereof. 
     
     
         14 . A recombinant gene comprising a nucleic acid sequence encoding the chimeric protein of any of  claims 1 ,  2 , and  5 , which is constructed to express the chimeric protein in a cell. 
     
     
         15 . A cell transformed with the recombinant gene of  claim 14 . 
     
     
         16 . The cell of  claim 15 , wherein the cell is a eukaryotic cell. 
     
     
         17 . The cell of  claim 16 , wherein the eukaryotic cell is a plant cell. 
     
     
         18 . A method for analyzing the activity of a protease in vivo, comprising
 (a) transforming a cell with the recombinant gene of  claim 14 ;   (b) transforming the cell to express the protease before, after, or at the same time as step (a);   (c) incubating the transformed cell to express the proteins;   (d) observing an image of the distribution of the fluorescence signal in the incubated cell; and   (e) determining the activity of the protease by comparing the fluorescence image observed in step (d) with that of a control transformed cell prepared without step (b).   
     
     
         19 . A method for screening protease inhibitors in vivo, comprising
 (a) transforming a cell with the recombinant gene of  claim 14 ;   (b) transforming the cell to express the protease before, after, or at the same time as step (a);   (c) treating the transformed cell with a drug candidate for inhibiting the protease before, after, or at the same time as step (b);   (d) incubating the transformed cell to express the proteins;   (e) observing an image of the distribution of the fluorescent signal in the incubated cell; and   (f) determining the protease inhibition activity of the drug candidate by comparing the fluorescence image observed in step (e) with at least one of the fluorescent images of a control transformed cell prepared without step (b), a control transformed cell prepared without step (c), and a control transformed cell prepared without steps (b) and (c).   
     
     
         20 . The method of any of  claims 18  and  19 , wherein the step of transforming the cell to express the protease is a step of inserting a recombinant gene for expression of the protease, or a step of infecting with a virus that can express the protease. 
     
     
         21 . The method of any of  claims 18  and  19 , wherein the cell to be transformed is a eukaryotic cell. 
     
     
         22 . The method of any of  claims 18  and  19 , wherein the cell to be transformed is a plant cell. 
     
     
         23 . A system for detecting a protease inside a cell, wherein the system comprises a chimeric protein comprising as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. 
     
     
         24 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) the signal protein; 2) the protease-specific cleavage site; and 3) the detectable amino acid sequence. 
     
     
         25 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) a masking sequence; 2) the protease cleavage site; 3) the signal protein; and 4) the detectable amino acid sequence. 
     
     
         26 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) the signal protein; 2) the protease cleavage site; 3) the masking sequence; and 4) the detectable amino acid sequence. 
     
     
         27 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) a first signal protein; 2) the protease cleavage site; and 3) a second signal protein; and 4) the detectable amino acid sequence. 
     
     
         28 . The system of  claim 27 , wherein the chimeric protein comprises covalently linked in sequence: 1) the first signal protein; 2) a first protease cleavage site; 3) the masking sequence; 4) the second signal protein; and 5) the detectable amino acid sequence. 
     
     
         29 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) the masking sequence; 2) a first protease cleavage site; 3) a first signal protein; 4) a second protease cleavage site; 5) a second signal protein; and 6) the detectable amino acid sequence. 
     
     
         30 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) a first signal protein; 2) a first protease cleavage site; 3) a second signal protein; 4) a second protease cleavage site; 5) a masking sequence; and 6) the detectable amino acid sequence. 
     
     
         31 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) the protease-specific cleavage site; 2) the signal protein; and 3) the detectable amino acid sequence. 
     
     
         32 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) a first signal protein; 2) a first detectable sequence; 3) the protease cleavage site; and 4) a second detectable sequence. 
     
     
         33 . The system of  claim 32 , wherein the chimeric protein further comprises a second signal protein covalently linked between the C-terminus of the protease cleavage site and the N-terminus of the second detectable sequence. 
     
     
         34 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) a first signal protein; 2) the protease cleavage site; 3) a second signal protein; and 4) a second detectable sequence. 
     
     
         35 . The system of  claim 23 , wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable sequence; 2) the protease cleavage site; 3) the signal protein; and 4) a second detectable sequence. 
     
     
         36 . The system of  claim 23 , wherein any one of the components comprises the N-terminus of the chimeric protein. 
     
     
         37 . The system of  claim 23 , wherein any one of the components comprises the C-terminus of the chimeric protein. 
     
     
         38 . The system of  claim 23 , wherein the signal protein is sufficient to localize the chimeric protein or at least one of its components to a plant or animal cell organelle. 
     
     
         39 . The system of  claim 38 , wherein the signal protein localizes the chimeric protein or at least one of its components to the nucleus, golgi body, lytic vacuole, storage vacuole, peroxisome, mitochondrion, endoplasmic reticulum, plasma membrane, or chloroplast of a plant cell. 
     
     
         40 . The system of  claim 39 , wherein the signal protein is one of AtOEP7; RbcS; Cab; RA; SKL; F1-ATPase; PH; FAPP; H + -ATPase; or a functional fragment thereof. 
     
     
         41 . The system of  claim 38 , wherein the signal protein localizes the chimeric protein to the nucleus, golgi body, storage vacuole, lysosome, peroxisome, endoplasmic reticulum, plasma membrane, or mitochondrion of an animal cell. 
     
     
         42 . The system of  claim 41 , wherein the signal protein is one of human peptide methionine sulfoxide reductase (MSRA), cytochrome b2,11-beta-hydroxysteroid dehydrogenase (11β-HSD), G9-AKL, peroxisomal integral membrane protein 47 (PMP47); or a functional fragment thereof. 
     
     
         43 . The system of  claim 23 , wherein the cleavage site is specifically cleaved by a mammalian or viral protease. 
     
     
         44 . The system of  claim 43 , wherein cleavage site is specifically cleaved by a protease associated with a human pathogen. 
     
     
         45 . The system of  claim 44 , wherein the protease is expressed by a cytomegalovirus (CMV); herpes simplex virus (HSV); hepatitis virus; a  plasmodium , human immunodeficiency virus (HIV), Kaposi's sarcoma-associated herpes virus (KSHV), yellow fever virus, flavivirus, or rhinovirus. 
     
     
         46 . The system of  claim 43 , wherein the protease is a serine-type protease. 
     
     
         47 . The system of  claim 45 , wherein the  plasmodium  is  P. falciparum  and the protease is one of plasmepsin I and plasmepsin II. 
     
     
         48 . The system of  claim 45 , wherein cleavage site is specifically cleaved by a maturational protease of HSV. 
     
     
         49 . The system of  claim 45 , wherein the hepatitis virus is type C. 
     
     
         50 . The system of  claim 44 , wherein the human pathogen is yeast, bacterium, fungi, nematode, virus, or protozoa. 
     
     
         51 . The system of  claim 43 , wherein the cleavage site is specifically cleaved by a mammalian protease associated with blood coagulation, apoptosis, or the extracellular matrix. 
     
     
         52 . The system of  claim 23 , wherein at least one of the detectable sequences is a fluorescent, phosphorescent, or chemiluminescent sequence. 
     
     
         53 . The system of  claim 52 , wherein the emission wavelength of one of the detectable sequences is different from at least one other of the detectable sequences. 
     
     
         54 . The system of  claim 52 , wherein the detectable sequence is a jellyfish fluorescent protein or a derivative thereof. 
     
     
         55 . A substantially pure chimeric protein comprising as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. 
     
     
         56 . A nucleic acid comprising sequence that encodes a chimeric protein for detecting protease activity in a cell, wherein the chimeric protein comprises as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. 
     
     
         57 . A vector comprising the nucleic acid of  claim 56 . 
     
     
         58 . A cell transformed with the vector of  claim 57 . 
     
     
         59 . A kit for detecting a protease inside a cell, the kit comprising at least one of:
 a) a chimeric protein comprising as covalently linked components: i) at least one optionally masked signal protein; ii) at least one protease-specific cleavage site; and iii) at least one detectable amino acid sequence; and b) a vector comprising a nucleic acid comprising sequence encoding the chimeric protein.   
     
     
         60 . A method for detecting a protease inside a cell or tissue, the method comprising:
 a) introducing, into a subject cell or tissue, a first vector comprising nucleic acid encoding a chimeric protein comprising as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence,   b) incubating the cell or tissue under conditions conducive to expressing the chimeric protein encoded by the first vector; and   c) detecting a change in the subcellular localization of the chimeric protein as being indicative of the presence of the protease inside the cell.   
     
     
         61 . The method of  claim 60 , wherein the method further comprises introducing, into the subject cell or tissue, a second vector comprising nucleic acid sequence encoding the protease; and expressing the second vector in the cell or tissue to produce the protease therein. 
     
     
         62 . A method for detecting a protease inhibitor in vivo, the method comprising:
 a) introducing, into a subject cell or tissue, a first vector comprising nucleic acid encoding a chimeric protein comprising as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence,   b) introducing into the cell or tissue a second vector encoding a subject protease,   c) contacting the cell or tissue with candidate compound,   d) incubating the cell or tissue under conditions conducive to expressing the chimeric protein encoded by the first vector and the protease encoded by the second vector; and   e) detecting a change in the subcellular localization of the chimeric protein as being indicative of the presence of the protease inhibitor.   
     
     
         63 . The method of  claim 62 , wherein the method further comprises use of an automated or semi-automated device for detecting the change in subcellular localization of the chimeric protein. 
     
     
         64 . The method of  claim 63 , wherein the automated or semi-automated device comprises an optical system adapted to detect the detectable sequence inside the cell or tissue.