Claims for Patent: 7,855,072

Summary for Patent: 7,855,072

Title:	System for detecting 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.
Inventor(s):	Hwang; Inhwan (Seoul, KR), Kim; Dae Heon (Seoul, KR), Lee; Yong Jik (Seoul, KR)
Assignee:	Ahram Biosystems Inc. (Seoul, KR)
Application Number:	11/506,143
Patent Claims:	1. An isolated nucleic acid comprising sequence encoding a chimeric protein, the protein comprising at least one signal protein that has a trafficking signal targeting to a subcellular organelle or plasma membrane 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 and at least one detectable amino acid sequence is transported to the subcellular organelle or plasma membrane; and c) the chimeric protein is labeled with the at least one detectable amino acid sequence and the position and intensity distribution of the detectable amino acid sequence signal in the cell is altered depending on the cleavage by the protease, provided that when the detectable amino acid sequence is targeted to the plasma membrane, it remains on the plasma membrane or is translocated from the plasma membrane to the cytosol. 2. An isolated nucleic acid comprising sequence encoding a chimeric protein, the protein comprising at least two signal proteins that have trafficking signals targeting to subcellular organelles or plasma membrane 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 or plasma membrane 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 or plasma membrane; (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 or plasma membrane that is different from the subcellular organelle or plasma membrane to which the chimeric protein was transported; and (d) the chimeric protein is labeled with at least one detectable amino acid sequence and the position and intensity distribution of the detectable amino acid sequence in the cell is altered depending on the cleavage by the protease. 3. The isolated nucleic acid according to claim 1 or 2, wherein among the fragment proteins produced by the proteolytic cleavage, at least two fragment proteins with different cellular localization characteristics includes different detectable amino acid sequences. 4. The isolated nucleic acid according to claim 1 or 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 the detectable amino acid sequence. 5. The isolated nucleic acid according to claim 1 or 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. 6. The isolated nucleic acid according to claim 1 or 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. 7. The isolated nucleic acid according to claim 2, 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. 8. The isolated nucleic acid according to claim 2, wherein the signal protein remaining active is a protein that binds specifically to a specific phospholipid. 9. The isolated nucleic acid according to claim 2, 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.sup.+-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. 10. The isolated nucleic acid according to claim 1 or 2, wherein the signal masking protein is selected from the group consisting of amino acids, peptides, and proteins. 11. The isolated nucleic acid according to claim 1 or 2 wherein the detectable amino acid sequence is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), mutants thereof, and derivatives thereof. 12. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the masked or unmasked signal protein comprises a trafficking signal targeting to a subcellular organelle or plasma membrane, provided that when the detectable amino acid sequence is targeted to the plasma membrane, it remains on the plasma membrane or is translocated from the plasma membrane to the cytosol. 13. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable amino acid sequence; 2) the masked signal protein; 3) the protease-specific cleavage site; and 4) a second detectable amino acid sequence. 14. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a masking sequence; 2) the protease cleavage site; 3) the masked signal protein; and 4) the detectable amino acid sequence. 15. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable amino acid sequence; 2) the masked signal protein; 3) the protease cleavage site; 4) a masking sequence; and 5) a second detectable amino acid sequence. 16. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) the unmasked signal protein; 2) the protease cleavage site; and 3) the masked signal protein; and 4) the detectable amino acid sequence. 17. The isolated nucleic acid of claim 16, wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable amino acid sequence; 2) a first masked signal protein; 3) a first protease cleavage site; 4) a masking sequence; 5) a second masked signal protein; and 6) a second detectable amino acid sequence. 18. The isolated nucleic acid of claim 16, wherein the chimeric protein comprises covalently linked in sequence: 1) a masking sequence; 2) a first protease cleavage site; 3) a first masked signal protein; 4) a second protease cleavage site; 5) a second masked signal protein; and 6) the detectable amino acid sequence. 19. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) the unmasked signal protein; 2) a first protease cleavage site; 3) the masked signal protein; 4) a second protease cleavage site; 5) a masking sequence; and 6) the detectable amino acid sequence. 20. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) the protease-specific cleavage site; 2) the masked signal protein; and 3) the detectable amino acid sequence. 21. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a first masked signal protein; 2) a first detectable sequence; 3) the protease cleavage site; and 4) a second detectable sequence. 22. The isolated nucleic acid of claim 21, 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. 23. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable sequence; 2) the protease cleavage site; 3) the masked signal protein; and 4) a second detectable sequence. 24. The isolated nucleic acid of claim 12, wherein any one of the components comprises the N-terminus of the chimeric protein. 25. The isolated nucleic acid of claim 12, wherein any one of the components comprises the C-terminus of the chimeric protein. 26. The isolated nucleic acid of claim 12, wherein the masked or unmasked signal protein is sufficient to localize the chimeric protein or at least one of its components to a plant, human or animal cell organelle or plasma membrane. 27. The isolated nucleic acid of claim 26, wherein the masked or unmasked 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. 28. The isolated nucleic acid of claim 27, wherein the masked or unmasked signal protein is one of AtOEP7; RbcS; Cab; RA; SKL; F1-ATPase; PH; FAPP; H.sup.+-ATPase; or a functional fragment thereof. 29. The isolated nucleic acid of claim 26, wherein the masked or unmasked signal protein localizes the chimeric protein to the nucleus, golgi body, storage vacuole, lysosome, peroxisome, endoplasmic reticulum, plasma membrane, or mitochrondrion of a human cell or an animal cell. 30. The isolated nucleic acid of claim 29, wherein the masked or unmasked signal protein is one of human peptide methionine sulfoxide reductase (MSRA), cytochrome b2, 11-beta-hydroxysteroid dehydrogenase (11.theta.-HSD), G9-AKL, peroxisomal integral membrane protein 47 (PMP47); or a functional fragment thereof. 31. The isolated nucleic acid of claim 12, wherein the cleavage site is specifically cleaved by a mammalian or viral protease. 32. The isolated nucleic acid of claim 31, wherein cleavage site is specifically cleaved by a protease associated with a human pathogen. 33. The isolated nucleic acid of claim 32, 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. 34. The isolated nucleic acid of claim 31, wherein the protease is a serine-type protease. 35. The isolated nucleic acid of claim 33, wherein the plasmodium is P. falciparum and the protease is one of plasmepsin I and plasmepsin II. 36. The isolated nucleic acid of claim 33, wherein cleavage site is specifically cleaved by a maturational protease of HSV. 37. The isolated nucleic acid of claim 33, wherein the hepatitis virus is type C. 38. The isolated nucleic acid of claim 32, wherein the human pathogen is yeast, bacterium, fungi, nematode, virus, or protozoa. 39. The isolated nucleic acid of claim 31, wherein the cleavage site is specifically cleaved by a mammalian protease associated with blood coagulation, apoptosis, or the extracellular matrix. 40. The isolated nucleic acid of claim 12, wherein at least one of the detectable sequences is directly or indirectly fluorescent, phosphorescent, or chemiluminescent. 41. The isolated nucleic acid of claim 40, wherein the mission wavelength of one of the detectable sequences is different from at least one other of the detectable sequences. 42. The isolated nucleic acid of claim 10, wherein the detectable sequence is a jellyfish fluorescent protein or a derivative thereof. 43. A nucleic acid encoding a chimeric protein for detecting protease activity in a cell, wherein the chimeric protein comprises as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the masked or unmasked signal protein comprises a trafficking signal targeting to a subcellular organelle or plasma membrane, provided that when the detectable amino acid sequence is targeted to the plasma membrane, it remains on the plasma membrane or is translocated from the plasma membrane to the cytosol. 44. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a masking sequence, 2) the protease-specific cleavage site, 3) a mitochondrial targeting sequence as the masked signal protein, and 4) the detectable amino acid sequence. 45. The isolated nucleic acid of claim 44, wherein the mitochondrial targeting sequence is human peptide methionine sulfoxide reductase (MSRA) or a functional fragment thereof. 46. The isolated nucleic acid of claim 44, wherein the masking sequence is one of a signal protein, a fluorescent protein; or a functional fragment thereof. 47. The isolated nucleic acid of claim 12, wherein the chimeric protein comprises covalently linked in sequence: 1) a Pleckstrin homology domain (PH) as the unmasked signal protein, 2) the protease-specific cleavage site, 3) a mitochondrial targeting sequence as the masked signal protein, and 4) the detectable amino acid sequence. 48. The isolated nucleic acid of claim 47, wherein the mitochondrial targeting sequence is human peptide methionine sulfoxide reductase (MSRA) or a functional fragment thereof. 49. The isolated nucleic acid of claim 12 or 43, wherein the nucleic acid further encodes at least one peptide linker sequence. 50. A vector comprising any one of the isolated nucleic acids of claim 1, 2, 12, or 43. 51. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable amino acid sequence; 2) the masked signal protein; 3) the protease cleavage site; 4) a masking sequence; and 5) a second detectable amino acid sequence. 52. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the chimeric protein comprises covalently linked in sequence: 1) the unmasked signal protein; 2) the protease cleavage site; and 3) the masked signal protein; and 4) the detectable amino acid sequence, wherein the masked or unmasked signal protein comprises a trafficking signal targeting to a subcellular organelle or plasma membrane, provided that when the detectable amino acid sequence is targeted to the plasma membrane, it remains on the plasma membrane or is translocated from the plasma membrane to the cytosol. 53. The isolated nucleic acid of claim 52, wherein the chimeric protein comprises covalently linked in sequence: 1) a first detectable amino acid sequence; 2) a first masked signal protein; 3) a first protease cleavage site; 4) a masking sequence; 5) a second masked signal protein; and 6) a second detectable amino acid sequence. 54. The isolated nucleic acid of claim 52, wherein the chimeric protein comprises covalently linked in sequence: 1) a masking sequence; 2) a first protease cleavage site; 3) a first masked signal protein; 4) a second protease cleavage site; 5) a second masked signal protein; and 6) the detectable amino acid sequence. 55. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the chimeric protein comprises covalently linked in sequence: 1) the unmasked signal protein; 2) a first protease cleavage site; 3) the masked signal protein; 4) a second protease cleavage site; 5) a masking sequence; and 6) the detectable amino acid sequence. 56. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the masked or unmasked signal protein is sufficient to localize the chimeric protein or at least one detectable amino acid sequence thereof to a subcellular organelle or plasma membrane of a plant, human or animal cell, provided that when the detectable amino acid sequence is localized to the plasma membrane, it remains on the plasma membrane or is translocated from the plasma membrane to the cytosol. 57. The isolated nucleic acid of claim 56, wherein the masked or unmasked 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. 58. The isolated nucleic acid of claim 57, wherein the masked or unmasked signal protein is one of AtOEP7; RbcS; Cab; RA; SKL; F1-ATPase; PH; FAPP; H.sup.+-ATPase; or a functional fragment thereof. 59. The isolated nucleic acid of claim 56, wherein the masked or unmasked signal protein localizes the chimeric protein or at least one of the detectable amino acid sequence thereof to the nucleus, golgi body, storage vacuole, lysosome, peroxisome, endoplasmic reticulum, plasma membrane, or mitochrondrion of a human cell or an animal cell. 60. The isolated nucleic acid of claim 59, wherein the masked or unmasked signal protein is one of human peptide methionine sulfoxide reductase (MSRA), cytochrome b2, 11-beta-hydroxysteroid dehydrogenase (11.theta.-HSD), G9-AKL, peroxisomal integral membrane protein 47 (PMP47); or a functional fragment thereof. 61. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the cleavage site is specifically cleaved by a protease selected from plasmepsin I and plasmepsin II. 62. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the cleavage site is specifically cleaved by a maturational protease of HSV. 63. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein at least one of the detectable sequences is directly or indirectly fluorescent, phosphorescent, or chemiluminescent, wherein the masked or unmasked signal protein comprises a trafficking signal targeting to a subcellular organelle or plasma membrane, provided that when the detectable amino acid sequence is targeted to the plasma membrane, it remains on the plasma membrane or is translocated from the plasma membrane to the cytosol. 64. The isolated nucleic acid of claim 63, wherein the emission wavelength of one of the detectable sequences is different from at least one other of the detectable sequences. 65. The isolated nucleic acid of claim 63, wherein the detectable sequence is a jellyfish fluorescent protein or a derivative thereof. 66. The isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the chimeric protein comprises covalently linked in sequence: 1) a masking sequence, 2) the protease-specific cleavage site, 3) a mitochondrial targeting sequence as the masked signal protein, and 4) the detectable amino acid sequence. 67. The isolated nucleic acid of claim 66, wherein the mitochondrial targeting sequence is human peptide methionine sulfoxide reductase (MSRA) or a functional fragment thereof. 68. The isolated nucleic acid of claim 66, wherein the masking sequence is one of a signal protein, a fluorescent protein; or a functional fragment thereof. 69. An isolated nucleic acid comprising sequence encoding a chimeric protein, the chimeric protein comprising as covalently linked components: 1) at least one masked signal protein; 2) at least one protease-specific cleavage site; 3) at least one detectable amino acid sequence; and 4) optionally, at least one unmasked signal protein, wherein the chimeric protein comprises covalently linked in sequence: 1) a Pleckstrin homology domain (PH) as the unmasked signal protein, 2) the protease-specific cleavage site, 3) a mitochondrial targeting sequence as the masked signal protein, and 4) the detectable amino acid sequence. 70. The isolated nucleic acid of claim 69, wherein the mitochondrial targeting sequence is human peptide methionine sulfoxide reductase (MSRA) or a functional fragment thereof.

Details for Patent 7,855,072

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	ACTIVASE	alteplase	For Injection	103172	11/13/1987		2021-08-10
Genentech, Inc.	CATHFLO ACTIVASE	alteplase	For Injection	103172	09/04/2001		2021-08-10