Claims
- 1. An isolated nucleic acid comprising:
(a) a first polynucleotide encoding a boiling stable protein, said boiling stable protein having a chaperone-like activity; and (b) a second polynucleotide including a promoter sequence being operably linked to said first polynucleotide for directing an expression of said boiling stable protein.
- 2. The isolated nucleic acid of claim 1, wherein said promoter sequence is a eukaryote promoter.
- 3. The isolated nucleic acid of claim 2, wherein said eukaryote promoter is a constitutive promoter.
- 4. The isolated nucleic acid of claim 1, wherein said promoter is a plant promoter selected from the group consisting of a constitutive plant promoter, a tissue specific plant promoter and an inducible plant promoter.
- 5. The isolated nucleic acid of claim 4, wherein:
(i) said constitutive plant promoter is selected from the group consisting of CaMV35S plant promoter, CaMV19S plant promoter, FMV34S plant promoter, sugarcane bacilliform badnavirus plant promoter, CsVMV plant promoter, Arabidopsis ACT2/ACT8 actin plant promoter, Arabidopsis ubiquitin UBQ1 plant promoter, barley leaf thionin BTH6 plant promoter, and rice actin plant promoter; (ii) said tissue specific plant promoter is selected from the group consisting of bean phaseolin storage protein plant promoter, DLEC plant promoter, PHSβ plant promoter, zein storage protein plant promoter, conglutin gamma plant promoter from soybean, AT2S1 gene plant promoter, ACT11 actin plant promoter from Arabidopsis, napA plant promoter from Brassica napus and potato patatin gene plant promoter; and (iii) said inducible plant promoter is selected from the group consisting of a light-inducible plant promoter derived from the pea rbcS gene, a plant promoter from the alfalfa rbcS gene, DRE, MYC and MYB plant promoters which are active in drought; INT, INPS, prxEa, Ha hsp17.7G4 and RD21 plant promoters active in high salinity and osmotic stress, and hsr203J and str246C plant promoters active in pathogenic stress.
- 6. The isolated nucleic acid of claim 1, wherein said promoter sequence is a prokaryote promoter.
- 7. The isolated nucleic acid of claim 1, wherein said first polynucleotide has a sequence at least 60% identical with SEQ ID NOs:1, 5, 6, 34, 39 or 40 as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals −9.
- 8. The isolated nucleic acid of claim 1, wherein said stable protein has a sequence at least 60% homologous to SEQ ID NOs:2 or 35, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
- 9. The isolated nucleic acid of claim 1, wherein said stable protein is natively an oligomer.
- 10. The isolated nucleic acid of claim 1, wherein said chaperone-like activity includes heat stabilization of proteins.
- 11. A nucleic acid construct comprising the nucleic acid of claim 1
- 12. A cell transformed with the nucleic acid of claim 1.
- 13. An organism transformed with the nucleic acid of claim 1.
- 14. The isolated nucleic acid of claim 1, further comprising
(c) a third polynucleotide encoding an additional protein, said third polynucleotide being adjacent and in frame to said first polynucleotide, said first and third polynucleotides encoding, in combination, a fusion protein of said stable protein and said additional protein.
- 15. A method of isolating a gene encoding a boiling stable protein having chaperone-like activity from a biological source, the method comprising screening an expression library with a polynucleotide encoding a boiling stable protein, said boiling stable protein having a chaperone-like activity.
- 16. An isolated nucleic acid comprising:
(a) a first polynucleotide encoding a detergent stable protein, said detergent stable protein having a chaperone-like activity; and (b) a second polynucleotide including a promoter sequence being operably linked to said first polynucleotide for directing an expression of said detergent stable protein.
- 17. The isolated nucleic acid of claim 16, wherein said promoter sequence is a eukaryote promoter.
- 18. The isolated nucleic acid of claim 17, wherein said eukaryote promoter is a constitutive promoter.
- 19. The isolated nucleic acid of claim 16, wherein said promoter is a plant promoter selected from the group consisting of a constitutive plant promoter, a tissue specific plant promoter and an inducible plant promoter.
- 20. The isolated nucleic acid of claim 19, wherein:
(i) said constitutive plant promoter is selected from the group consisting of CaMV35S plant promoter, CaMV19S plant promoter, FMV34S plant promoter, sugarcane bacilliform badnavirus plant promoter, CsVMV plant promoter, Arabidopsis ACT2/ACT8 actin plant promoter, Arabidopsis ubiquitin UBQ1 plant promoter, barley leaf thionin BTH6 plant promoter, and rice actin plant promoter; (ii) said tissue specific plant promoter is selected from the group consisting of bean phaseolin storage protein plant promoter, DLEC plant promoter, PHSβ plant promoter, zein storage protein plant promoter, conglutin gamma plant promoter from soybean, AT2S1 gene plant promoter, ACT11 actin plant promoter from Arabidopsis, napA plant promoter from Brassica napus and potato patatin gene plant promoter; and (iii) said inducible plant promoter is selected from the group consisting of a light-inducible plant promoter derived from the pea rbcS gene, a plant promoter from the alfalfa rbcS gene, DRE, MYC and MYB plant promoters which are active in drought; INT, INPS, prxEa, Ha hsp17.7G4 and RD21 plant promoters active in high salinity and osmotic stress, and hsr203J and str246C plant promoters active in pathogenic stress
- 21. The isolated nucleic acid of claim 16, wherein said promoter sequence is a prokaryote promoter.
- 22. The isolated nucleic acid of claim 16, wherein said first polynucleotide has a sequence at least 60% identical with SEQ ID NOs:1, 5, 6, 34, 39 or 40, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals −9.
- 23. The isolated nucleic acid of claim 16, wherein said stable protein has a sequence at least 60% homologous to SEQ ID NOs:2 or 35, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
- 24. The isolated nucleic acid of claim 16, wherein said stable protein is natively an oligomer.
- 25. The isolated nucleic acid of claim 16, wherein said chaperone-like activity includes heat stabilization of proteins.
- 26. A nucleic acid construct comprising the isolated nucleic acid of claim 16
- 27. A cell transformed with the isolated nucleic acid of claim 16.
- 28. An organism transformed with the isolated nucleic acid of claim 16.
- 29. The isolated nucleic acid of claim 16, further comprising (c) a third polynucleotide encoding an additional protein, said third polynucleotide being adjacent and in frame to said first polynucleotide, said first and third polynucleotides encoding, in combination, a fusion protein of said stable protein and said additional protein.
- 30. A method of isolating a gene encoding a detergent stable protein having chaperone-like activity from a biological source, the method comprising screening an expression library with a polynucleotide encoding a detergent stable protein, said detergent stable protein having a chaperone-like activity.
- 31. An isolated nucleic acid comprising:
(a) a first polynucleotide encoding a protease resistant protein, said protease resistant protein having a chaperone-like activity; and (b) a second polynucleotide including a promoter sequence being operably linked to said first polynucleotide for directing an expression of said protease resistant protein.
- 32. The isolated nucleic acid of claim 31, wherein said promoter sequence is a eukaryote promoter.
- 33. The isolated nucleic acid of claim 32, wherein said eukaryote promoter is a constitutive promoter.
- 34. The isolated nucleic acid of claim 31, wherein said promoter is a plant promoter selected from the group consisting of a constitutive plant promoter, a tissue specific plant promoter and an inducible plant promoter.
- 35. The isolated nucleic acid of claim 34, wherein:
(i) said constitutive plant promoter is selected from the group consisting of CaMV35S plant promoter, CaMV19S plant promoter, FMV34S plant promoter, sugarcane bacilliform badnavirus plant promoter, CsVMV plant promoter, Arabidopsis ACT2/ACT8 actin plant promoter, Arabidopsis ubiquitin UBQ1 plant promoter, barley leaf thionin BTH6 plant promoter, and rice actin plant promoter; (ii) said tissue specific plant promoter is selected from the group consisting of bean phaseolin storage protein plant promoter, DLEC plant promoter, PHSβ plant promoter, zein storage protein plant promoter, conglutin gamma plant promoter from soybean, AT2S1 gene plant promoter, ACT11 actin plant promoter from Arabidopsis, napA plant promoter from Brassica napus and potato patatin gene plant promoter; and (iii) said inducible plant promoter is selected from the group consisting of a light-inducible plant promoter derived from the pea rbcS gene, a plant promoter from the alfalfa rbcS gene, DRE, MYC and MYB plant promoters which are active in drought; INT, INPS, prxEa, Ha hsp17.7G4 and RD21 plant promoters active in high salinity and osmotic stress, and hsr203J and str246C plant promoters active in pathogenic stress.
- 36. The isolated nucleic acid of claim 31, wherein said promoter sequence is a prokaryote promoter.
- 37. The isolated nucleic acid of claim 31, wherein said first polynucleotide has a sequence at least 60% identical with SEQ ID NOs:1, 5, 6, 34, 39 or 40, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals −9.
- 38. The isolated nucleic acid of claim 31, wherein said resistant protein has a sequence at least 60% homologous to SEQ ID NOs:2 or 35, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
- 39. The isolated nucleic acid of claim 31, wherein said resistant protein is natively an oligomer.
- 40. The isolated nucleic acid of claim 31, wherein said chaperone-like activity includes heat stabilization of proteins.
- 41. A nucleic acid construct comprising The isolated nucleic acid of claim 31
- 42. A cell transformed with the isolated nucleic acid of claim 31.
- 43. An organism transformed with the isolated nucleic acid of claim 31.
- 44. The isolated nucleic acid of claim 31, further comprising
(c) a third polynucleotide encoding an additional protein, said third polynucleotide being adjacent and in frame to said first polynucleotide, said first and third polynucleotides encoding, in combination, a fusion protein of said resistant protein and said additional protein.
- 45. A method of isolating a gene encoding a protease resistant protein having chaperone-like activity from a biological source, the method comprising screening an expression library with a polynucleotide encoding a protease resistant protein, said a protease resistant protein having a chaperone-like activity.
- 46. An isolated boiling stable polypeptide having a chaperone-like activity.
- 47. The polypeptide of claim 46, encoded by a polynucleotide having a sequence at least 60% identical with SEQ ID NOs:1, 5, 6, 34, 39 or 40, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals −9.
- 48. The polypeptide of claim 46, having a sequence at least 60% homologous to SEQ ID NOs:2 or 35, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
- 49. The polypeptide of claim 46 which is natively an oligomer.
- 50. The polypeptide of claim 46, wherein said chaperone-like activity includes heat stabilization of proteins.
- 51. An antibody recognizing at least one epitope of the polypeptide of claim 46.
- 52. A method of isolating a gene encoding a boiling stable protein comprising screening an expression library with the antibody of claim 51.
- 53. A method of preventing an aggregating protein from aggregating into an aggregate comprising causing an effective amount of the polypeptide of claim 46 to become in contact with said aggregating protein.
- 54. A method of de-aggregating aggregates of an aggregating protein comprising causing an effective amount of the polypeptide of claim 46 to become in contact with said aggregate.
- 55. A method of stabilizing a protein against denaturing conditions comprising causing an effective amount of the polypeptide of claim 46 to become in contact with said protein.
- 56. An isolated detergent stable polypeptide having a chaperone-like activity.
- 57. The polypeptide of claim 56 encoded by a polynucleotide having a sequence at least 60% identical with SEQ ID NOs:1, 5, 6, 34, 39 or 40, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals −9.
- 58. The polypeptide of claim 56, having a sequence at least 60% homologous to SEQ ID NOs:2 or 35, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
- 59. The polypeptide of claim 56 which is natively an oligomer.
- 60. The polypeptide of claim 56, wherein said chaperone-like activity includes heat stabilization of proteins.
- 61. An antibody recognizing at least one epitope of the polypeptide of claim 56.
- 62. A method of isolating a gene encoding a detergent stable protein comprising screening an expression library with the antibody of claim 61.
- 63. A method of preventing an aggregating protein from aggregating into an aggregate comprising causing an effective amount of the polypeptide of claim 56 to become in contact with said aggregating protein.
- 64. A method of de-aggregating aggregates of an aggregating protein comprising causing an effective amount of the polypeptide of claim 56 to become in contact with said aggregate.
- 65. A method of stabilizing a protein against denaturing conditions comprising causing an effective amount of the polypeptide of claim 56 to become in contact with said protein.
- 66. An isolated protease resistant polypeptide having a chaperone-like activity.
- 67. The polypeptide of claim 66 encoded by a polynucleotide having a sequence at least 60% identical with SEQ ID NOs:1, 5, 6, 34, 39 or 40, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals −9.
- 68. The polypeptide of claim 66, having a sequence at least 60% homologous to SEQ ID NOs:2 or 35, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.
- 69. The polypeptide of claim 66 which is natively an oligomer.
- 70. The polypeptide of claim 66, wherein said chaperone-like activity includes heat stabilization of proteins.
- 71. An antibody recognizing at least one epitope of the polypeptide of claim 66.
- 72. A method of isolating a gene encoding a detergent stable protein comprising screening an expression library with the antibody of claim 71.
- 73. A method of preventing an aggregating protein from aggregating into an aggregate comprising causing an effective amount of the polypeptide of claim 66 to become in contact with said aggregating protein.
- 74. A method of de-aggregating aggregates of an aggregating protein comprising causing an effective amount of the polypeptide of claim 66 to become in contact with said aggregate.
- 75. A method of stabilizing a protein against denaturing conditions comprising causing an effective amount of the polypeptide of claim 66 to become in contact with said protein.
- 76. A method of enriching or isolating a denaturant stable and/or protease resistant protein having chaperone-like activity from a biological source, the method comprising:
(a) extracting total proteins from the biological source so as to obtain a proteins extract; (b) boiling said proteins extract; (c) collecting soluble proteins; and optionally (d) assaying for chaperone-like activity of the soluble proteins and enriching or isolating the stable protein having chaperone-like activity.
- 77. The method of claim 76, further comprising size fractionating said soluble proteins.
- 78. A method of isolating a gene encoding a denaturant stable and/or protease resistant protein having chaperone-like activity from a biological source, the method comprising:
(a) extracting total proteins from the biological source, so as to obtain a proteins extract; (b) boiling said proteins extract; (c) collecting soluble proteins; (d) assaying for chaperone-like activity of the soluble proteins and isolating a stable protein having chaperone-like activity; (e) raising antibodies recognizing said stable protein having said chaperone-like activity; and (f) screening an expression library with said antibodies.
- 79. A method of isolating a gene encoding a denaturant stable and/or protease resistant protein having chaperone-like activity from a biological source, the method comprising:
(a) extracting total proteins from the biological source, so as to obtain a proteins extract; (b) boiling said proteins extract; (c) collecting soluble proteins; (d) assaying for chaperone-like activity of the soluble proteins and enriching or isolating a stable protein having chaperone-like activity; (f) microsequencing said stable protein so as to obtain at least a partial amino acid sequence thereof; (g) designing an oligonucleotide corresponding to said amino acid sequence; and (h) screening a library with said oligonucleotide.
- 80. A method of isolating a nucleic acid potentially encoding a denaturant stable and/or protease resistant protein having chaperone-like activity, the method comprising screening a cDNA or genomic library with a polynucleotide of at least 17 bases at least 60% identical to a contiguous portion of SEQ ID NOs:1, 5, 6, 34, 39 or 40.
- 81. A method of identifying a nucleic acid potentially encoding a denaturant stable and/or protease resistant protein having chaperone-like activity, the method comprising searching an electronic library containing a plurality of nucleic acid and/or amino acid sequences for sequences having a predetermined degree of identity or homology to any of SEQ ID NOs:1, 2, 5-35 or 39-40 or portions thereof of, or corresponding to, at least 15 bases.
- 82. A method of isolating a nucleic acid potentially encoding a denaturant stable and/or protease resistant protein having chaperone-like activity, the method comprising:
(a) providing at least one pair of oligonucleotides each being at least 15 bases in length, said at least one pair of oligonucleotides including at least one oligonucleotide corresponding to SEQ ID NOs:1, 2, 5-35 or 39-40, said at least one pair of oligonucleotides being selected for amplifying a nucleic acid having a degree of identity with, or encoding proteins homologous, to SEQ ID NOs:1, 2, 5-35 or 39-40; (b) contacting said at least one pair of oligonucleotides with a sample of nucleic acid and amplifying said nucleic acid having said degree of identity with, or encoding proteins homologous to, SEQ ID NOs:1, 2, 5-35 or 39-40; and (c) using said nucleic acid having said degree of identity with or encoding proteins homologous to SEQ ID NOs:1, 2, 5-35 or 39-40 for isolating a nucleic acid potentially encoding a denaturant stable and/or protease resistant protein.
- 83. A method of detergent-free isolation of a protease-resistant protein having chaperone-like activity from a biological source, the method comprising:
(a) extracting total proteins from the biological source, so as to obtain a proteins extract; (b) contacting said protein extract with a protease; and (c) isolating a protease-resistant protein; and optionally (d) assaying said protease-resistant protein for chaperone-like activity.
- 84. A fusion protein comprising a denaturant stable and/or protease resistant polypeptide having a chaperone-like activity fused to an additional polypeptide.
- 85. The fusion protein of claim 84, wherein said denaturant stable and/or protease resistant polypeptide having said chaperone-like activity is fused to said additional polypeptide via a peptide bond.
- 86. The fusion protein of claim 84, wherein said denaturant stable and/or protease resistant polypeptide having said chaperone-like activity is fused to said additional polypeptide via a cross-linker.
- 87. The fusion protein of claim 84, having an oligomeric form.
- 88. A method of immunization comprising subjecting an immune system of a mammal to the fusion protein of claim 84.
- 89. A method of protecting an enzyme preparation from reduction in enzymatic activity, the method comprising adding to the enzyme preparation a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, in an amount sufficient for protecting the enzyme preparation from reduction in enzymatic activity.
- 90. A method of repairing at least a portion of lost enzymatic activity of an enzyme preparation, the method comprising adding to the enzyme preparation a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, in an amount sufficient for repairing at least said portion of said lost enzymatic activity of the enzyme preparation.
- 91. A method of administering to an animal having an immune system a polypeptide, while reducing an immune response against said polypeptide, the method comprising administering the polypeptide to the animal, said polypeptide being fused to a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, thereby reducing said immune response against said polypeptide, as compared to an immune response that develops by administering to the animal the polypeptide alone.
- 92. A transgenic plant expressing a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity above a natural amount of said denaturant stable and/or protease resistant protein having said chaperone-like activity in said plant.
- 93. A method of rendering a plant more tolerant to a biotic or abiotic stress, the method comprising engineering the plant to express a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, above a natural amount of said denaturant stable and/or protease resistant protein having said chaperone-like activity in said plant.
- 94. A method of rendering a plant more recoverable from a biotic or abiotic stress, the method comprising engineering the plant to express a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, above a natural amount of said denaturant stable and/or protease resistant protein having said chaperone-like activity in said plant.
- 95. A method of increasing cell migration, the method comprising exposing said cells to an amount of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, sufficient for increasing cell migration.
- 96. A method of accelerating wound healing, the method comprising administering onto a wound an amount of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, sufficient for accelerating wound healing.
- 97. A method of inducing wound healing, the method comprising administering onto a wound an amount of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, sufficient for inducing wound healing.
- 98. A method of strengthening hair, nail or skin, the method comprising administering onto the hair, nail or skin an amount of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, sufficient for strengthening the hair, nail or skin.
- 99. A method of grooming hair, nail or skin, the method comprising administering onto the hair, nail or skin an amount of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, sufficient for grooming the hair, nail or skin.
- 100. A pharmaceutical composition, comprising, as an active ingredient, a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, and a pharmaceutically acceptable carrier.
- 101. The pharmaceutical composition of claim 100, wherein said pharmaceutical composition is packaged in a package and identified in print for use in a wound healing application.
- 102. The pharmaceutical composition of claim 100, wherein said pharmaceutical composition is packaged in a package and identified in print for use in a strengthening and/or grooming hair, nail or skin application.
- 103. A method of isolating a boiling stable protein having chaperone-like activity from a biological source, the method comprising:
(a) extracting total proteins from the biological source, so as to obtain a proteins extract; (b) boiling said protein extract; (c) recovering soluble protein fraction; and optionally (d) assaying said protease-resistant protein for chaperone-like activity.
- 104. The method of claim 103, further comprising digesting said protein extract with a protease.
- 105. A method of treating a disease associated with protein aggregation of an aggregating protein, the method comprising administering to a subject in need thereof a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, in an amount sufficient for de-aggregating and/or preventing aggregation of said aggregating protein.
- 106. The method of claim 105, wherein said aggregating protein is selected from the group consisting of beta-amyloid and prion.
- 107. A method of increasing a binding avidity of a binding molecule, the method comprising displaying multiple copies of the binding molecule on a surface of an oligomer of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity.
- 108. The method of claim 107, wherein said binding molecule is selected from the group consisting of a receptor, a ligand, an enzyme, a substrate, an inhibitor, an antibody and an antigen.
- 109. A hetero complex comprising an oligomer including a plurality of a denaturant stable and/or protease resistant protein, said denaturant stable and/or protease resistant protein having a chaperone-like activity, and at least two different molecules being fused to said oligomer.
- 110. The hetero complex of claim 109, wherein said at least two different molecules comprise at least a first enzyme and a second enzyme.
- 111. The hetero complex of claim 110, wherein said first enzyme and said second enzyme catalyze sequential reactions in a synthesis or degradation pathway.
- 112. The hetero complex of claim 110, wherein said first enzyme and said second enzyme catalyze different reactions in a synthesis or degradation pathway.
- 113. The hetero complex of claim 109, wherein said at least two different molecules comprise at least a binding molecule and a reporter molecule.
- 114. An isolated denaturant stable and/or protease resistant protein having chaperone-like activity having an HRP protection activity, as determined using an HRP protection assay, of at lest 10 Units/mg protein, wherein said HRP protection assay comprises mixing the isolated denaturant stable and/or protease resistant protein having chaperone-like activity at different final protein concentrations at a predetermined volume with 100 μl of 5 nM HRP present in 40 mM HEPES buffer at pH 7.5, thus forming a first reaction mixture, and following incubation of said reaction mixture at 25° C. for 16 hours, determining HRP remaining enzymatic activity by mixing 5 μl of said first reaction mixture with 100 μl of 3 3′ 5 5′-tetramethylbenzidiine, thus forming a second reaction mixture, incubating said second reaction mixture for 10 minutes, stopping a reaction of said second reaction mixture by an addition of 100 μl of 1 M sulfuric acid and recording calorimetric change in said second reaction mixture at 435 nm, whereby said units are defined as a dilution factor of said denaturant stable and/or protease resistant protein having chaperone-like activity at a concentration of 1 mg/ml that confers 50% protection of HRP activity in said HRP protection assay.
- 115. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 500 Units/mg protein.
- 116. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 1000 Units/mg protein.
- 117. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 1500 Units/mg protein.
- 118. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 2000 Units/mg protein.
- 119. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 2500 Units/mg protein.
- 120. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 3000 Units/mg protein.
- 121. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 3500 Units/mg protein.
- 122. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 4000 Units/mg protein.
- 123. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 4500 Units/mg protein.
- 124. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 5000 Units/mg protein.
- 125. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 5500 Units/mg protein.
- 126. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 6000 Units/mg protein.
- 127. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 8000 Units/mg protein.
- 128. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 10000 Units/mg protein.
- 129. The isolated denaturant stable and/or protease resistant protein of claim 114, wherein said HRP protection activity is of at lest 15000 Units/mg protein.
- 130. A method of increasing a specific activity of a pre-isolated denaturant stable and/or protease resistant protein having chaperone-like activity as determined in Units of protecting activity per mg protein, the method comprising autoclaving said pre-isolated denaturant stable and/or protease resistant protein.
- 131. A method of increasing a specific activity of a pre-isolated denaturant stable and/or protease resistant protein having chaperone-like activity as determined in Units of protecting activity per mg protein, the method comprising treating said pre-isolated denaturant stable and/or protease resistant protein with a protease.
Parent Case Info
[0001] This is a Continuation-In-Part of PCT/IL02/00174, filed Mar. 5, 2002, which claims priority from U.S. Provisional Patent Application No. 60/272,771, filed Mar. 5, 2001.
Provisional Applications (1)
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Number |
Date |
Country |
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60272771 |
Mar 2001 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
PCT/IL02/00174 |
Mar 2002 |
US |
Child |
10233409 |
Sep 2002 |
US |