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US20030083467A1 - Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity - Google Patents

Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity Download PDF

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US20030083467A1
US20030083467A1 US10/300,757 US30075702A US2003083467A1 US 20030083467 A1 US20030083467 A1 US 20030083467A1 US 30075702 A US30075702 A US 30075702A US 2003083467 A1 US2003083467 A1 US 2003083467A1
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fusion protein
protease
host cell
protein
dna
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Ursula Germann
Thomas Hoock
Ann Kwong
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035—Herpes simplex virus I or II
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones

Definitions

  • the invention relates to fusion proteins, DNA molecules encoding the fusion proteins, vectors comprising the DNA molecules, host cells transformed with the vectors, and methods and kits for using them to determine the activity of a protease.
  • the invention relates to a fusion protein having a protease cleavage site, a ligand-binding domain, and a DNA-binding domain, wherein (1) the association of a ligand with the ligand-binding domain of said fusion protein mediates the binding of the DNA-binding domain of said fusion protein to a ligand-response element (“LRE”) that is operatively linked to a reporter gene; and wherein (2) the fusion protein comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates transcription of the reporter gene.
  • LRE ligand-response element
  • the invention also relates to kits for assaying protease activity comprising DNA molecules encoding the fusion proteins, an appropriate ligand, and DNA molecules comprising a promoter-reporter gene construct and at least one LRE recognized by the DNA binding domain of the fusion protein.
  • the DNA molecules in these kits may be isolated or present in host cells.
  • Proteases play an important role in the regulation of biological processes in almost every life form from bacteria to virus to mammals. They perform critical functions in, for example, digestion, blood clotting, apoptosis, activation of immune responses, zymogen activation, viral maturation, protein secretion and protein trafficking.
  • Proteases have been implicated as the cause of or as contributors to several diseases such as Alzheimer's disease, cystic fibrosis, emphysema, hypertension, tumor invasion and metastasis and viral-associated diseases [e.g., Kim, T. W., et al., (1997) “Alternative Cleavage of Alzheimer-associated Presinilins During Apoptosis by a Caspase-3 Family Protease,” Science 277: 373-6; Lacana, E.
  • HIV Human Immunodeficiency Virus
  • the hepatitis C virus produces a long polypeptide translation product, NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, which is cleaved to produce at least 10 proteins.
  • C, E1 and E2 are putative structural proteins, and the remainder are known as the nonstructural (NS) proteins.
  • NS3 a 70 kilodalton protein having serine protease activity. It is been shown that the protease activity of NS3 resides exclusively in the N-terminal 180 amino acids of the enzyme.
  • NS3 protease cleavages at four sites in the nonstructural region of the HCV polypeptide (3/4A, 4A/4B, 4B/5A, and 5A/5B).
  • Another protein, NS4A has 54 amino acids and has been characterized as a cofactor for the NS3 protease [C. Failla, et al., (1994) J. Virology 68:3753-3760].
  • the C-terminal 33 amino acids of NS4A are required for cleavage at the 3/4A site and 4B/5A sites and accelerate the rate of cleavage at the 5A/5B site.
  • NS3 serine protease-dependent cleavage site sequences have been identified in various strains of HCV [A. Grakoui, et al., (1993) J. Virology 67:2832-2843 incorporated by reference herein].
  • protease assays are known in the art. T. A. Smith et al., Proc. Natl. Acad. Sci. USA, 88, pp. 5159-62 (1991); B. Dasmahapatra et al., Proc. Natl. Acad. Sci. USA, 89, pp. 4159-62 (1992); and M. G. Murray et al., Gene, 134, pp. 123-128 (1993) each describe protease assay systems utilizing the yeast GAL4 protein. Each of these documents describe inserting a protease cleavage site in between the DNA binding domain and the transcriptional activating domain of GAL4. Cleavage of that site by a coexpressed protease renders GAL4 transcriptionally inactive leading to the inablility of the transformed yeast to metabolize galactose.
  • Y. Hirowatari et al., Anal. Biochem., 225, pp. 113-120 (1995) describes an assay to detect HCV protease activity.
  • the substrate is a fusion protein consisting of (HCV NS2)-(DHFR)-(HCV NS3 cleavage site)-Taxi.
  • the reporter gene is chloramphenicol transferase (CAT) under control of the HTLV-1 long terminal repeat (LTR) and resides in the cell nucleus following expression.
  • the uncleaved substrate is expressed as a membrane-bound protein on the surface of the endoplasmic reticulum due to the HCV NS2 portion.
  • the released Tax1 protein Upon cleavage, the released Tax1 protein translocates to the nucleus and activates CAT expression by binding to the HTLV-1 LTR. Protease activity is determined by measuring CAT activity in a cell lysate.
  • the present invention fulfills this need by providing novel fusion proteins, DNA molecules encoding them, vectors comprising the DNA molecules, and host cells containing the vectors useful in a fusion protein ligand-dependent transcriptional assay to determine the activity of a protease.
  • the novel fusion protein comprises a protease cleavage site, a ligand-binding domain, and a DNA-binding domain, wherein (1) the association of a ligand with the ligand-binding domain of said fusion protein mediates the binding of the DNA-binding domain of said fusion protein to a LRE that is operatively linked to a reporter gene; and wherein (2) the fusion protein comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates the transcription of the reporter gene.
  • the binding of a ligand to the ligand binding domain of the uncleaved fusion protein initiates the activation or repression of transcription of the reporter gene at a discrete point in time. This inducibility allows the assay to be better controlled and therefore, produces more accurate results.
  • Cleavage of the fusion protein at the protease cleavage site deregulates transcription of the reporter gene by preventing the expression modulator domain from modulating transcription positively or negatively.
  • the amount of cleaved fusion protein is quantitated by assaying an increase or decrease in transactivation of a reporter gene, whose expression is driven by a promoter which is modulated by the expression modulating domain of the fusion protein.
  • This invention also relates to a method for measuring the inhibitory activity of a compound against a protease comprising the steps of incubating the fusion protein with a protease in the presence or absence of a compound whose activity is being tested, adding a ligand to the incubation and quantifying the gene product produced from a reporter gene.
  • Yet another embodiment of this invention relates to a method for comparing the activity of two proteases or mutants of a protease which recognize the same cleavage site.
  • the invention also relates to kits for assaying protease activity comprising DNA molecules encoding the fusion protein, the fusion protein or host cells containing the DNA molecules, wherein the kit optionally includes DNA molecules encoding a protease, DNA molecules comprising a reporter gene whose expression is regulated by said fusion protein, a ligand which associates and regulates the activity of said fusion protein, and instructions for using said kit.
  • the DNA molecules in the kit have been engineered into a vector that allows their expression.
  • a kit according to this invention may comprise any one or all of the following DNA molecules transformed into a single host cell selected from the group consisting of: a DNA molecule encoding a fusion protein of this invention, a DNA molecule encoding a protease, a DNA molecule encoding a protein binding partner for the fusion protein and a DNA molecule comprising a promoter-reporter gene construct.
  • FIG. 1 depicts the structure of the vectors pVgRXR, pVgRXR-5A/5B, and pVgRXR-5A(Stop)5B.
  • the backslash positioned between the cysteine and the serine of the pVgRxR-5A/5B encoded protein indicates where the cleavage will take place.
  • the two asterisks near the cleavage site of the VgRXR-5A(Stop)5B encoded protein indicate the placement of two stop codons.
  • FIG. 2 depicts the structure of a DNA vector encoding the HCV NS3-4A protease, pSR ⁇ -NS3-4A.
  • FIG. 3 depicts the structure of a control vector PIND and a reporter vector for Ecdysone-Inducible Luciferase Expression, pIND-luc.
  • FIG. 4 graphically depicts the results of transfections experiments into COS cells with any one of the following: (1) a DNA vector encoding a mammalian RXR receptor protein and a fusion protein comprising the DNA-binding domain of the Drosophila ecdysone receptor fused to the activation domain of the Herpes simplex virus VP16 protein (pVgRXR); (2) a DNA vector encoding an RXR receptor protein and a fusion protein comprising the DNA-binding domain of the Drosophila ecdysone receptor fused to the 5A/5B proteolytic cleavage site of HCV fused to the activation domain of the VP16 protein (pVgRXR-5A/5B); and (3) a DNA vector encoding a RXR receptor protein and a fusion protein as described above except that the DNA encoding the 5A/5B cleavage site is modified to contain tandem stop codons in the cleavage site (pVgRXR
  • a plasmid comprising a luciferase reporter gene was included in each transfection (pIND-luc). After the transfection, transcription of the reporter gene was induced by administering 1 ⁇ M muristerone A (an ecdysone analog) to the transfected cells. The data was obtained by measuring the luciferase activity present in the lysates from the transfected cells as described in Example 2.
  • FIG. 5 depicts the results of cotransfecting plasmids pVgRXR-5A/5B and pIND-luc into COS cells with increasing amounts of a plasmid encoding HCV NS3-4A protease (pSR ⁇ Ns3-4A) or of a control plasmid (pSR ⁇ ). After the transfection, transcription of the reporter plasmid was induced by administering 1 ⁇ M muristerone A to the transfected cells.
  • FIG. 6 graphically depicts the results of cotransfecting plasmids pVgRXR-5A/5B and pIND-luc into COS cells with increasing amounts of a plasmid encoding HCV NS3-4A protease or an inactivate mutant thereof (pSR ⁇ Ns3-4A or pSR ⁇ Ns3-4A(S1165A), respectively). After the transfection, transcription of the reporter gene was induced by administering 1 ⁇ M muristerone A to the transfected cells.
  • FIG. 7 graphically depicts the results of administering either 1, 3.3, 10, 33 or 100 ⁇ M of ponasterone A (an ecdysone analog) to COS cells after cotransfection with plasmids pVgRXR-5A/5B and pIND-Luc in the presence or absence of a plasmid encoding the HCV-NS3-4A protease (pSR ⁇ Ns3-4A).
  • ponasterone A an ecdysone analog
  • FIG. 8 graphically depicts the results of a control experiment demonstrating DMSO tolerance.
  • Plasmids pVgRXR-5A/5B and pIND-Luc were cotransfected into COS cells with or without a plasmid encoding the HCV-NS3-4A protease (pSR ⁇ Ns3-4A). After the transfection, transcription of the reporter plasmid was induced by administering 5 ⁇ M ponasterone A in the presence of increasing concentrations of dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • FIG. 9 graphically depicts the results of cotransfecting plasmids pVgRXR-5A/5B, pIND-Luc and pSR ⁇ NS3-4A into COS cells and then administering 5 ⁇ M ponasterone A and varying amounts of a protease inhibitor (VH-25531).
  • the present invention provides novel tools and methods for measuring protease activity.
  • the activity of a protease is measured by using a novel fusion protein as a substrate in a transcriptional assay wherein transcription is activated or repressed at a discrete time point by addition of a ligand after a protease and a fusion protein of this invention are incubated together.
  • the present invention provides a fusion protein comprising: a protease cleavage site, a ligand binding domain, and a DNA binding domain, wherein (1) the association of a ligand with the ligand-binding domain of said fusion protein mediates the binding of the DNA-binding domain of said fusion protein to a ligand response element (“LRE”) operatively linked to a reporter gene; and wherein (2) the fusion protein comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates transcription of the reporter gene.
  • LRE ligand response element
  • a protease cleavage site (hereinafter, “PCS”) according to this invention is a peptide incorporated into the primary sequence of the fusion protein of this invention. It may be located in any of the DNA binding domain, ligand binding domain, or, optionally, the expression modulator domain, if one exists, of the protein, as well as in between any two of these domains.
  • the presence of the cleavage site in the protein should not substantially interfere with the activity of the fusion protein's DNA binding domain, ligand binding domain, or, if present, expression modulator domain.
  • the activity of a fusion protein with the PCS may be compared with the activity of the same fusion protein without the PCS by gel shift assay to assess DNA binding or by transcriptional assay (e.g., D. Latchman, (1995) Eukaryotic Transcription Factors, 2nd Ed. Academic Press:London).
  • the protease cleavage site should be engineered into the fusion protein such that, when cleaved, neither of the resulting fusion protein fragments are capable of modulating the expression of a target or reporter gene.
  • the protease cleavage site is situated between the DNA binding domain and the effector modulator domain of the fusion protein.
  • the PCS has an amino acid sequence comprising a processing/protease cleavage site in the HIV gag and gag/pol polyproteins or in the HCV polyprotein.
  • the HIV PCS site selected from the group consisting of Seq ID Nos. 1-10 [S.
  • the PCS has the amino acid sequence of a cleavage site for the HCV NS3 serine protease or HIV aspartyl protease.
  • the PCS has the amino acid sequence of an HCV 5A/5B protease cleavage site.
  • the 5A/5B protease cleavage site is Seq ID No. 10.
  • the next portion of the fusion protein of this invention is the ligand binding domain (hereinafter, “LBD”).
  • LBD is a peptide domain which binds to a ligand, wherein said ligand binding is required for said fusion protein to (1) bind to a LRE, wherein the element is operatively linked to a reporter gene; and/or (2) to bind to a protein binding partner as an interim step preceding the binding to the resulting fusion protein-protein binding partner complex to a LRE.
  • ligand binding to the fusion protein ligand binding domain is necessary for the transcriptional regulation of the expression of said reporter gene.
  • Ligand binding domains suitable for use in this invention may be derived from ligand binding domains of transcription factors that initiate or repress of transcription upon ligand binding.
  • transcription factors include hormone receptors [Freedman, L. P., 1998, “Molecular Biology of Steroid and Nuclear Hormone Receptors,” Progress in Gene Expression, Boston: Birkhauser; Tsai, M-J., 1994, “Mechanism of Steroid Hormone Regulation of Gene Transcription,” Molecular Biology Intelligence Unit, Austin: R. G. Austin Co.; Eggert, M. et al., “The Glucocorticoid Hormone Receptor,” Inducible Gene Expression, vol. 2 (1995) Birkhauser: Boston, Mass. Ed. P. A.
  • the ligand binding domain is derived from the ligand binding domains of the steroid/thyroid hormone receptor superfamily.
  • Amino acid sequences encoding steroid and nuclear receptor ligand binding domains are well known in the art [See, for example, S. Simons, (1998) “Structure and Function of the Steroid and Nuclear Receptor Ligand Binding Domain,” Molecular Biology of Steroid and Nuclear Hormone Receptors: Progress in Gene Expression, Birkhauser: Boston, Mass., the disclosure of which is herein incorporated by reference].
  • the ligand binding domain is derived from an ecdysone receptor.
  • a ligand binding domain from a transcription factor may be modified in the fusion protein of this invention such that it binds to a different ligand.
  • a human progesterone receptor ligand binding domain may be mutated so that it is capable of binding the anti-progesterone compound RU486 (Wang, Y., et al., (1997) “Positive and Negative Regulation of Gene Expression in Eukaryotic Cells with an Inducible Transcriptional Regulator,” Gene Ther. 4:432-41, the disclosure of which is herein incorporated by reference).
  • RU486-inducible GAL4 DNA binding domain fusion protein is contemplated for use in this invention.
  • a DNA binding domain refers to a peptide sequence in the fusion protein according to this invention which recognizes and binds to a specific nucleotide sequence (e.g., a DNA element or a LRE).
  • DNA binding domains useful according to this invention may be derived from the DNA binding domains of DNA binding proteins, such as transcription factors.
  • these DNA binding proteins are transcription factors that directly bind DNA and initiate or repress transcription.
  • transcription factors are well known in the art [McKnight, S. L. and K. R. Yamamoto, 1992, “Transcriptional Regulation,” Cold Spring Harbor Monograph Series, Plainview, N.Y.: Cold Spring Harbor Laboratory Press; Latchman, D. S., 1995, Eukaryotic Transcription Factors, San Diego: Academic Press, 2.ed.; Latchman, D.
  • Preferred DNA binding domains contemplated for use in this invention include the DNA binding domains of the following transcription factors: homeobox proteins, zinc finger proteins (Sluyser, M., 1993, Zinc Finger Proteins in Oncogenesis: DNA Binding and Gene Regulation, New York: New York Academy of Sciences), helix-turn-helix proteins, helix-loop-helix proteins (e.g., Littlewood, Trevor D., 1998, Helix - Loop - Helix Transcription Factors, 3rd. Ed., New York: Oxford University Press), leucine zipper proteins (e.g., Hurst, H. C., 1996, Leucine Zippers: Transcription Factors, 3rd. ed., San Diego: Academic Press), GAL4 protein, hormone receptors, orphan receptors, and E. coli transcription factors such as the lactose operon repressor, tetracycline-controlled transactivator, and FadR.
  • homeobox proteins zinc finger proteins
  • the present invention also contemplates the use of the DBD's of metal-binding DNA-binding proteins that regulate the transcription of metallothionein (MT) genes.
  • metal-binding DNA-binding proteins include MTF-1, ACE1, and AMT1.
  • the DNA-binding domain is obtained from a member of the steroid/thyroid superfamily of receptors.
  • the members of the steroid/thyroid superfamily of receptors are known in the art as hormone binding proteins that function as ligand-dependent transcription factors. They include identified members of the steroid/thyroid superfamily of receptors for which specific natural ligands have not yet been identified (referred to herein as “orphan receptors”) [B. M. Forman, (1998) “Orphan Nuclear Receptors and Their Ligands,” Molecular Biology of Steroid and Nuclear Hormone Receptors, L. P. Freedman, Ed., Birhauser: Boston, pp. 281-305.]
  • HNF4 the COUP family of receptors and COUP-like receptors, peroxisome proliferator-activated receptors (PPARs), insect derived knirps and knirps-related receptors, and various isoforms thereof.
  • PPARs peroxisome proliferator-activated receptors
  • the DNA binding domains of this invention may be modified from the sequence present in the source transcription factor so that it recognizes a different LRE.
  • the amino acid sequence of the DNA binding domain of a steroid/thyroid family members may be modified such that the DNA binding domain binds to a LRE recognized by another steroid/thyroid family member.
  • modification of the “P-box” amino acid sequence of a steroid/thyroid family member i.e., a region in the DNA-binding domain that is typically located at the junction of the first zinc finger and the linker region, is contemplated by this invention (Umesono et al., (1989) Cell 57:1139-1146, particularly FIG. 2 and Table 1; the disclosure of which is herein incorporated by reference).
  • the DNA binding domain is derived from the Drosophila ecdysone receptor, wherein the P-box of the ecdysone receptor having the amino acid sequence EGCKG (SEQ. ID. NO. 23) is replaced with the amino acid sequence GSCKV (SEQ. ID. NO. 24).
  • the new P-box sequence GSCKV causes the ecdysone receptor to recognize the LRE -AGAACA- instead of the LRE -AGGTCA-.
  • steroid receptors such as the glucocorticoid receptor (GR), the mineralcorticoid receptor (MR), the estrogen receptor (ER), the progesterone receptor (PR) such as hPR-A or hPR-B, the androgen receptor (AR), the vitamin D3 receptor (VDR); retinoid receptors such as retinoic acid receptors (e.g., RAR ⁇ , RAR ⁇ , or RAR ⁇ ) and retinoid X receptors (e.g., RXR ⁇ , RXR ⁇ , or RXR ⁇ ); thyroid receptors (TR) such as TR ⁇ and TR ⁇ ; insect-derived receptors such as the ecdysone receptors; and isoforms thereof.
  • GR glucocorticoid receptor
  • MR mineralcorticoid receptor
  • ER estrogen receptor
  • PR progesterone receptor
  • AR the vitamin D3 receptor
  • VDR vitamin D3 receptor
  • retinoid receptors such as retinoic acid receptors (e
  • a fusion protein of this invention contains a DNA binding domain which is engineered near or next to a ligand binding domain and neither of the two domains contains a PCS, nor is a PCS located between the two domains.
  • An optional component of the fusion protein of this invention is an expression modulator domain. As stated above, if the expression modulator domain is not present on the fusion protein, it must be present on a protein that associates with the fusion protein.
  • Expression modulator domains contemplated for use in this invention are typically derived from DNA binding proteins, such as transcription factors. These sequences are known in the art to activate or to repress transcription through their interaction with other transcription factors necessary for transcription.
  • the expression modulator domain is present within a second protein that binds to the fusion protein, that second protein should not increase or decrease transcription in the absence of a fusion protein of this invention and its ligand.
  • the expression modulator domain is present within the fusion protein it is preferably located at the terminus opposite to the location of the DNA binding domain.
  • the expression modulator domain activates transcription as opposed to repressing that activity.
  • activation domains include the N-terminal regions encoding activation domains in the steroid/thyroid superfamily of receptors, the activation domains of viral transcription factors, the yeast GCN4 activation domain or the GAL4 activation domain [K. Struhl, “Yeast GCN4 Transcriptional Activator Protein,” Transcriptional Regulation, Eds. S. L. McKnight and K. R. Yamamoto, Cold Spring Harbor Laboratory Press (1992), pp. 833-859; A. A. F. Gann et al., “GAL11, GAL11P, and the Action of GAL4,” Transcriptional Regulation, Eds.
  • the expression modulating domain is part of the fusion protein.
  • the expression modulating domain is the N-terminal activation domain of the VP16 protein.
  • ligand binding domain DNA binding domain and expression modulator domain need not be derived from the same transcription factor.
  • a chimera comprising the DNA-binding domain of the retinoic acid receptor and the ligand-binding domain of the vitamin D receptor (VDR) may be made (Pemrick, S., et al., (1998) “Characterization of the Chimeric Retinoic Acid Receptor RAR ⁇ /VDR,” Leukemia 12:554-562) or a chimera comprising the Jun DNA binding domain may be fused to the estrogen receptor ligand binding domain and expression modulator activation domain (Kruse, U., et al., (1997) “Hormone-regulatable Neoplastic Transformation Induced by a Jun-Estrogen Receptor Chimera,” PNAS USA 94:12396-12400).
  • lacR DNA-binding domain and the VP16 activation domain.
  • a tetR-VP16 fusion protein according to this invention may contain a protease cleavage site in its DNA binding domain such that DNA binding is eliminated when the PCS is cleaved.
  • uncleaved fusion protein would continue to transactivate from tetO, whereas cleaved fusion proteins would be inactive.
  • a preferred embodiment of this invention is the fusion of the VP16 activation domain of herpes simplex virus, the ligand binding domain of a steroid/thyroid receptor superfamily member and the DBD of a steroid/thyroid receptor superfamily member.
  • the steroid/thyroid receptor superfamily member is the ecdysone receptor.
  • a protein binding partner is a protein that binds to a fusion protein of this invention and increases the affinity or specificity of binding of the DNA-binding domain of the fusion protein to a specific LRE.
  • the protein binding partner is a protein that also binds to a particular DNA element that is operatively linked to the reporter gene. If the fusion protein of this invention requires a protein binding partner for increased affinity or specificity of DNA binding, then it is desirable to ensure that the fusion protein includes a dimerization domain for interaction with the PBP.
  • Protein binding partners that may be suitable for interacting with the dimerization domains of the fusion proteins of this invention are known in the art. [e.g., T. D. Littlewood and G. I. Evan (1998) “Structure/Function Relationships of HLH Proteins,” Helix - Loop - Helix Transcription Factors, 3rd. Ed., New York: Oxford University Press, pp. 27-41; Hurst, H. C., 1996, Leucine Zippers: Transcription Factors, 3rd. ed., San Diego: Academic Press, pp. 27-29; and L. P. Freedman, Ed., (1998) Molecular Biology of Steroid an Nuclear Hormone Receptors: Progress in Gene Expression, Birkhauser: Boston, Mass.].
  • bHLH transcription factors such as the ligand-dependent dioxin receptors and aryl hydrocarbon receptors (AHRs) heterodimerize with the AHR nuclear translocator protein (ARNT) [Whitelaw, M. L.
  • Another example is the steroid/thyroid family of receptors which have the ability to heterodimerize with each other or with other non-steroid/thyroid receptor family members (e.g., Rhee, et al., (1995) “Retinoid X-Receptor-alpha and Apolipoprotein AI Regulatory Protein 1 Differentially Modulate 3,5,3′-Triiodothyronine-Induced Transcription,” Endocrinology 136:2697-704; Li, et al., (1997) “Coexpression of Nuclear Receptors Partners Increases Their Solubility and Biological Activities,” PNAS USA 94:2278-83).
  • Rhee, et al. (1995) “Retinoid X-Receptor-alpha and Apolipoprotein AI Regulatory Protein 1 Differentially Modulate 3,5,3′-Triiodothyronine-Induced Transcription,” Endocrinology 136:2697-704; Li, et
  • a P-box modified ecdysone receptor heterodimerizes with mammalian RXR.
  • a fusion protein of this invention for use in cellular transcription assays should localize to the nucleus of the host cell.
  • a nuclear localization signal already present within a domain of a fusion protein of this invention may provide the nuclear localization signal.
  • a nuclear localization signal known in the art may be engineered into the fusion protein to ensure nuclear localization.
  • the fusion protein lacks a ligand binding domain.
  • the fusion protein comprises (1) a DNA-binding domain and (2) a protease cleavage site.
  • the transcriptional activity of the fusion protein is inducible by altering the temperature of the transcription environment or causing stress to the cells in which the transcription event takes place.
  • high affinity binding of a heat shock factor (“HSF”) to its response element, HSE is dependent on a heat shock stimulus.
  • a protease cleavage site of interest may be engineered into the HSF protein such that the transcriptional activity of the cleaved HSF fusion protein is lower or negligible compared to uncleaved HSF fusion protein after stimulus by heat.
  • fusion proteins, DNA molecules and vectors of this invention contemplated by the methods of this invention include cellular and in vitro transcription reactions.
  • DNA encoding a fusion protein of this invention a reporter plasmid, a protease and, optionally, a second protein having a expression modulator domain will be introduced into a cell.
  • the transcriptional activity of the fusion protein will be induced by adding a ligand or by altering the environment of the cell (e.g., changing the temperature of the environment of the cell).
  • the quantity of mRNA, the quantity of reporter protein, or the activity of the reporter protein will be measured using standard techniques known in the art. Transcription in the absence of protease can be compared with transcription in the presence of protease.
  • cellular extracts prepared by protocols which have been shown to support in vitro transcription reactions would be prepared.
  • Purified or partially purified proteins would be added to the cellular extract together with a reporter plasmid.
  • Such purified or partially purified proteins such as the fusion protein of this invention may be produced by overexpression in a cell introduced to the DNA encoding the fusion protein, e.g., SF9 cells via baculovirus infection, plant cells, yeast cells, mammalian cells, and bacterial cells.
  • Proteins such as the protease and, optionally, the second protein having an expression modulator domain and/or a protein binding partner may also be prepared from their original sources using methods known in the art to purify them.
  • the transcriptional activity of the fusion protein will be induced by adding a ligand.
  • the quantity of mRNA produced, the quantity of reporter protein produced, or the activity of the reporter protein will be measured using standard techniques known in the art. Transcription in the presence of protease can be compared with transcription in the absence of protease.
  • the invention also provides a method for screening compounds for inhibitory protease activity.
  • a compound to be screened may be added at the same time the ligand is added or added during the initial expression/incubation of the protease with a fusion protein.
  • the optimal level of compound needed to achieve the greatest inhibition of the protease may be determined by titration of the compound.
  • the invention also provides for a method for comparing the activity of two proteases which recognize the same protease cleavage site.
  • the method is useful for comparing the activity of mutant and non-mutant proteases, e.g., HIV aspartyl proteases from patients.
  • the invention also provides a method for comparing the activity of a protease against different protease cleavage sites. This method is useful for determining substrate specificity of a given protease
  • the invention also provides kits for assaying protease activity. If the kit is to be used in an in vitro transcription assay of a reporter gene, then it may comprise an in vitro transcription extract, a vector as described above containing a reporter gene, a supply of ligand, and instructions, optionally, it may provide a supply of a fusion protein of this invention, a second protein comprising an expression modulator domain, and/or a protease expressed from a source including bacteria, insect cells, mammalian cells, and yeast.
  • the kit may include a DNA sequence encoding a fusion protein of this invention and a vector encoding a reporter plasmid as described above, a ligand which associates with and regulates the activity of said fusion protein encoded by the DNA sequence of the protein; and instructions for using said kit to assay protease activity.
  • proteases that are particularly useful in the methods of this invention are proteases that contribute to the symptoms or to the onset of a disease. For example, those proteases involved in digestion, blood clotting, apoptosis, activation of immune responses, zymogen activation, viral maturation, protein secretion and protein trafficking.
  • the proteases of interest are those involved in Alzheimer's disease, cystic fibrosis, emphysema, hypertension, tumor invasion and metastasis and viral-associated diseases.
  • the proteases are HIV aspartyl protease and HCV NS3 protease, and active fragments or fusion proteins thereof.
  • any one or all of the DNA molecules encoding the fusion proteins, PBP's, proteases and second proteins comprising the expression modulator domain may be further modified to provide an additional polypeptide sequence for ease in purification or identifying of the expression of the protein.
  • polypeptide sequences may include an epitope for binding an antibody, an Fc portion of an antibody for binding protein A beads, glutathione S transferase, maltose binding protein, His(n), FLAG, and Strep-tag.
  • screenable tags are all well known in the art and are fully available to the person skilled in the art.
  • Host cells useful according to the methods of this invention are well known in the art. If the activity of an exogenous protease is being studied, it is desirable that the host cell does not express high levels of endogenous proteases which recognize the protease cleavage site(s) being targetted.
  • Suitable host cells may include CHO cells; HeLa cells; liver cells; CV-1 cells; P19 cells; NT2/D1 cells; mouse L cells; African Green monkey kidney cells, such as COS-7 cells or other COS cells; human embryonic kidney cells, such as HEK 293; DG44 cells, ltk-cells, mouse NIH 3T3 cells and yeast cells.
  • the host cells may be transiently transfected or stably transformed cell lines. In a preferred embodiment of this invention, the host cells are COS cell or cells from a liver cell line.
  • Ligands suitable for use in the methods of this invention are well known in the art.
  • the ligands should bind to the ligand binding domains of the transcription factors from which the ligand binding domains of the fusion proteins have been derived, or to modified ligand binding domains thereof (see above).
  • the ligand does not substantially enhance or repress transcription from the promoter unless a LRE for the fusion protein is spliced or inserted into an vector comprising a promoter and a reporter gene, wherein the LRE is linked to a promoter in a manner which makes transcriptional activity from the promoter operatively responsive to ligand.
  • ligand to use will necessarily depend on the ligand binding domain of the fusion protein being used.
  • suitable ligands for use with metal-binding DNA fusion proteins may include zinc, cadmium, and copper.
  • suitable ligands for use with steroid binding or modified steroid binding fusion proteins may include androgen, glucocorticoid, mineralocorticoids, thyroid hormones, estrogen, progesterone, progestogen, retinoic acid, vitamin D3, 20-hydroxy-ecdysone, ponasterone A, 26-iodoponasterone A, muristerone A, inokosterone, 26-mesylinokosterone, mifepristone (RU 486) and analogs thereof.
  • androgens include dihydroxytestosterone and analogs thereof including methyltrienolone.
  • glucocorticoid hormones include cortisol, hydrocortisone, and corticosterone, and analogs therof including dexamethasone, deoxycorticosterone, and triamcinolone acetonide.
  • mineralcorticoids include aldosterone, coricosterone and deoxycorticosterone.
  • thyroid hormones include thyroxine (T4) and triodothyronine (T3).
  • estrogens include estradiol-17 beta, and analogs thereof indulcing diethylstilbestrol.
  • progestogens include analogs of progesterone including promegestrone.
  • Suitable ligands for tetracycline-inducible fusion protein include tetracycline and analogs thereof.
  • Suitable ligands for carbohydrate-inducible fusion proteins include arabinose, lactose, and isopropyl ⁇ -D-thiogalactoside (IPTG).
  • Suitable ligands for aryl hydrocarbon inducible fusion proteins include dioxin.
  • Suitable ligands for orphan receptors include the following: phytenic acid, 9-cis retinoic acid, and LG100268 for the RXR receptor; 8S-HETE and Wy 14,643 for the PPAR ⁇ receptor; 15-deoxy- ⁇ 12,14 -PGJ2 and BRL 49653 for the PPAR ⁇ receptor; linoleic acid and carba-prostacyclin for the PPAR ⁇ receptor; farnesol for the FXR receptor; and 24(S),25-epoxycholesterol for the LXR receptor.
  • the LREs and PBP-DNA elements in this invention are nucleic acid molecules that provide DNA binding sites for the fusion proteins and the protein binding partners, respectively. Both elements should be operatively linked to a promoter to control the activation or repression of transcription of a reporter gene.
  • operatively linked refers to linking a nucleic acid molecule (e.g., a reporter gene encoding a reporter protein) to a LRE and a PBP-DNA element (if PBP binding is necessary for transcription) and to transcription control sequences in a manner such that the nucleic acid molecule molecule is able to be expressed when introduced (e.g., transfected, transformed, transduced, conjugated, or by recombination or by infection) into a host cell.
  • a nucleic acid molecule e.g., a reporter gene encoding a reporter protein
  • Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as the promoter.
  • the promoter is a minimal promoter.
  • the term “minimal promoter” is intended to describe a partial promoter sequence which defines the start site of transcription for the linked sequence to be transcribed, but which by itself is not capable of initiating transcription efficiently, if at all, in a certain cell environment. Thus, the activity of such a minimal promoter is dependent upon the binding of a fusion protein of this invention.
  • the minimal promoter is from the drosophila heat shock promoter and the target LRE for the fusion protein is AGAACA generated as described in PCT/US97/05330 (incorporated by reference herein).
  • LREs according to this invention should be operative to confer responsiveness to a ligand.
  • DNA elements that bind to PBP's of this invention may also be ligand-responsive if the PBP used is a ligand-binding protein.
  • Choice of LRE and DNA element (as necessary) and the arrangement of those elements with respect to each other will depend upon the the fusion protein or the PBP used. For example, if the DNA-binding domain of the fusion protein is from a basic-helix-loop-helix protein, the LRE would likely comprise the consensus DNA hexamer CANNTG (also known as an “E box”) (e.g., Littlewood, et al., supra, page 31).
  • the promoter controlling the expression of the fusion proteins, PBP's, and proteases in a mammalian cell may be constitutive such as the cytomegalovirus (CMV) promoter, the SV40 early promoter, and the Rous Sarcoma Virus (RSV) promoter.
  • CMV cytomegalovirus
  • RSV Rous Sarcoma Virus
  • the fusion protein, PBP's, and proteases may be under the control of an inducible promoter. It is desireable that the inducible promoter not be the same as the promoter that drives transcription of the reporter gene.
  • Reporter genes useful according to the methods of this invention are well known in the art. Such reporter genes include the luciferase gene, green fluorescent protein gene (U.S. Pat. No. 5,491,084), ⁇ -galactosidase, secreted alkaline phosphatase gene and the chloramphenicol acetyl transferase gene. Also, the invention contemplates reporter genes encoding a marker protein with a signal sequence for secretion out of the cell such as the IL-1 beta gene.
  • reporter plasmid may be prepared from materials available in the art.
  • Such reporter plasmids preferably also include, for example, the following: an origin of replication and a selectable marker(s).
  • the reporter plasmid may optionally also include other DNA sequences, such as long terminal repeats (“LTR's”), to cause the insertion of the vector into the genome of a cell line.
  • LTR's long terminal repeats
  • Vectors suitable for expressing the fusion proteins, PBP's, proteins having an expression modulator domain or proteases in bacteria or mammalian cells are well known in the art.
  • Such vectors may for example include the following: an origin of replication, a selectable marker and transcription control sequences such as a promoter and a polyadenylation sequence.
  • the fusion proteins, PBP's, proteins having expression modulator domains and proteases should be operatively linked to a constitutive or inducible promoter as described above.
  • the vectors may be constructed such that two or more of the following proteins: the fusion protein, the protease, and optionally, the PBP and/or protein having the expression modulator domain (if necessary for fusion protein function) are present in the same vector.
  • the transcription of these genes may be controlled by the same or different promoters. For example, each gene may be controlled by a different inducible promoter or each gene may be controlled by the same constitutive promoter.
  • the reporter plasmid and the vectors for expressing the fusion proteins, PBP's, proteases, and second proteins comprising the expression modulator domain may comprise two selectable markers—one that confers growth in prokaryotic cells such as bacteria and one that confers for growth in eukaryotic cells such as yeast, mammalian or plant cells in the presence of specific compounds.
  • Selectable markers useful in this invention may confer resistance to drugs such as ampicillin, kanamycin, hygromycin, neomycin or G418.
  • the vector encodes the fusion protein having the DNA sequence Seq. ID. No. 19.
  • a vector encoding the fusion protein having the DNA sequence, Seq. ID. No. 21, is used as a control.
  • the reporter plasmid is derived from PIND from Invitrogen.
  • the vectors and reporter plasmids are to be used in a transcriptional assay in a cell, they may be introduced into the host cells by techniques known in the art such as transfection, lipofectin, cytofectin, particle bead bombardment, electroporation, microinjection, or viral infection [e.g., F. M. Ausubel et al., Eds. Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley-Interscience: New York (1991); Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd. Ed., Cold Spring Harbor Laboratory Press (1989)].
  • the DNA of interest may be cloned into a viral vector between two retroviral LTRs, used to generate retrovirus, and infect cells with the virus.
  • viruses useful according to this invention include adenovirus, adeno-associated virus, and vaccinia virus.
  • Methods for assaying for the presence of a reporter gene product are well known in the art.
  • methods for assaying the mRNA resulting from the transcription of the reporter gene are known [Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press:Plainview, N.Y.), 2nd Ed.; Eds. F. M. Ausubel et al., (1991) Current Protocols in Molecular Biology (John Wiley & Sons: New York, 5th Ed.]
  • Methods for assaying the protein encoded by the reporter gene are also known, and kits are available from companies for determining the same (e.g., Promega).
  • pVgRXR The plasmid pVgRXR was obtained from Invitrogen.
  • pVgRXR is a 8728 kb vector that expresses both a modified Ecdysone Receptor (VgEcR) and a Retinoid X Receptor (RXR) to form a heterodimeric nuclear receptor (FIG. 1).
  • VgEcR modified Ecdysone Receptor
  • RXR Retinoid X Receptor
  • FOG. 1 heterodimeric nuclear receptor
  • Transcription of the VgEcR gene is driven by the cytomegalovirus (CMV) immediate early promoter.
  • Transcription of the RXR gene is driven by the Rous sarcoma virus (RSV) promoter.
  • Stable cell lines expressing the genes may be made by selection on the antibiotic Zeocin.
  • the plasmids PIND was obtained from Invitrogen (FIG. 3).
  • the plasmid PIND is a 5024 bp vector based on the pcDNA3.1 vector. It has five hybrid LREs, i.e., five ecdysone/glucocorticoid response elements (E/GRE), and the heat shock minimal promoter.
  • the E/GRE's can be recognized by a modified ecdysone receptor expressed from pVgRXR.
  • the plasmid pIND-Luc was made by digesting the luciferase gene out of another plasmid, including its Kozak sequence and methionine start site and cloning it into the poly linker of PIND, i.e., downstream of the minimal heat shock promoter (FIG. 3). Both plasmids have neomycin and ampicillin resistance genes for selection purposes. Product information regarding the features and the maintenance of the pIND and pIND-Luc vectors are incorporated by reference from the Invitrogen technical manual.
  • the mammalian expression plasmid pSRa has been described in the art [Y. Takebe et al., Mol. Cell. Biol., 8, pp. 466-72 (1988) incorporated by reference herein].
  • the plasmid contains a promoter system composed of the simian virus 40 (SV40) early promoter and the R segment and part of the U5 sequence (R-U5′) of the long terminal repeat of human T-cell leukemia virus type 1 (HTLV-1).
  • the plasmid also contains a 16S splice junction, an SV40 polyadenylation signal and an ampicillin resistance gene.
  • the HTLV LTR enhancer/promoter sequence drives high level expression of genes cloned downstream. Genes of interest may be cloned into the Pst I and EcoRl cites located 3′ to the 16S splice junction.
  • the VP16/5A5BF primer (Seq. ID. No. 12) is a 28-mer designed to hybridize to an Xba I site upstream of the VP16 coding region of the pVgRXR plasmid.
  • the VP16/5A5BR primer (Seq. ID. No. 13) is a 86-mer designed to hybridize in part to the antisense strand in the 3′ region of the DNA encoding the VP16 activation domain of the pVgRXR plasmid.
  • the primer also comprises nucleotides complementary to the coding sequences for the 5A/5B cleavage site of the HCV NS3-4A protease and an Xba I site.
  • the VP16/5A**5BR primer (Seq. ID. No. 14) is a 92-mer designed to hybridize in part to the antisense strand in the 3′ region of the DNA encoding the VP16 activation domain of the pVgRXR plasmid.
  • the primer also has nucleotides sequences complementary to the coding sequences for the 5A/5B cleavage site of the HCV NS3-4A protease.
  • the DNA encoding the 5A/5B cleavage site additionally has a six nucleotide insertion between the DNA sequences encoding a cysteine and a serine codon. The six nucleotide insertion encodes two stop codons.
  • the NS3-4AF primer (Seq. ID. No. 15) is a 47-mer designed to bind partly to the start of the NS3 coding region (corresponding to amino acid #1027-1032 in the HCV polypeptide). The remainder of the oligonucleotide contains multiple restriction sites for ease of cloning.
  • the NS3-4AB primer (Seq. ID. No. 16) is a 44-mer designed to bind partly to the 3′ end of the NS4A coding region (corresponding to amino acid #1705-1711 in the HCV polypeptide). The remainder of the oligonucleotide contains multiple restriction sites for ease of cloning.
  • Each primer was synthesized under standard conditions known in the art using an oligonucleotide synthesis machine.
  • the first step towards preparing the expression vector encoding VgRXR-5A/5B involved a PCR reaction using the VP16/5A5BF primer (Seq. ID. No. 12), the VP16/5A5BR primer (Seq. ID. No. 13) and the pVgRXR plasmid as the template.
  • the PCR product was digested with Xba I.
  • a vector for ligation with the PCR product was prepared by digesting the pVgRXR vector with Xba I to remove the DNA encoding the VP16 activation domain.
  • the vector backbone was isolated and the digested, PCR insert was ligated into the Xba I site in the backbone. The ligations were screened by sequencing to ensure the proper orientation of the insert and correctness of its nucleotide sequence.
  • the first step towards preparing the expression vector encoding VgRXR-5A**5B involved a PCR reaction using the VP16/5A5BF primer (Seq. ID. No. 12), the VP16/5A**5BR primer (Seq. ID. No. 14) and the pVgRXR plasmid as the template.
  • the PCR product was digested with Xba I.
  • a vector for ligating with the PCR product was prepared by digesting the pVgRXR vector with Xba I to remove the DNA encoding the VP16 activation domain.
  • the vector backbone was isolated and the digested PCR insert was ligated into the Xba I site in the backbone. The ligations were screened by sequencing to ensure the proper orientation of the insert and the correctness of its nucleotide sequence.
  • the first step towards preparing the pSR ⁇ -NS3-4A vector encoding the NS3-4A cleavage site involved a PCR reaction using the NS3-4AF primer (Seq. ID. No. 15), the NS3-4AB primer (Seq. ID. No. 16) and full length HCV H strain cDNA as a template [Inchauspe et al., (1991), PNAS USA 88:10292-10296 incorporated by reference herein].
  • the PCR product was digested with Pst1 and EcoR1.
  • a vector for ligating with the PCR product was prepared by digesting the pSR ⁇ vector with Pst 1 and EcoR1.
  • the vector backbone was isolated and the PCR insert was ligated into the Pst1-EcoR1 site in the backbone.
  • the first step towards preparing the pSR ⁇ -NS3-4A(S1165A) vector encoding the NS3-4A mutant protease involved a PCR reaction using the NS3-4AF primer (Seq. ID. No. 15), the NS3-4AB primer (Seq. ID. No. 16) and the cDNA of the NS3 active site mutant S1165A [A. Grakoui et al., (1993) J. Virology 67: 2832-43 incorporated by reference herein].
  • the PCR product was digested with Pst1 and EcoR1.
  • a vector for ligating with the PCR product was prepared by digesting the pSRa vector with Pst1 and EcoR1.
  • the vector backbone was isolated and the PCR insert was ligated into the Pst1-EcoR1 site in the backbone.
  • the NS3-4A(S1165A) has a serine to alanine substitution at amino acid number 1165, which makes the protease inactive. Compare Seq ID Nos. 10 and Seq ID No. 11.
  • HCV NS3-4A protease ecdysone-inducible luciferase assays were generally carried out as described below.
  • COS African green monkey kidney cells were plated at approximately 150,000 cells/well in a 6-well plate. The next day, approximately 3.6 ⁇ g plasmid DNA was dissolved in 100 ⁇ l of Dulbecco's modified Eagle's media-Gibco BRL (DMEM), combined with 29 ⁇ l of Superfect reagent (Qiagen), and then mixed by pipetting. The mixture was incubated at room temperature for ten minutes.
  • DMEM Dulbecco's modified Eagle's media-Gibco BRL
  • Qiagen Superfect reagent
  • the amounts of DNA typically used in each experiment was as follows: 0.6 ⁇ g of pIND-Luc, 0.6 ⁇ g of pVgRXR-5A/5B or pVgRXR-5A**5B, and 0-2.4 ⁇ g of pSR ⁇ -NS3-4A or pSR ⁇ -NS3-4A (S1165A) or pSR ⁇ .
  • an exogenous ligand was added to the cells to induce transcription of the reporter gene.
  • the 10% FBS-DMEM was aspirated off the cells and replaced with a 10% FBS-DMEM solution containing a concentration of 1-10 ⁇ M muristerone A or 1-10 ⁇ M ponasterone A (Invitrogen, United Kingdom).
  • a protease inhibitor dissolved in dimethylsulfoxide(DMSO) was also added to the cells for a final concentration of 1-40 ⁇ M.
  • the cells were incubated with muristerone A or ponasterone A for 24 hours at 37° C. in a 5% CO 2 incubator.
  • the cells were washed with PBS.
  • the activity of the luciferase protein was measured using a luciferase assay kit (Promega). Specifically, the cells were lysed in 250 ⁇ l of Cell Culture Lysis Reagent. The cells were scraped from the plate and transferred to a microfuge tube. The extract was spun at 12,000 ⁇ g for 5 seconds. Twenty microliters of each sample was added to 100 ⁇ l of Luciferase Assay Reagent. The light produced by the reaction of the luciferase with the Reagent was measured in a luminometer and was reported as relative light units (RLU).
  • RLU relative light units
  • Vectors encoding fusion proteins (VgRXR, VgRXR-5A/5B, or VgRXR-5A(Stop)5B) and pIND-Luc were transfected into COS cells as described above (Example 2). On the following day, some of the cells were incubated with 1 ⁇ M of muristerone for 24 hours. The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 4.
  • the control fusion protein expressed by pVgRXR-5A(Stop)5B which lacks the ecdysone DNA binding domain shows negligible activity. Little or no difference is observed between the activities of the VgRXR and the VgRXR-5A/5B expressed fusion proteins. The results indicate that the insertion of the 5A/5B junction in the VgRXR expressed protein does not interfere with muristerone A-induced activity.
  • the reporter plasmid pIND-Luc was cotransfected with either pVgRXR-5A/5B or pVgRXR and increasing amounts ( ⁇ gs) of the DNA encoding the protease NS3-4A as indicated in FIG. 5. On the following day, the cells were incubated in 1 ⁇ M muristerone A. Next, the cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 5.
  • the fusion protein encoded by VgRXR-5A/5B demonstrated a high level of activity in the absence of the NS3-4A protease.
  • the activity of VgRXR-5A/5B-mediated luciferase activity decreased in a dosage-dependent manner with the cotransfection of DNA encoding the NS3-4A protease, but not with the DNA encoding the mutant protease NS3-4A(S1165A). This is presumably due to the inability of the mutant NS3-4A protease to cleave the ecdysone/VP16 fusion protein.
  • the plasmid pIND-Luc (0.6 ⁇ g) was cotransfected with pVgRXR-5A/5B (0.6 ⁇ g) in the absence or presence of 1.8 ⁇ g of pSR ⁇ NS3-4A.
  • the cells were incubated with varying amounts of ponasterone A, a ligand which induces the heterodimerization of ecdysone receptor with the mammalian retinoic acid receptor, RXR.
  • the cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 7.
  • ponasterone A is effective at activating the fusion protein EdR5A/5B (i.e., the protein encoded by VgRXR-5A/5B) in the absence of the protease NS3-4A.
  • EdR5A/5B i.e., the protein encoded by VgRXR-5A/5B
  • the greatest activation of the fusion protein occurred when ponasterone A was present in a concentration between 3.3-10 ⁇ M.
  • ponasterone A was found to be equally effective at inducing transcription as muristerone A in these assays.
  • pIND-Luc The plasmid pIND-Luc (0.6 ⁇ g) was cotransfected with pVgRXR-5A/5B (0.6 ⁇ g) in the absence or presence of 1.8 ⁇ g of pSR ⁇ NS3-4A. On the following day, the cells were incubated with 5 ⁇ M ponasterone A. Dimethylsulfoxide (DMSO) was also added to the cells to a final concentration of 0-1% DMSO (v/v). The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 8.
  • DMSO Dimethylsulfoxide
  • DMSO was added to cells to determine whether the presence of it interfered with the activation of the fusion protein by ponasterone A.
  • DMSO is a solvent that was sometimes used to dissolve protease inhibitor compounds prior to addition to these assays.
  • the DMSO concentration in cell culture did not exceed 0.1%. The results indicate that DMSO concentrations up to 1% had little or no effect on the ponasterone A-induced luciferase activity in this assay.
  • the plasmid pIND-Luc (0.6 ⁇ g) was cotransfected with pVgRXR-5A/5B (0.6 ⁇ g) in the presence of 1.8 ⁇ g of pSR ⁇ NS3-4A (1:3 ratio).
  • the cells were incubated with 5 ⁇ M ponasterone A and varying amounts of a compound VH-25531 as indicated in FIG. 9.
  • the cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 9.
  • VH-25531 in the absence of the NS3-4A protease does not significantly increase or decrease the activity of the fusion protein (compare lanes 1 and 3, FIG. 9).
  • VH-25531 does inhibit the activity of the NS3-4A protease especially at 20 ⁇ M (compare lanes 4-11 with claim 2, FIG. 9).
  • the results indicate that VH-25531 may inhibit the NS3-4A protease as much as 55.5% at a 20 ⁇ M concentration.

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Abstract

The present invention relates to novel fusion proteins, DNA molecules encoding the same, vectors comprising the DNA molecules, and host cells containing the vectors for use in measuring protease activity using a novel transcriptional assay. This invention also relates to a method for determining the inhibitory activity of a compound against a protease and to a method for comparing the activity of two proteases which recognize the same cleavage site. Kits for assaying protease activity comprising DNA molecules encoding the fusion protein substrates of this invention are also contemplated.

Description

    TECHNICAL FIELD OF THE INVENTION
  • The invention relates to fusion proteins, DNA molecules encoding the fusion proteins, vectors comprising the DNA molecules, host cells transformed with the vectors, and methods and kits for using them to determine the activity of a protease. Specifically, the invention relates to a fusion protein having a protease cleavage site, a ligand-binding domain, and a DNA-binding domain, wherein (1) the association of a ligand with the ligand-binding domain of said fusion protein mediates the binding of the DNA-binding domain of said fusion protein to a ligand-response element (“LRE”) that is operatively linked to a reporter gene; and wherein (2) the fusion protein comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates transcription of the reporter gene. The invention also relates to kits for assaying protease activity comprising DNA molecules encoding the fusion proteins, an appropriate ligand, and DNA molecules comprising a promoter-reporter gene construct and at least one LRE recognized by the DNA binding domain of the fusion protein. The DNA molecules in these kits may be isolated or present in host cells. [0001]
    • BACKGROUND OF THE INVENTION
  • Proteases play an important role in the regulation of biological processes in almost every life form from bacteria to virus to mammals. They perform critical functions in, for example, digestion, blood clotting, apoptosis, activation of immune responses, zymogen activation, viral maturation, protein secretion and protein trafficking. [0002]
  • Proteases have been implicated as the cause of or as contributors to several diseases such as Alzheimer's disease, cystic fibrosis, emphysema, hypertension, tumor invasion and metastasis and viral-associated diseases [e.g., Kim, T. W., et al., (1997) “Alternative Cleavage of Alzheimer-associated Presinilins During Apoptosis by a Caspase-3 Family Protease,” [0003] Science 277: 373-6; Lacana, E. et al., (1997) “Disassociation of Apoptosis and Activation of IL-1 beta-converting Enzyme/Ced-3 Proteases by ALG-2 and the Truncated Alzheimer's gene ALG-3,” J. Immunol. 158:5129-35; Birrer, P., (1995) “Proteases and Antiproteases in Cystic Fibrosis: Pathogenic Considerations and Therapeutic Strategies,” Respiration 62:25-8; Patel, T., et al., (1996) “The Role of Proteases During Apoptosis,” FASEB J. 10:587-97].
  • Several viral genomes also encode proteases that are important in the viral maturation process. For example, the viral aspartyl protease of the Human Immunodeficiency Virus (HIV) cleaves a HIV polypeptide containing the Gag and Pol polyproteins. [0004]
  • In another example, the hepatitis C virus (HCV) produces a long polypeptide translation product, NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, which is cleaved to produce at least 10 proteins. C, E1 and E2 are putative structural proteins, and the remainder are known as the nonstructural (NS) proteins. One of those proteins is NS3, a 70 kilodalton protein having serine protease activity. It is been shown that the protease activity of NS3 resides exclusively in the N-terminal 180 amino acids of the enzyme. The NS3 protease cleavages at four sites in the nonstructural region of the HCV polypeptide (3/4A, 4A/4B, 4B/5A, and 5A/5B). Another protein, NS4A, has 54 amino acids and has been characterized as a cofactor for the NS3 protease [C. Failla, et al., (1994) J. Virology 68:3753-3760]. The C-[0005] terminal 33 amino acids of NS4A are required for cleavage at the 3/4A site and 4B/5A sites and accelerate the rate of cleavage at the 5A/5B site. Several other NS3 serine protease-dependent cleavage site sequences have been identified in various strains of HCV [A. Grakoui, et al., (1993) J. Virology 67:2832-2843 incorporated by reference herein].
  • The ability to detect viral, cellular, or microorganism protease activity in a quick and simple assay is important in the biochemical characterization of these proteases, in detecting viral infection, and in the screening and identification of potential inhibitors. [0006]
  • Several protease assays are known in the art. T. A. Smith et al., [0007] Proc. Natl. Acad. Sci. USA, 88, pp. 5159-62 (1991); B. Dasmahapatra et al., Proc. Natl. Acad. Sci. USA, 89, pp. 4159-62 (1992); and M. G. Murray et al., Gene, 134, pp. 123-128 (1993) each describe protease assay systems utilizing the yeast GAL4 protein. Each of these documents describe inserting a protease cleavage site in between the DNA binding domain and the transcriptional activating domain of GAL4. Cleavage of that site by a coexpressed protease renders GAL4 transcriptionally inactive leading to the inablility of the transformed yeast to metabolize galactose.
  • Y. Hirowatari et al., [0008] Anal. Biochem., 225, pp. 113-120 (1995) describes an assay to detect HCV protease activity. In this assay, the substrate, HCV protease and a reporter gene are cotransfected into COS cells. The substrate is a fusion protein consisting of (HCV NS2)-(DHFR)-(HCV NS3 cleavage site)-Taxi. The reporter gene is chloramphenicol transferase (CAT) under control of the HTLV-1 long terminal repeat (LTR) and resides in the cell nucleus following expression. The uncleaved substrate is expressed as a membrane-bound protein on the surface of the endoplasmic reticulum due to the HCV NS2 portion. Upon cleavage, the released Tax1 protein translocates to the nucleus and activates CAT expression by binding to the HTLV-1 LTR. Protease activity is determined by measuring CAT activity in a cell lysate.
  • Each of the assays described above requires simultaneous (1) expression of an active protease and a substrate and (2) transcription of a reporter gene construct. The constitutive nature of these assays can often produce uncontrollable and undesirable effects. These effects may give rise to misleading or inaccurate conclusions regarding the activity of the protease. Thus, there is a need for a sensitive and quantitative protease assay that is inducible or can be readily controlled by the user. [0009]
    • SUMMARY OF THE INVENTION
  • The present invention fulfills this need by providing novel fusion proteins, DNA molecules encoding them, vectors comprising the DNA molecules, and host cells containing the vectors useful in a fusion protein ligand-dependent transcriptional assay to determine the activity of a protease. [0010]
  • The novel fusion protein comprises a protease cleavage site, a ligand-binding domain, and a DNA-binding domain, wherein (1) the association of a ligand with the ligand-binding domain of said fusion protein mediates the binding of the DNA-binding domain of said fusion protein to a LRE that is operatively linked to a reporter gene; and wherein (2) the fusion protein comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates the transcription of the reporter gene. [0011]
  • According to the methods of this invention, the binding of a ligand to the ligand binding domain of the uncleaved fusion protein initiates the activation or repression of transcription of the reporter gene at a discrete point in time. This inducibility allows the assay to be better controlled and therefore, produces more accurate results. [0012]
  • Cleavage of the fusion protein at the protease cleavage site deregulates transcription of the reporter gene by preventing the expression modulator domain from modulating transcription positively or negatively. The amount of cleaved fusion protein is quantitated by assaying an increase or decrease in transactivation of a reporter gene, whose expression is driven by a promoter which is modulated by the expression modulating domain of the fusion protein. [0013]
  • This invention also relates to a method for measuring the inhibitory activity of a compound against a protease comprising the steps of incubating the fusion protein with a protease in the presence or absence of a compound whose activity is being tested, adding a ligand to the incubation and quantifying the gene product produced from a reporter gene. [0014]
  • Yet another embodiment of this invention relates to a method for comparing the activity of two proteases or mutants of a protease which recognize the same cleavage site. [0015]
  • The invention also relates to kits for assaying protease activity comprising DNA molecules encoding the fusion protein, the fusion protein or host cells containing the DNA molecules, wherein the kit optionally includes DNA molecules encoding a protease, DNA molecules comprising a reporter gene whose expression is regulated by said fusion protein, a ligand which associates and regulates the activity of said fusion protein, and instructions for using said kit. Preferably, the DNA molecules in the kit have been engineered into a vector that allows their expression. A kit according to this invention, may comprise any one or all of the following DNA molecules transformed into a single host cell selected from the group consisting of: a DNA molecule encoding a fusion protein of this invention, a DNA molecule encoding a protease, a DNA molecule encoding a protein binding partner for the fusion protein and a DNA molecule comprising a promoter-reporter gene construct.[0016]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts the structure of the vectors pVgRXR, pVgRXR-5A/5B, and pVgRXR-5A(Stop)5B. The backslash positioned between the cysteine and the serine of the pVgRxR-5A/5B encoded protein indicates where the cleavage will take place. The two asterisks near the cleavage site of the VgRXR-5A(Stop)5B encoded protein indicate the placement of two stop codons. [0017]
  • FIG. 2 depicts the structure of a DNA vector encoding the HCV NS3-4A protease, pSRα-NS3-4A. [0018]
  • FIG. 3 depicts the structure of a control vector PIND and a reporter vector for Ecdysone-Inducible Luciferase Expression, pIND-luc. [0019]
  • FIG. 4 graphically depicts the results of transfections experiments into COS cells with any one of the following: (1) a DNA vector encoding a mammalian RXR receptor protein and a fusion protein comprising the DNA-binding domain of the Drosophila ecdysone receptor fused to the activation domain of the Herpes simplex virus VP16 protein (pVgRXR); (2) a DNA vector encoding an RXR receptor protein and a fusion protein comprising the DNA-binding domain of the Drosophila ecdysone receptor fused to the 5A/5B proteolytic cleavage site of HCV fused to the activation domain of the VP16 protein (pVgRXR-5A/5B); and (3) a DNA vector encoding a RXR receptor protein and a fusion protein as described above except that the DNA encoding the 5A/5B cleavage site is modified to contain tandem stop codons in the cleavage site (pVgRXR-5A(Stop)5B). A plasmid comprising a luciferase reporter gene was included in each transfection (pIND-luc). After the transfection, transcription of the reporter gene was induced by administering 1 μM muristerone A (an ecdysone analog) to the transfected cells. The data was obtained by measuring the luciferase activity present in the lysates from the transfected cells as described in Example 2. [0020]
  • FIG. 5 depicts the results of cotransfecting plasmids pVgRXR-5A/5B and pIND-luc into COS cells with increasing amounts of a plasmid encoding HCV NS3-4A protease (pSRαNs3-4A) or of a control plasmid (pSRα). After the transfection, transcription of the reporter plasmid was induced by administering 1 μM muristerone A to the transfected cells. [0021]
  • FIG. 6 graphically depicts the results of cotransfecting plasmids pVgRXR-5A/5B and pIND-luc into COS cells with increasing amounts of a plasmid encoding HCV NS3-4A protease or an inactivate mutant thereof (pSRαNs3-4A or pSRαNs3-4A(S1165A), respectively). After the transfection, transcription of the reporter gene was induced by administering 1 μM muristerone A to the transfected cells. [0022]
  • FIG. 7 graphically depicts the results of administering either 1, 3.3, 10, 33 or 100 μM of ponasterone A (an ecdysone analog) to COS cells after cotransfection with plasmids pVgRXR-5A/5B and pIND-Luc in the presence or absence of a plasmid encoding the HCV-NS3-4A protease (pSRαNs3-4A). [0023]
  • FIG. 8 graphically depicts the results of a control experiment demonstrating DMSO tolerance. Plasmids pVgRXR-5A/5B and pIND-Luc were cotransfected into COS cells with or without a plasmid encoding the HCV-NS3-4A protease (pSRαNs3-4A). After the transfection, transcription of the reporter plasmid was induced by administering 5 μM ponasterone A in the presence of increasing concentrations of dimethylsulfoxide (DMSO). [0024]
  • FIG. 9 graphically depicts the results of cotransfecting plasmids pVgRXR-5A/5B, pIND-Luc and pSRαNS3-4A into COS cells and then administering 5μM ponasterone A and varying amounts of a protease inhibitor (VH-25531).[0025]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides novel tools and methods for measuring protease activity. The activity of a protease is measured by using a novel fusion protein as a substrate in a transcriptional assay wherein transcription is activated or repressed at a discrete time point by addition of a ligand after a protease and a fusion protein of this invention are incubated together. [0026]
  • The present invention provides a fusion protein comprising: a protease cleavage site, a ligand binding domain, and a DNA binding domain, wherein (1) the association of a ligand with the ligand-binding domain of said fusion protein mediates the binding of the DNA-binding domain of said fusion protein to a ligand response element (“LRE”) operatively linked to a reporter gene; and wherein (2) the fusion protein comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates transcription of the reporter gene. [0027]
  • A protease cleavage site (hereinafter, “PCS”) according to this invention is a peptide incorporated into the primary sequence of the fusion protein of this invention. It may be located in any of the DNA binding domain, ligand binding domain, or, optionally, the expression modulator domain, if one exists, of the protein, as well as in between any two of these domains. [0028]
  • The presence of the cleavage site in the protein should not substantially interfere with the activity of the fusion protein's DNA binding domain, ligand binding domain, or, if present, expression modulator domain. [0029]
  • To ensure that the protease cleavage site does not substantially interfere with the activity of the above domains of the fusion protein, the activity of a fusion protein with the PCS may be compared with the activity of the same fusion protein without the PCS by gel shift assay to assess DNA binding or by transcriptional assay (e.g., D. Latchman, (1995) [0030] Eukaryotic Transcription Factors, 2nd Ed. Academic Press:London).
  • Also, the protease cleavage site should be engineered into the fusion protein such that, when cleaved, neither of the resulting fusion protein fragments are capable of modulating the expression of a target or reporter gene. [0031]
  • Preferably, there is only one target protease cleavage site located within the fusion protein substrate. [0032]
  • According to a preferred embodiment, the protease cleavage site is situated between the DNA binding domain and the effector modulator domain of the fusion protein. [0033]
  • In a preferred embodiment of this invention, the PCS has an amino acid sequence comprising a processing/protease cleavage site in the HIV gag and gag/pol polyproteins or in the HCV polyprotein. In a more preferred embodiment of this invention the HIV PCS site selected from the group consisting of Seq ID Nos. 1-10 [S. Pichuantes, et al., (1989) “Recombinant HIV1 Protease Secreted by [0034] Saccharomyces cervisiae Correctly Processes Myristylated gag Polyprotein,” Proteins: Structure, Function, and Genetics 6:324-337 is incorporated by reference.] In a more preferred embodiment of this invention, the PCS has the amino acid sequence of a cleavage site for the HCV NS3 serine protease or HIV aspartyl protease. In a still more preferred embodiment of this invention the PCS has the amino acid sequence of an HCV 5A/5B protease cleavage site. In a still more preferred embodiment of this invention, the 5A/5B protease cleavage site is Seq ID No. 10.
  • The next portion of the fusion protein of this invention is the ligand binding domain (hereinafter, “LBD”). The LBD is a peptide domain which binds to a ligand, wherein said ligand binding is required for said fusion protein to (1) bind to a LRE, wherein the element is operatively linked to a reporter gene; and/or (2) to bind to a protein binding partner as an interim step preceding the binding to the resulting fusion protein-protein binding partner complex to a LRE. Thus, ligand binding to the fusion protein ligand binding domain is necessary for the transcriptional regulation of the expression of said reporter gene. [0035]
  • Ligand binding domains suitable for use in this invention may be derived from ligand binding domains of transcription factors that initiate or repress of transcription upon ligand binding. Such transcription factors include hormone receptors [Freedman, L. P., 1998, “Molecular Biology of Steroid and Nuclear Hormone Receptors,” [0036] Progress in Gene Expression, Boston: Birkhauser; Tsai, M-J., 1994, “Mechanism of Steroid Hormone Regulation of Gene Transcription,” Molecular Biology Intelligence Unit, Austin: R. G. Landes Co.; Eggert, M. et al., “The Glucocorticoid Hormone Receptor,” Inducible Gene Expression, vol. 2 (1995) Birkhauser: Boston, Mass. Ed. P. A. Baeuerle. pp. 131-156; Piedrafita, F. J. and M. Pfahl, “The Thyroid Hormone Receptors,” Inducible Gene Expression, vol. 2 (1995) Birkhauser: Boston, Mass. Ed. P. A. Baeuerle. pp. 157-185; Keaveney M. and H. G. Stunnenberg, “Retinoic Acid Receptors,” Inducible Gene Expression, vol. 2 (1995) Birkhauser: Boston, Mass. Ed. P. A. Baeuerle. pp. 187-242), each of which is herein incorporated by reference]; carbohydrate-responsive transcription factors; metallothionein (MT) genes; orphan receptors; tetracycline-inducible transcription factors; the IPTG-inducible transcription factors; dioxin receptors; and aryl hydrocarbon receptors.]
  • In a preferred embodiment of this invention, the ligand binding domain is derived from the ligand binding domains of the steroid/thyroid hormone receptor superfamily. Amino acid sequences encoding steroid and nuclear receptor ligand binding domains are well known in the art [See, for example, S. Simons, (1998) “Structure and Function of the Steroid and Nuclear Receptor Ligand Binding Domain,” [0037] Molecular Biology of Steroid and Nuclear Hormone Receptors: Progress in Gene Expression, Birkhauser: Boston, Mass., the disclosure of which is herein incorporated by reference]. In a more preferred embodiment, the ligand binding domain is derived from an ecdysone receptor.
  • In a further embodiment of this invention, a ligand binding domain from a transcription factor may be modified in the fusion protein of this invention such that it binds to a different ligand. For example, a human progesterone receptor ligand binding domain may be mutated so that it is capable of binding the anti-progesterone compound RU486 (Wang, Y., et al., (1997) “Positive and Negative Regulation of Gene Expression in Eukaryotic Cells with an Inducible Transcriptional Regulator,” [0038] Gene Ther. 4:432-41, the disclosure of which is herein incorporated by reference). Such RU486-inducible GAL4 DNA binding domain fusion protein is contemplated for use in this invention.
  • A DNA binding domain (hereinafter, “DBD”) refers to a peptide sequence in the fusion protein according to this invention which recognizes and binds to a specific nucleotide sequence (e.g., a DNA element or a LRE). DNA binding domains useful according to this invention may be derived from the DNA binding domains of DNA binding proteins, such as transcription factors. [0039]
  • In a preferred embodiment, these DNA binding proteins are transcription factors that directly bind DNA and initiate or repress transcription. Such transcription factors are well known in the art [McKnight, S. L. and K. R. Yamamoto, 1992, “Transcriptional Regulation,” [0040] Cold Spring Harbor Monograph Series, Plainview, N.Y.: Cold Spring Harbor Laboratory Press; Latchman, D. S., 1995, Eukaryotic Transcription Factors, San Diego: Academic Press, 2.ed.; Latchman, D. S., 1993, “Transcription Factors: A Practical Approach,” The Practical Approach Series, New York: IRL Press at Oxford University Press; Papavassiliou, A., (1997) “Transcription Factors in Eukaryotes,” Molecular Biology Intelligence Unit, New York: Chapman & Hall; Eckstein, F., and D. M. J. Lilley, 1997, “Mechanisms of Transcription,” Nucleic Acids and Molecular Biology, Vol. 11, New York: Springer-Verlag, the disclosures of which are herein incorporated by reference].
  • Preferred DNA binding domains contemplated for use in this invention include the DNA binding domains of the following transcription factors: homeobox proteins, zinc finger proteins (Sluyser, M., 1993, [0041] Zinc Finger Proteins in Oncogenesis: DNA Binding and Gene Regulation, New York: New York Academy of Sciences), helix-turn-helix proteins, helix-loop-helix proteins (e.g., Littlewood, Trevor D., 1998, Helix-Loop-Helix Transcription Factors, 3rd. Ed., New York: Oxford University Press), leucine zipper proteins (e.g., Hurst, H. C., 1996, Leucine Zippers: Transcription Factors, 3rd. ed., San Diego: Academic Press), GAL4 protein, hormone receptors, orphan receptors, and E. coli transcription factors such as the lactose operon repressor, tetracycline-controlled transactivator, and FadR.
  • The present invention also contemplates the use of the DBD's of metal-binding DNA-binding proteins that regulate the transcription of metallothionein (MT) genes. Such metal-binding DNA-binding proteins include MTF-1, ACE1, and AMT1. [Heuchel, R., et al., “Transcriptional Regulation by Heavy Metals, Exemplified at the Metallothionein Genes,” [0042] Inducible Gene Expression, vol. 1 (1995) Birkhauser: Boston, Mass. Ed. P. A. Baeuerle. pp. 206-240.]
  • In a more preferred embodiment of this invention, the DNA-binding domain is obtained from a member of the steroid/thyroid superfamily of receptors. The members of the steroid/thyroid superfamily of receptors are known in the art as hormone binding proteins that function as ligand-dependent transcription factors. They include identified members of the steroid/thyroid superfamily of receptors for which specific natural ligands have not yet been identified (referred to herein as “orphan receptors”) [B. M. Forman, (1998) “Orphan Nuclear Receptors and Their Ligands,” [0043] Molecular Biology of Steroid and Nuclear Hormone Receptors, L. P. Freedman, Ed., Birhauser: Boston, pp. 281-305.]
  • Members of the orphan receptors useful in this invention include HNF4, the COUP family of receptors and COUP-like receptors, peroxisome proliferator-activated receptors (PPARs), insect derived knirps and knirps-related receptors, and various isoforms thereof. [0044]
  • The amino acid sequences encoding such DBD's are well known in the art [F. Rastinejad, (1998) “Structure and Function of the Steroid and Nuclear Receptor DNA Binding Domains,” [0045] Molecular Biology of Steroid and Nuclear Hormone Receptors: Progress in Gene Expression Birkhauser: Boston, Mass., the disclosure of which is herein incorporated by reference].
  • Like the LBD used in the fusion proteins of this invention, the DNA binding domains of this invention may be modified from the sequence present in the source transcription factor so that it recognizes a different LRE. For example, the amino acid sequence of the DNA binding domain of a steroid/thyroid family members may be modified such that the DNA binding domain binds to a LRE recognized by another steroid/thyroid family member. For example, modification of the “P-box” amino acid sequence of a steroid/thyroid family member, i.e., a region in the DNA-binding domain that is typically located at the junction of the first zinc finger and the linker region, is contemplated by this invention (Umesono et al., (1989) [0046] Cell 57:1139-1146, particularly FIG. 2 and Table 1; the disclosure of which is herein incorporated by reference).
  • In a preferred embodiment, the DNA binding domain is derived from the Drosophila ecdysone receptor, wherein the P-box of the ecdysone receptor having the amino acid sequence EGCKG (SEQ. ID. NO. 23) is replaced with the amino acid sequence GSCKV (SEQ. ID. NO. 24). The new P-box sequence GSCKV causes the ecdysone receptor to recognize the LRE -AGAACA- instead of the LRE -AGGTCA-. [0047]
  • Members of the steroid/thyroid superfamily of receptors that are particularly useful for providing the LBD and/or the DBD of a fusion protein of this invention include steroid receptors such as the glucocorticoid receptor (GR), the mineralcorticoid receptor (MR), the estrogen receptor (ER), the progesterone receptor (PR) such as hPR-A or hPR-B, the androgen receptor (AR), the vitamin D3 receptor (VDR); retinoid receptors such as retinoic acid receptors (e.g., RARα, RARβ, or RARγ) and retinoid X receptors (e.g., RXRα, RXRβ, or RXRγ); thyroid receptors (TR) such as TRα and TRβ; insect-derived receptors such as the ecdysone receptors; and isoforms thereof. [0048]
  • According to one preferred embodiment, a fusion protein of this invention contains a DNA binding domain which is engineered near or next to a ligand binding domain and neither of the two domains contains a PCS, nor is a PCS located between the two domains. [0049]
  • An optional component of the fusion protein of this invention is an expression modulator domain. As stated above, if the expression modulator domain is not present on the fusion protein, it must be present on a protein that associates with the fusion protein. [0050]
  • Expression modulator domains contemplated for use in this invention, either as part of the fusion protein or a separate protein which associates with the fusion protein, are typically derived from DNA binding proteins, such as transcription factors. These sequences are known in the art to activate or to repress transcription through their interaction with other transcription factors necessary for transcription. [0051]
  • In embodiments where the expression modulator domain is present within a second protein that binds to the fusion protein, that second protein should not increase or decrease transcription in the absence of a fusion protein of this invention and its ligand. In embodiments where the expression modulator domain is present within the fusion protein it is preferably located at the terminus opposite to the location of the DNA binding domain. [0052]
  • In a preferred embodiment of this invention, the expression modulator domain activates transcription as opposed to repressing that activity. Such activation domains include the N-terminal regions encoding activation domains in the steroid/thyroid superfamily of receptors, the activation domains of viral transcription factors, the yeast GCN4 activation domain or the GAL4 activation domain [K. Struhl, “Yeast GCN4 Transcriptional Activator Protein,” [0053] Transcriptional Regulation, Eds. S. L. McKnight and K. R. Yamamoto, Cold Spring Harbor Laboratory Press (1992), pp. 833-859; A. A. F. Gann et al., “GAL11, GAL11P, and the Action of GAL4,” Transcriptional Regulation, Eds. S. L. McKnight and K. R. Yamamoto, Cold Spring Harbor Laboratory Press (1992), pp. 931-946; K. J. Martin and M. R. Green, “Transcriptional Activation by Viral Immediate-Early Proteins: Variations on a Common Theme,” Transcriptional Regulation, Eds. S. L. McKnight and K. R. Yamamoto, Cold Spring Harbor Laboratory Press (1992), pp. 695-725, the disclosures of which are herein incorporated by reference].
  • In another preferred embodiment, the expression modulating domain is part of the fusion protein. An even more preferred is when the expression modulating domain is the N-terminal activation domain of the VP16 protein. [0054]
  • It should be understood that the ligand binding domain, DNA binding domain and expression modulator domain need not be derived from the same transcription factor. [0055]
  • For example, a chimera comprising the DNA-binding domain of the retinoic acid receptor and the ligand-binding domain of the vitamin D receptor (VDR) may be made (Pemrick, S., et al., (1998) “Characterization of the Chimeric Retinoic Acid Receptor RARα/VDR,” [0056] Leukemia 12:554-562) or a chimera comprising the Jun DNA binding domain may be fused to the estrogen receptor ligand binding domain and expression modulator activation domain (Kruse, U., et al., (1997) “Hormone-regulatable Neoplastic Transformation Induced by a Jun-Estrogen Receptor Chimera,” PNAS USA 94:12396-12400).
  • Other examples include the fusion of the lacR DNA-binding domain and the VP16 activation domain. This creates a fusion protein that requires IPTG to bind to the LRE, lacO [Labow, M. et al., (1990) [0057] Mol. Cell. Biol. 10:3343-3356]. In another example, the fusion of the tetR protein and the VP16 activation domain creates a fusion protein which releases from the LRE, tetO, in the presence of tetracycline in cells and in transgenic animals [Gossen, M. and H. Bujard, (1992) “Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters,” PNAS USA 89:5547-5551; Furth, P. A., et al., (1994) “Temporal Control of Gene Expression in Transgenic Mice by a Tetracycline-Responsive Promoter,” PNAS USA 89:9302-9306]. A tetR-VP16 fusion protein according to this invention may contain a protease cleavage site in its DNA binding domain such that DNA binding is eliminated when the PCS is cleaved. Thus, upon addition of tetracycline, uncleaved fusion protein would continue to transactivate from tetO, whereas cleaved fusion proteins would be inactive.
  • A preferred embodiment of this invention is the fusion of the VP16 activation domain of herpes simplex virus, the ligand binding domain of a steroid/thyroid receptor superfamily member and the DBD of a steroid/thyroid receptor superfamily member. In a more preferred embodiment, the steroid/thyroid receptor superfamily member is the ecdysone receptor. [0058]
  • As described above, the invention also contemplates fusion proteins which require contact with a protein binding partner (“PBP”) in order to bind DNA or to increase specificity of binding or affinity of binding of the fusion protein to its LRE. Thus, a protein binding partner according to this invention is a protein that binds to a fusion protein of this invention and increases the affinity or specificity of binding of the DNA-binding domain of the fusion protein to a specific LRE. The protein binding partner is a protein that also binds to a particular DNA element that is operatively linked to the reporter gene. If the fusion protein of this invention requires a protein binding partner for increased affinity or specificity of DNA binding, then it is desirable to ensure that the fusion protein includes a dimerization domain for interaction with the PBP. [0059]
  • Protein binding partners that may be suitable for interacting with the dimerization domains of the fusion proteins of this invention are known in the art. [e.g., T. D. Littlewood and G. I. Evan (1998) “Structure/Function Relationships of HLH Proteins,” [0060] Helix-Loop-Helix Transcription Factors, 3rd. Ed., New York: Oxford University Press, pp. 27-41; Hurst, H. C., 1996, Leucine Zippers: Transcription Factors, 3rd. ed., San Diego: Academic Press, pp. 27-29; and L. P. Freedman, Ed., (1998) Molecular Biology of Steroid an Nuclear Hormone Receptors: Progress in Gene Expression, Birkhauser: Boston, Mass.].
  • For example, several heterodimeric partners of basic-helix-loop-helix (bHLH) transcription factors are known in the art. [Littlewood, Trevor D., 1998, [0061] Helix-Loop-Helix Transcription Factors, 3rd. Ed., New York: Oxford University Press) pp. 37-41] In a more specific example, bHLH transcription factors such as the ligand-dependent dioxin receptors and aryl hydrocarbon receptors (AHRs) heterodimerize with the AHR nuclear translocator protein (ARNT) [Whitelaw, M. L. et al., (1994) “Identification of Transactivation and Repression Functions of the Dioxin Receptor and Its Basic Helix-Loop-Helix/PAS Partner Factor Arnt: Inducible Versus Constitutive Modes of Regulation,” Mol. Cell. Bio. 14:8343-8355].
  • Another example is the steroid/thyroid family of receptors which have the ability to heterodimerize with each other or with other non-steroid/thyroid receptor family members (e.g., Rhee, et al., (1995) “Retinoid X-Receptor-alpha and Apolipoprotein [0062] AI Regulatory Protein 1 Differentially Modulate 3,5,3′-Triiodothyronine-Induced Transcription,” Endocrinology 136:2697-704; Li, et al., (1997) “Coexpression of Nuclear Receptors Partners Increases Their Solubility and Biological Activities,” PNAS USA 94:2278-83).
  • Yet another example are the bZIP factors which heterodimerize with several other proteins. [e.g., H. C. Hurst, [0063] Leucine Zippers: Transcription Factors, 3rd Ed. London: Academic Press, (1996), page, 28.] Such heterodimers and homodimers comprising a fusion protein of this invention are contemplated.
  • In a preferred embodiment of this invention, a P-box modified ecdysone receptor heterodimerizes with mammalian RXR. [0064]
  • A fusion protein of this invention for use in cellular transcription assays should localize to the nucleus of the host cell. A nuclear localization signal already present within a domain of a fusion protein of this invention may provide the nuclear localization signal. Alternatively, a nuclear localization signal known in the art may be engineered into the fusion protein to ensure nuclear localization. [e.g., D. B. DeFranco, (1998) “Subcellular and Subnuclear Trafficking of Steroid Receptors,” [0065] Molecular Biology of Steroid and Nuclear Hormone Receptors: Progress in Gene Expression, Birkhauser: Boston, Mass.; Guiochon-Mantel A, et al., (1996) “The Ernst Schering Poster Award. Intracellular Traffic of Steroid Hormone Receptors,” J. Steroid Biochem. Mol. Biol. 56:3-9, the disclosures of which are herein incorporated by reference].
  • In an alternative embodiment of the methods of this invention, the fusion protein lacks a ligand binding domain. Specifically, in this embodiment, the fusion protein comprises (1) a DNA-binding domain and (2) a protease cleavage site. In these methods, the transcriptional activity of the fusion protein is inducible by altering the temperature of the transcription environment or causing stress to the cells in which the transcription event takes place. For example, high affinity binding of a heat shock factor (“HSF”) to its response element, HSE, is dependent on a heat shock stimulus. [e.g., J. Lis and C. Wu, “Heat Shock Factor,” [0066] Transcriptional Regulation, Eds. S. L. McKnight and K. R. Yamamoto, Cold Spring Harbor Laboratory Press (1992), pp. 907-930; D. S. Latchman, “Transcription Factors and Inducible Gene Expression,” Eukaryotic Transcription Factors, 2nd Ed. London: Academic Press Limited, (1995) pp. 71-78.]
  • A protease cleavage site of interest may be engineered into the HSF protein such that the transcriptional activity of the cleaved HSF fusion protein is lower or negligible compared to uncleaved HSF fusion protein after stimulus by heat. [0067]
  • The use of the fusion proteins, DNA molecules and vectors of this invention contemplated by the methods of this invention include cellular and in vitro transcription reactions. [0068]
  • Methods for conducting transcription assays in cells are known. [e.g., R. White and M. Parker, “Analysis of Cloned Factors,” [0069] Transcription Factors: A Practical Approach, Ed. D. S. Latchman, IRL Press: Oxford (1993) pp. 145-152; F. M. Ausubel et al., Eds. Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley-Interscience: New York (1991); Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd. Ed., Cold Spring Harbor Laboratory Press (1989)]. Briefly, DNA encoding a fusion protein of this invention, a reporter plasmid, a protease and, optionally, a second protein having a expression modulator domain will be introduced into a cell. Next, the transcriptional activity of the fusion protein will be induced by adding a ligand or by altering the environment of the cell (e.g., changing the temperature of the environment of the cell). The quantity of mRNA, the quantity of reporter protein, or the activity of the reporter protein will be measured using standard techniques known in the art. Transcription in the absence of protease can be compared with transcription in the presence of protease.
  • Methods for conducting in vitro transcription reactions are also known [Sierra, F., et al., “In vitro Transcription with Nuclear Extracts from Differentiated Tissues,” [0070] Gene Transcription: A Practical Approach (1993) Oxford University Press: Walton Street, Oxford. Ed. D. Hames and S. Higgins. pp. 125-152; Snoek, R. et al., (1996) “Induction of Cell-Free, In Vitro Transcription by Recombinant Androgen Receptor Peptides,” J. Steroid Biochem. Molec. Biol. 59:243-250; Dignam, J. D., et al., (1992) “Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells,” In Current Protocols in Molecular Biology (Ed. by F. A. Ausubel et al.) John Wiley and Sons: New York pp. 12.1.1-12.1.9.; Klein-Hitpaiss, L., et al., (1990) “The Progesterone Receptor Stimulates Cell-Free Transcription by Enhancing the Formation of a Stable Preinitiation Complex,” Cell 60:247-257].
  • Briefly, cellular extracts prepared by protocols which have been shown to support in vitro transcription reactions would be prepared. Purified or partially purified proteins would be added to the cellular extract together with a reporter plasmid. Such purified or partially purified proteins, such as the fusion protein of this invention may be produced by overexpression in a cell introduced to the DNA encoding the fusion protein, e.g., SF9 cells via baculovirus infection, plant cells, yeast cells, mammalian cells, and bacterial cells. Proteins such as the protease and, optionally, the second protein having an expression modulator domain and/or a protein binding partner may also be prepared from their original sources using methods known in the art to purify them. [0071]
  • Next, the transcriptional activity of the fusion protein will be induced by adding a ligand. The quantity of mRNA produced, the quantity of reporter protein produced, or the activity of the reporter protein will be measured using standard techniques known in the art. Transcription in the presence of protease can be compared with transcription in the absence of protease. [0072]
  • The invention also provides a method for screening compounds for inhibitory protease activity. A compound to be screened may be added at the same time the ligand is added or added during the initial expression/incubation of the protease with a fusion protein. The optimal level of compound needed to achieve the greatest inhibition of the protease may be determined by titration of the compound. [0073]
  • The invention also provides for a method for comparing the activity of two proteases which recognize the same protease cleavage site. The method is useful for comparing the activity of mutant and non-mutant proteases, e.g., HIV aspartyl proteases from patients. [0074]
  • The invention also provides a method for comparing the activity of a protease against different protease cleavage sites. This method is useful for determining substrate specificity of a given protease The invention also provides kits for assaying protease activity. If the kit is to be used in an in vitro transcription assay of a reporter gene, then it may comprise an in vitro transcription extract, a vector as described above containing a reporter gene, a supply of ligand, and instructions, optionally, it may provide a supply of a fusion protein of this invention, a second protein comprising an expression modulator domain, and/or a protease expressed from a source including bacteria, insect cells, mammalian cells, and yeast. [0075]
  • If the kit is to be used in a cellular transcription assay, the kit may include a DNA sequence encoding a fusion protein of this invention and a vector encoding a reporter plasmid as described above, a ligand which associates with and regulates the activity of said fusion protein encoded by the DNA sequence of the protein; and instructions for using said kit to assay protease activity. [0076]
  • Proteases that are particularly useful in the methods of this invention are proteases that contribute to the symptoms or to the onset of a disease. For example, those proteases involved in digestion, blood clotting, apoptosis, activation of immune responses, zymogen activation, viral maturation, protein secretion and protein trafficking. In one embodiment of the invention, the proteases of interest are those involved in Alzheimer's disease, cystic fibrosis, emphysema, hypertension, tumor invasion and metastasis and viral-associated diseases. According to a preferred embodiment of this invention, the proteases are HIV aspartyl protease and HCV NS3 protease, and active fragments or fusion proteins thereof. [0077]
  • In one embodiment of this invention, any one or all of the DNA molecules encoding the fusion proteins, PBP's, proteases and second proteins comprising the expression modulator domain may be further modified to provide an additional polypeptide sequence for ease in purification or identifying of the expression of the protein. For example, such polypeptide sequences may include an epitope for binding an antibody, an Fc portion of an antibody for binding protein A beads, glutathione S transferase, maltose binding protein, His(n), FLAG, and Strep-tag. These screenable tags are all well known in the art and are fully available to the person skilled in the art. [0078]
  • Several vectors useful for overexpression of proteins of this invention are commercially available or known for expression in yeast, insect cells, bacteria, yeast, plant cells and mammalian cells. [0079]
  • Host cells useful according to the methods of this invention are well known in the art. If the activity of an exogenous protease is being studied, it is desirable that the host cell does not express high levels of endogenous proteases which recognize the protease cleavage site(s) being targetted. Suitable host cells may include CHO cells; HeLa cells; liver cells; CV-1 cells; P19 cells; NT2/D1 cells; mouse L cells; African Green monkey kidney cells, such as COS-7 cells or other COS cells; human embryonic kidney cells, such as HEK 293; DG44 cells, ltk-cells, mouse NIH 3T3 cells and yeast cells. The host cells may be transiently transfected or stably transformed cell lines. In a preferred embodiment of this invention, the host cells are COS cell or cells from a liver cell line. [0080]
  • Ligands suitable for use in the methods of this invention are well known in the art. The ligands should bind to the ligand binding domains of the transcription factors from which the ligand binding domains of the fusion proteins have been derived, or to modified ligand binding domains thereof (see above). Preferably, the ligand does not substantially enhance or repress transcription from the promoter unless a LRE for the fusion protein is spliced or inserted into an vector comprising a promoter and a reporter gene, wherein the LRE is linked to a promoter in a manner which makes transcriptional activity from the promoter operatively responsive to ligand. [0081]
  • Choice of ligand to use will necessarily depend on the ligand binding domain of the fusion protein being used. For example, suitable ligands for use with metal-binding DNA fusion proteins may include zinc, cadmium, and copper. [0082]
  • For example, suitable ligands for use with steroid binding or modified steroid binding fusion proteins may include androgen, glucocorticoid, mineralocorticoids, thyroid hormones, estrogen, progesterone, progestogen, retinoic acid, vitamin D3, 20-hydroxy-ecdysone, ponasterone A, 26-iodoponasterone A, muristerone A, inokosterone, 26-mesylinokosterone, mifepristone (RU 486) and analogs thereof. [0083]
  • As used herein, androgens include dihydroxytestosterone and analogs thereof including methyltrienolone. [0084]
  • As used herein, glucocorticoid hormones include cortisol, hydrocortisone, and corticosterone, and analogs therof including dexamethasone, deoxycorticosterone, and triamcinolone acetonide. [0085]
  • As used herein, mineralcorticoids include aldosterone, coricosterone and deoxycorticosterone. [0086]
  • As used herein, thyroid hormones include thyroxine (T4) and triodothyronine (T3). [0087]
  • As used herein estrogens include estradiol-17 beta, and analogs thereof indulcing diethylstilbestrol. [0088]
  • As used herein progestogens include analogs of progesterone including promegestrone. [0089]
  • Suitable ligands for tetracycline-inducible fusion protein include tetracycline and analogs thereof. [0090]
  • Suitable ligands for carbohydrate-inducible fusion proteins include arabinose, lactose, and isopropyl β-D-thiogalactoside (IPTG). [0091]
  • Suitable ligands for aryl hydrocarbon inducible fusion proteins include dioxin. [0092]
  • Suitable ligands for orphan receptors include the following: phytenic acid, 9-cis retinoic acid, and LG100268 for the RXR receptor; 8S-HETE and Wy 14,643 for the PPARα receptor; 15-deoxy-Δ[0093] 12,14-PGJ2 and BRL 49653 for the PPARγ receptor; linoleic acid and carba-prostacyclin for the PPARδ receptor; farnesol for the FXR receptor; and 24(S),25-epoxycholesterol for the LXR receptor. [B. M. Forman, (1998) “Orphan Nuclear Receptors and Their Ligands,” Molecular Biology of Steroid and Nuclear Hormone Receptors, L. P. Freedman, Ed., Birhauser: Boston, pp. 281-305.]
  • The LREs and PBP-DNA elements in this invention are nucleic acid molecules that provide DNA binding sites for the fusion proteins and the protein binding partners, respectively. Both elements should be operatively linked to a promoter to control the activation or repression of transcription of a reporter gene. The phrase “operatively linked” refers to linking a nucleic acid molecule (e.g., a reporter gene encoding a reporter protein) to a LRE and a PBP-DNA element (if PBP binding is necessary for transcription) and to transcription control sequences in a manner such that the nucleic acid molecule molecule is able to be expressed when introduced (e.g., transfected, transformed, transduced, conjugated, or by recombination or by infection) into a host cell. [0094]
  • Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as the promoter. Preferably, the promoter is a minimal promoter. The term “minimal promoter” is intended to describe a partial promoter sequence which defines the start site of transcription for the linked sequence to be transcribed, but which by itself is not capable of initiating transcription efficiently, if at all, in a certain cell environment. Thus, the activity of such a minimal promoter is dependent upon the binding of a fusion protein of this invention. [0095]
  • In a preferred embodiment, the minimal promoter is from the drosophila heat shock promoter and the target LRE for the fusion protein is AGAACA generated as described in PCT/US97/05330 (incorporated by reference herein). [0096]
  • LREs according to this invention should be operative to confer responsiveness to a ligand. In a further embodiment, DNA elements that bind to PBP's of this invention may also be ligand-responsive if the PBP used is a ligand-binding protein. Choice of LRE and DNA element (as necessary) and the arrangement of those elements with respect to each other will depend upon the the fusion protein or the PBP used. For example, if the DNA-binding domain of the fusion protein is from a basic-helix-loop-helix protein, the LRE would likely comprise the consensus DNA hexamer CANNTG (also known as an “E box”) (e.g., Littlewood, et al., supra, page 31). [0097]
  • The preferred DNA binding sites (i.e., LREs and DNA elements) for many transcription factors are known. [D. J. Mangelsdorf and R. M. Evans, (1992) “Retinoid Receptors as Transcription Factors,” [0098] Transcriptional Regulation, Eds. S. L. McKnight and K. R. Yamamoto, pp. 1137-1167; L. P. Freedman, Ed., (1998) Molecular Biology of Steroid and Nuclear Hormone Receptors: Progress in Gene Expression, Birkhauser: Boston, Mass., pp. 111-113; and PCT/US97/05330 incorporated by reference herein.]
  • The promoter controlling the expression of the fusion proteins, PBP's, and proteases in a mammalian cell may be constitutive such as the cytomegalovirus (CMV) promoter, the SV40 early promoter, and the Rous Sarcoma Virus (RSV) promoter. For added control, the fusion protein, PBP's, and proteases may be under the control of an inducible promoter. It is desireable that the inducible promoter not be the same as the promoter that drives transcription of the reporter gene. [0099]
  • Reporter genes useful according to the methods of this invention are well known in the art. Such reporter genes include the luciferase gene, green fluorescent protein gene (U.S. Pat. No. 5,491,084), β-galactosidase, secreted alkaline phosphatase gene and the chloramphenicol acetyl transferase gene. Also, the invention contemplates reporter genes encoding a marker protein with a signal sequence for secretion out of the cell such as the IL-1 beta gene. [0100]
  • Vectors containing the reporter gene operatively linked to an LRE and DNA element (i.e., the reporter plasmid) may be prepared from materials available in the art. Such reporter plasmids preferably also include, for example, the following: an origin of replication and a selectable marker(s). The reporter plasmid may optionally also include other DNA sequences, such as long terminal repeats (“LTR's”), to cause the insertion of the vector into the genome of a cell line. [0101]
  • Vectors suitable for expressing the fusion proteins, PBP's, proteins having an expression modulator domain or proteases in bacteria or mammalian cells are well known in the art. Such vectors may for example include the following: an origin of replication, a selectable marker and transcription control sequences such as a promoter and a polyadenylation sequence. Thus, the fusion proteins, PBP's, proteins having expression modulator domains and proteases should be operatively linked to a constitutive or inducible promoter as described above. The vectors may be constructed such that two or more of the following proteins: the fusion protein, the protease, and optionally, the PBP and/or protein having the expression modulator domain (if necessary for fusion protein function) are present in the same vector. The transcription of these genes may be controlled by the same or different promoters. For example, each gene may be controlled by a different inducible promoter or each gene may be controlled by the same constitutive promoter. [0102]
  • The reporter plasmid and the vectors for expressing the fusion proteins, PBP's, proteases, and second proteins comprising the expression modulator domain may comprise two selectable markers—one that confers growth in prokaryotic cells such as bacteria and one that confers for growth in eukaryotic cells such as yeast, mammalian or plant cells in the presence of specific compounds. Selectable markers useful in this invention may confer resistance to drugs such as ampicillin, kanamycin, hygromycin, neomycin or G418. [0103]
  • In a preferred embodiment of this invention, the vector encodes the fusion protein having the DNA sequence Seq. ID. No. 19. In a further preferred embodiment of this invention, a vector encoding the fusion protein having the DNA sequence, Seq. ID. No. 21, is used as a control. In a preferred embodiment of this invention, the reporter plasmid is derived from PIND from Invitrogen. [0104]
  • If the vectors and reporter plasmids are to be used in a transcriptional assay in a cell, they may be introduced into the host cells by techniques known in the art such as transfection, lipofectin, cytofectin, particle bead bombardment, electroporation, microinjection, or viral infection [e.g., F. M. Ausubel et al., Eds. Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley-Interscience: New York (1991); Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd. Ed., Cold Spring Harbor Laboratory Press (1989)]. [0105]
  • In the case of viral infection, the DNA of interest may be cloned into a viral vector between two retroviral LTRs, used to generate retrovirus, and infect cells with the virus. Other viruses useful according to this invention include adenovirus, adeno-associated virus, and vaccinia virus. [0106]
  • Methods for assaying for the presence of a reporter gene product are well known in the art. For example, methods for assaying the mRNA resulting from the transcription of the reporter gene are known [Sambrook et al., (1989) [0107] Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press:Plainview, N.Y.), 2nd Ed.; Eds. F. M. Ausubel et al., (1991) Current Protocols in Molecular Biology (John Wiley & Sons: New York, 5th Ed.] Methods for assaying the protein encoded by the reporter gene are also known, and kits are available from companies for determining the same (e.g., Promega).
  • In order that the invention described herein may be more fully understood, the following examples are set forth. These examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. [0108]
    • EXAMPLE 1 Construction of DNA Vectors
  • A. Materials [0109]
  • (1) Vectors [0110]
  • The plasmid pVgRXR was obtained from Invitrogen. pVgRXR is a 8728 kb vector that expresses both a modified Ecdysone Receptor (VgEcR) and a Retinoid X Receptor (RXR) to form a heterodimeric nuclear receptor (FIG. 1). Transcription of the VgEcR gene is driven by the cytomegalovirus (CMV) immediate early promoter. Transcription of the RXR gene is driven by the Rous sarcoma virus (RSV) promoter. Stable cell lines expressing the genes may be made by selection on the antibiotic Zeocin. Product information regarding the features and the maintenance of the pVgRXR vector and the Ecdysone System kit are incorporated by reference from the Invitrogen technical manual. [0111]
  • The plasmids PIND was obtained from Invitrogen (FIG. 3). The plasmid PIND is a 5024 bp vector based on the pcDNA3.1 vector. It has five hybrid LREs, i.e., five ecdysone/glucocorticoid response elements (E/GRE), and the heat shock minimal promoter. The E/GRE's can be recognized by a modified ecdysone receptor expressed from pVgRXR. The plasmid pIND-Luc was made by digesting the luciferase gene out of another plasmid, including its Kozak sequence and methionine start site and cloning it into the poly linker of PIND, i.e., downstream of the minimal heat shock promoter (FIG. 3). Both plasmids have neomycin and ampicillin resistance genes for selection purposes. Product information regarding the features and the maintenance of the pIND and pIND-Luc vectors are incorporated by reference from the Invitrogen technical manual. [0112]
  • The mammalian expression plasmid pSRa has been described in the art [Y. Takebe et al., [0113] Mol. Cell. Biol., 8, pp. 466-72 (1988) incorporated by reference herein]. The plasmid contains a promoter system composed of the simian virus 40 (SV40) early promoter and the R segment and part of the U5 sequence (R-U5′) of the long terminal repeat of human T-cell leukemia virus type 1 (HTLV-1). The plasmid also contains a 16S splice junction, an SV40 polyadenylation signal and an ampicillin resistance gene. The HTLV LTR enhancer/promoter sequence drives high level expression of genes cloned downstream. Genes of interest may be cloned into the Pst I and EcoRl cites located 3′ to the 16S splice junction.
  • (2) Primers [0114]
  • The VP16/5A5BF primer (Seq. ID. No. 12) is a 28-mer designed to hybridize to an Xba I site upstream of the VP16 coding region of the pVgRXR plasmid. [0115]
  • The VP16/5A5BR primer (Seq. ID. No. 13) is a 86-mer designed to hybridize in part to the antisense strand in the 3′ region of the DNA encoding the VP16 activation domain of the pVgRXR plasmid. The primer also comprises nucleotides complementary to the coding sequences for the 5A/5B cleavage site of the HCV NS3-4A protease and an Xba I site. [0116]
  • The VP16/5A**5BR primer (Seq. ID. No. 14) is a 92-mer designed to hybridize in part to the antisense strand in the 3′ region of the DNA encoding the VP16 activation domain of the pVgRXR plasmid. The primer also has nucleotides sequences complementary to the coding sequences for the 5A/5B cleavage site of the HCV NS3-4A protease. However, the DNA encoding the 5A/5B cleavage site additionally has a six nucleotide insertion between the DNA sequences encoding a cysteine and a serine codon. The six nucleotide insertion encodes two stop codons. [0117]
  • The NS3-4AF primer (Seq. ID. No. 15) is a 47-mer designed to bind partly to the start of the NS3 coding region (corresponding to amino acid #1027-1032 in the HCV polypeptide). The remainder of the oligonucleotide contains multiple restriction sites for ease of cloning. [0118]
  • The NS3-4AB primer (Seq. ID. No. 16) is a 44-mer designed to bind partly to the 3′ end of the NS4A coding region (corresponding to amino acid #1705-1711 in the HCV polypeptide). The remainder of the oligonucleotide contains multiple restriction sites for ease of cloning. [0119]
  • Each primer was synthesized under standard conditions known in the art using an oligonucleotide synthesis machine. [0120]
  • B. Polymerase Chain Reactions (PCR) and Cloning Strategy [0121]
  • The PCR reactions were carried out under standard conditions (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). [0122] Molecular Cloning: A Laboratory Manual. Second Edition., Plainview, N.Y.: Cold Spring Harbor Laboratory Press).
  • (1) pVgRXR-5A/5B vector and pVgRXR-5A**5A vector [0123]
  • The first step towards preparing the expression vector encoding VgRXR-5A/5B (Seq. ID. No. 19) involved a PCR reaction using the VP16/5A5BF primer (Seq. ID. No. 12), the VP16/5A5BR primer (Seq. ID. No. 13) and the pVgRXR plasmid as the template. Next, the PCR product was digested with Xba I. A vector for ligation with the PCR product was prepared by digesting the pVgRXR vector with Xba I to remove the DNA encoding the VP16 activation domain. The vector backbone was isolated and the digested, PCR insert was ligated into the Xba I site in the backbone. The ligations were screened by sequencing to ensure the proper orientation of the insert and correctness of its nucleotide sequence. [0124]
  • The first step towards preparing the expression vector encoding VgRXR-5A**5B (Seq. ID. No. 21) involved a PCR reaction using the VP16/5A5BF primer (Seq. ID. No. 12), the VP16/5A**5BR primer (Seq. ID. No. 14) and the pVgRXR plasmid as the template. Next, the PCR product was digested with Xba I. A vector for ligating with the PCR product was prepared by digesting the pVgRXR vector with Xba I to remove the DNA encoding the VP16 activation domain. The vector backbone was isolated and the digested PCR insert was ligated into the Xba I site in the backbone. The ligations were screened by sequencing to ensure the proper orientation of the insert and the correctness of its nucleotide sequence. [0125]
  • (2) pSRα-NS3-4A and pSRα-NS3-4A(S1165A) [0126]
  • The first step towards preparing the pSRα-NS3-4A vector encoding the NS3-4A cleavage site (Seq. ID. No. 10) involved a PCR reaction using the NS3-4AF primer (Seq. ID. No. 15), the NS3-4AB primer (Seq. ID. No. 16) and full length HCV H strain cDNA as a template [Inchauspe et al., (1991), PNAS USA 88:10292-10296 incorporated by reference herein]. Next, the PCR product was digested with Pst1 and EcoR1. A vector for ligating with the PCR product was prepared by digesting the pSRα vector with [0127] Pst 1 and EcoR1. The vector backbone was isolated and the PCR insert was ligated into the Pst1-EcoR1 site in the backbone.
  • The first step towards preparing the pSRα-NS3-4A(S1165A) vector encoding the NS3-4A mutant protease involved a PCR reaction using the NS3-4AF primer (Seq. ID. No. 15), the NS3-4AB primer (Seq. ID. No. 16) and the cDNA of the NS3 active site mutant S1165A [A. Grakoui et al., (1993) [0128] J. Virology 67: 2832-43 incorporated by reference herein]. Next, the PCR product was digested with Pst1 and EcoR1. A vector for ligating with the PCR product was prepared by digesting the pSRa vector with Pst1 and EcoR1. The vector backbone was isolated and the PCR insert was ligated into the Pst1-EcoR1 site in the backbone. The NS3-4A(S1165A) has a serine to alanine substitution at amino acid number 1165, which makes the protease inactive. Compare Seq ID Nos. 10 and Seq ID No. 11.
    • EXAMPLE 2 The NS3-4A Protease Ecdysone-Inducible Luciferase Assay
  • HCV NS3-4A protease ecdysone-inducible luciferase assays were generally carried out as described below. [0129]
  • COS African green monkey kidney cells were plated at approximately 150,000 cells/well in a 6-well plate. The next day, approximately 3.6 μg plasmid DNA was dissolved in 100 μl of Dulbecco's modified Eagle's media-Gibco BRL (DMEM), combined with 29 μl of Superfect reagent (Qiagen), and then mixed by pipetting. The mixture was incubated at room temperature for ten minutes. [0130]
  • The amounts of DNA typically used in each experiment was as follows: 0.6 μg of pIND-Luc, 0.6 μg of pVgRXR-5A/5B or pVgRXR-5A**5B, and 0-2.4 μg of pSRα-NS3-4A or pSRα-NS3-4A (S1165A) or pSRα. [0131]
  • Next 600 μl of DMEM with v/[0132] v 10% fetal bovine serum (10% FBS-DMEM) was added to the DNA mixture and mixed by pipetting. The cells in the 6-well plate were washed with 2.5 ml phosphate-buffered saline (PBS). The PBS was removed from the cells and replaced by the DNA mixture. The cells were incubated in the DNA mixture for three hours at 37° C. in a 5% CO2 incubator.
  • After incubation, the cells were washed with PBS. The PBS was removed and 10% FBS-DMEM was added to the cells. The cells were incubated in 10% FBS-DMEM overnight at 37° C. in a 5% CO[0133] 2 incubator.
  • On the next day, an exogenous ligand was added to the cells to induce transcription of the reporter gene. Specifically, the 10% FBS-DMEM was aspirated off the cells and replaced with a 10% FBS-DMEM solution containing a concentration of 1-10 μM muristerone A or 1-10 μM ponasterone A (Invitrogen, United Kingdom). In some cases, a protease inhibitor dissolved in dimethylsulfoxide(DMSO) was also added to the cells for a final concentration of 1-40 μM. The cells were incubated with muristerone A or ponasterone A for 24 hours at 37° C. in a 5% CO[0134] 2 incubator.
  • The following day, the cells were washed with PBS. The activity of the luciferase protein was measured using a luciferase assay kit (Promega). Specifically, the cells were lysed in 250 μl of Cell Culture Lysis Reagent. The cells were scraped from the plate and transferred to a microfuge tube. The extract was spun at 12,000×g for 5 seconds. Twenty microliters of each sample was added to 100 μl of Luciferase Assay Reagent. The light produced by the reaction of the luciferase with the Reagent was measured in a luminometer and was reported as relative light units (RLU). [0135]
  • Most of the experiments were done several times in triplicate. Error bars reflecting the standard deviation of the data points are included in the Figures. [0136]
    • Example 3 The Insertion of a 5A/5B Junction Between the Activation Domain and the DNA-binding Domain Does Not Interfere with Muristerone A-Induced Transactivation
  • Vectors encoding fusion proteins (VgRXR, VgRXR-5A/5B, or VgRXR-5A(Stop)5B) and pIND-Luc were transfected into COS cells as described above (Example 2). On the following day, some of the cells were incubated with 1 μM of muristerone for 24 hours. The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 4. [0137]
  • The control fusion protein expressed by pVgRXR-5A(Stop)5B, which lacks the ecdysone DNA binding domain shows negligible activity. Little or no difference is observed between the activities of the VgRXR and the VgRXR-5A/5B expressed fusion proteins. The results indicate that the insertion of the 5A/5B junction in the VgRXR expressed protein does not interfere with muristerone A-induced activity. [0138]
    • EXAMPLE 4 Cotransfection of Increasing Amounts of pSRαNS3-4A With pVgRXR-5A/5B Leads to a Dose-Dependent Decrease of Muristerone A-Inducible Transactivation
  • The reporter plasmid pIND-Luc was cotransfected with either pVgRXR-5A/5B or pVgRXR and increasing amounts (μgs) of the DNA encoding the protease NS3-4A as indicated in FIG. 5. On the following day, the cells were incubated in 1 μM muristerone A. Next, the cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 5. [0139]
  • Transcription of the luciferase reporter construct when co-transfected with the VgRXR construct encoding mammalian RXR and the ecdysone receptor/VP16 fusion proteins showed little or no change when coexpressed with the protease NS3-4A. The VgRXR-5A/5B encoded protein, on the other hand, produced a dosage-dependent decrease in luciferase activity when coexpressed with the NS3-4A protein. These results suggest that the protease NS3-4A cleaves the 5A/5B junction but not other areas of the fusion protein necessary for activity. [0140]
    • EXAMPLE 5 Cotransfection of Increasing Amounts of pSRαNS3-4A(S1165A) with pVgRXR-5A/5B Does Not Affect Muristerone A-Inducible Transactivation
  • We used 0.6 μgs of pVgRXR-5A/5B and 0.6 μgs of pIND-Luc to cotransfect with varying amounts (μgs) of the vectors encoding NS3 protease or an inactive mutant thereof as indicated in FIG. 6. The total amount of DNA used for each transfection was 3.6 μgs with the addition of pSRα. On the following day, the cells were incubated in 1 μM of muristerone A. The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 6. [0141]
  • As seen in the previous results (Example 4), the fusion protein encoded by VgRXR-5A/5B demonstrated a high level of activity in the absence of the NS3-4A protease. The activity of VgRXR-5A/5B-mediated luciferase activity decreased in a dosage-dependent manner with the cotransfection of DNA encoding the NS3-4A protease, but not with the DNA encoding the mutant protease NS3-4A(S1165A). This is presumably due to the inability of the mutant NS3-4A protease to cleave the ecdysone/VP16 fusion protein. [0142]
  • Dosage-dependence studies such Examples 4 and 5 were useful for determining the optimal ratio of DNA encoding fusion protein and DNA encoding protease for use in future assays. [0143]
    • EXAMPLE 6 Ponasterone A Dose Response
  • The plasmid pIND-Luc (0.6 μg) was cotransfected with pVgRXR-5A/5B (0.6 μg) in the absence or presence of 1.8 μg of pSRαNS3-4A. On the following day, the cells were incubated with varying amounts of ponasterone A, a ligand which induces the heterodimerization of ecdysone receptor with the mammalian retinoic acid receptor, RXR. The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 7. [0144]
  • The results indicate that ponasterone A is effective at activating the fusion protein EdR5A/5B (i.e., the protein encoded by VgRXR-5A/5B) in the absence of the protease NS3-4A. For the purposes of these experiments, the greatest activation of the fusion protein occurred when ponasterone A was present in a concentration between 3.3-10 μM. Generally, ponasterone A was found to be equally effective at inducing transcription as muristerone A in these assays. [0145]
    • EXAMPLE 7 Ponasterone A Induction and % DMSO Control
  • The plasmid pIND-Luc (0.6 μg) was cotransfected with pVgRXR-5A/5B (0.6 μg) in the absence or presence of 1.8 μg of pSRαNS3-4A. On the following day, the cells were incubated with 5 μM ponasterone A. Dimethylsulfoxide (DMSO) was also added to the cells to a final concentration of 0-1% DMSO (v/v). The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 8. [0146]
  • In this control experiment, DMSO was added to cells to determine whether the presence of it interfered with the activation of the fusion protein by ponasterone A. DMSO is a solvent that was sometimes used to dissolve protease inhibitor compounds prior to addition to these assays. In most experiments, the DMSO concentration in cell culture did not exceed 0.1%. The results indicate that DMSO concentrations up to 1% had little or no effect on the ponasterone A-induced luciferase activity in this assay. [0147]
  • The Examples below demonstrate how this assay may be useful to screen compounds potentially useful as inhibitors of a protease. [0148]
    • EXAMPLE 8 Dose Dependent Inhibition of NS3-4A Activity by VRT-25,531
  • The plasmid pIND-Luc (0.6 μg) was cotransfected with pVgRXR-5A/5B (0.6 μg) in the presence of 1.8 μg of pSRαNS3-4A (1:3 ratio). On the following day, the cells were incubated with 5 μM ponasterone A and varying amounts of a compound VH-25531 as indicated in FIG. 9. The cells were lysed and assayed for luciferase activity as described previously. The results are depicted in FIG. 9. [0149]
  • The results indicate that VH-25531 in the absence of the NS3-4A protease does not significantly increase or decrease the activity of the fusion protein (compare [0150] lanes 1 and 3, FIG. 9). However, VH-25531 does inhibit the activity of the NS3-4A protease especially at 20 μM (compare lanes 4-11 with claim 2, FIG. 9). In fact, the results indicate that VH-25531 may inhibit the NS3-4A protease as much as 55.5% at a 20 μM concentration. The percent inhibition of protease activity at 20 μM VH-25531 was roughly calculated by subtracting the values of lanes 3 and 2 (19900−5177=14732), by subtracting the values of lanes 4 and 2 (13356=5177=8179), and then by dividing the two values (8179/14723=0.555).
  • While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims and specification rather than the specific embodiments which are exemplified here. [0151]
  • 1 24 1 10 PRT Human immunodeficiency virus 1 Val Ser Phe Asn Phe Pro Gln Ile Thr Leu 1 5 10 2 8 PRT Human immunodeficiency virus 2 Ser Gln Asn Tyr Pro Ile Val Gln 1 5 3 8 PRT Human immunodeficiency virus 3 Ala Arg Val Leu Ala Glu Ala Met 1 5 4 8 PRT Human immunodeficiency virus 4 Ala Asn Ile Met Met Gln Arg Gly 1 5 5 8 PRT Human immunodeficiency virus 5 Pro Gly Asn Phe Leu Gln Ser Arg 1 5 6 8 PRT Human immunodeficiency virus 6 Ser Phe Asn Phe Pro Gln Ile Thr 1 5 7 8 PRT Human immunodeficiency virus 7 Gln Ile Thr Leu Trp Gln Arg Pro 1 5 8 8 PRT Human immunodeficiency virus 8 Thr Leu Asn Phe Pro Ile Ser Pro 1 5 9 8 PRT Human immunodeficiency virus 9 Arg Lys Val Leu Phe Leu Asn Gly 1 5 10 18 PRT Hepatitis C virus 10 Gly Ala Asp Thr Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp 1 5 10 15 Thr Gly 11 10 PRT Artificial Sequence Description of Artificial Sequencemutated hepatitis C virus 11 Gly Ala Ala Thr Glu Asp Val Val Cys Cys 1 5 10 12 28 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 12 agctagctct agagtaccga gctcggat 28 13 86 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 13 tcgtctagag cctgtccagg tataagacat tgagcagcac acgacatctt ccgtgtcggc 60 gccggtacct agaagcttcc caccgt 86 14 92 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 14 tcgtctagag cctgtccagg tataagacat tgatcatcag cagcacacga catcttccgt 60 gtcggcgccg gtacctagaa gcttcccacc gt 92 15 47 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 15 ggactagtct gcagtctaga gctccatggc gcccatcacg gcgtacg 47 16 44 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 16 gaagatctga attctagatt ttagcactct tccatctcat cgaa 44 17 2241 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 17 atggcccccc cgaccgatgt cagcctgggg gacgaactcc acttagacgg cgaggacgtg 60 gcgatggcgc atgccgacgc gctagacgat ttcgatctgg acatgttggg ggacggggat 120 tccccaggtc cgggatttac cccccacgac tccgccccct acggcgctct ggatatggcc 180 gacttcgagt ttgagcagat gtttaccgat gcccttggaa ttgacgagta cggtgggaag 240 cttctaggta cctctagaag aatatcaaat tctatatctt caggtcgcga tgatctctcg 300 ccttcgagca gcttgaacgg atactcggcg aacgaaagct gcgatgcgaa gaagagcaag 360 aagggacctg cgccacgggt gcaagaggag ctgtgcctgg tttgcggcga cagggcctcc 420 ggctaccact acaacgccct cacctgtggg ggctgcaagg ggttctttcg acgcagcgtt 480 acgaagagcg ccgtctactg ctgcaagttc gggcgcgcct gcgaaatgga catgtacatg 540 aggcgaaagt gtcaggagtg ccgcctgaaa aagtgcctgg ccgtgggtat gcggccggaa 600 tgcgtcgtcc cggagaacca atgtgcgatg aagcggcgcg aagagaaggc ccagaaggag 660 aaggacaaaa tgaccacttc gccgagctct cagcatggcg gcaatggcag cttggcctct 720 ggtggcggcc aagactttgt taagaaggag attcttgacc ttatgacatg cgagccgccc 780 cagcatgcca ctattccgct actacctgat gaaatattgg ccaagtgtca agcgcgcaat 840 ataccttcct taacgtacaa tcagttggcc gttatataca agttaatttg gtaccaggat 900 ggctatgagc agccatctga agaggatctc aggcgtataa tgagtcaacc cgatgagaac 960 gagagccaaa cggacgtcag ctttcggcat ataaccgaga taaccatact cacggtccag 1020 ttgattgttg agtttgctaa aggtctacca gcgtttacaa agatacccca ggaggaccag 1080 atcacgttac taaaggcctg ctcgtcggag gtgatgatgc tgcgtatggc acgacgctat 1140 gaccacagct cggactcaat attcttcgcg aataatagat catatacgcg ggattcttac 1200 aaaatggccg gaatggctga taacattgaa gacctgctgc atttctgccg ccaaatgttc 1260 tcgatgaagg tggacaacgt cgaatacgcg cttctcactg ccattgtgat cttctcggac 1320 cggccgggcc tggagaaggc ccagctagtc gaagcgatcc agagctacta catcgacacg 1380 ctacgcattt atatactcaa ccgccactgc ggcgactcaa tgagcctcgt cttctacgca 1440 aagctgctct cgatcctcac cgagctgcgt acgctgggca accagaacgc cgagatgtgt 1500 ttctcactaa agctcaaaaa ccgcaaactg cccaagttcc tcgaggagat ctgggacgtt 1560 catgccatcc cgccatcggt ccagtcgcac cttcagatta cccaggagga gaacgagcgt 1620 ctcgagcggg ctgagcgtat gcgggcatcg gttgggggcg ccattaccgc cggcattgat 1680 tgcgactctg cctccacttc ggcggcggca gccgcggccc agcatcagcc tcagcctcag 1740 ccccagcccc aaccctcctc cctgacccag aacgattccc agcaccagac acagccgcag 1800 ctacaacctc agctaccacc tcagctgcaa ggtcaactgc aaccccagct ccaaccacag 1860 cttcagacgc aactccagcc acagattcaa ccacagccac agctccttcc cgtctccgct 1920 cccgtgcccg cctccgtaac cgcacctggt tccttgtccg cggtcagtac gagcagcgaa 1980 tacatgggcg gaagtgcggc cataggacca atcacgccgg caaccaccag cagtatcacg 2040 gctgccgtta ccgctagctc caccacatca gcggtaccga tgggcaacgg agttggagtc 2100 ggtgttgggg tgggcggcaa cgtcagcatg tatgcgaacg cccagacggc gatggccttg 2160 atgggtgtag ccctgcattc gcaccaagag cagcttatcg ggggagtggc ggttaagtcg 2220 gagcactcga cgactgcata g 2241 18 746 PRT Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 18 Met Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15 Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 20 25 30 Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 35 40 45 His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 50 55 60 Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly Lys 65 70 75 80 Leu Leu Gly Thr Ser Arg Arg Ile Ser Asn Ser Ile Ser Ser Gly Arg 85 90 95 Asp Asp Leu Ser Pro Ser Ser Ser Leu Asn Gly Tyr Ser Ala Asn Glu 100 105 110 Ser Cys Asp Ala Lys Lys Ser Lys Lys Gly Pro Ala Pro Arg Val Gln 115 120 125 Glu Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr 130 135 140 Asn Ala Leu Thr Cys Gly Gly Cys Lys Gly Phe Phe Arg Arg Ser Val 145 150 155 160 Thr Lys Ser Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys Glu Met 165 170 175 Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys 180 185 190 Leu Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys 195 200 205 Ala Met Lys Arg Arg Glu Glu Lys Ala Gln Lys Glu Lys Asp Lys Met 210 215 220 Thr Thr Ser Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser 225 230 235 240 Gly Gly Gly Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr 245 250 255 Cys Glu Pro Pro Gln His Ala Thr Ile Pro Leu Leu Pro Asp Glu Ile 260 265 270 Leu Ala Lys Cys Gln Ala Arg Asn Ile Pro Ser Leu Thr Tyr Asn Gln 275 280 285 Leu Ala Val Ile Tyr Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln 290 295 300 Pro Ser Glu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn 305 310 315 320 Glu Ser Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile 325 330 335 Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe 340 345 350 Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser 355 360 365 Ser Glu Val Met Met Leu Arg Met Ala Arg Arg Tyr Asp His Ser Ser 370 375 380 Asp Ser Ile Phe Phe Ala Asn Asn Arg Ser Tyr Thr Arg Asp Ser Tyr 385 390 395 400 Lys Met Ala Gly Met Ala Asp Asn Ile Glu Asp Leu Leu His Phe Cys 405 410 415 Arg Gln Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu 420 425 430 Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln 435 440 445 Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr 450 455 460 Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu Val Phe Tyr Ala 465 470 475 480 Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln Asn 485 490 495 Ala Glu Met Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys 500 505 510 Phe Leu Glu Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser Val Gln 515 520 525 Ser His Leu Gln Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala 530 535 540 Glu Arg Met Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp 545 550 555 560 Cys Asp Ser Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln 565 570 575 Pro Gln Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln Asn Asp 580 585 590 Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln Leu Pro Pro Gln 595 600 605 Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln Pro Gln Leu Gln Thr Gln 610 615 620 Leu Gln Pro Gln Ile Gln Pro Gln Pro Gln Leu Leu Pro Val Ser Ala 625 630 635 640 Pro Val Pro Ala Ser Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser 645 650 655 Thr Ser Ser Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr 660 665 670 Pro Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr 675 680 685 Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly Val Gly Val 690 695 700 Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala Met Ala Leu 705 710 715 720 Met Gly Val Ala Leu His Ser His Gln Glu Gln Leu Ile Gly Gly Val 725 730 735 Ala Val Lys Ser Glu His Ser Thr Thr Ala 740 745 19 2295 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 19 atggcccccc cgaccgatgt cagcctgggg gacgaactcc acttagacgg cgaggacgtg 60 gcgatggcgc atgccgacgc gctagacgat ttcgatctgg acatgttggg ggacggggat 120 tccccaggtc cgggatttac cccccacgac tccgccccct acggcgctct ggatatggcc 180 gacttcgagt ttgagcagat gtttaccgat gcccttggaa ttgacgagta cggtgggaag 240 cttctaggta ccggcgccga cacggaagat gtcgtgtgct gctcaatgtc ttatacctgg 300 acaggctcta gaagaatatc aaattctata tcttcaggtc gcgatgatct ctcgccttcg 360 agcagcttga acggatactc ggcgaacgaa agctgcgatg cgaagaagag caagaaggga 420 cctgcgccac gggtgcaaga ggagctgtgc ctggtttgcg gcgacagggc ctccggctac 480 cactacaacg ccctcacctg tgggggctgc aaggggttct ttcgacgcag cgttacgaag 540 agcgccgtct actgctgcaa gttcgggcgc gcctgcgaaa tggacatgta catgaggcga 600 aagtgtcagg agtgccgcct gaaaaagtgc ctggccgtgg gtatgcggcc ggaatgcgtc 660 gtcccggaga accaatgtgc gatgaagcgg cgcgaagaga aggcccagaa ggagaaggac 720 aaaatgacca cttcgccgag ctctcagcat ggcggcaatg gcagcttggc ctctggtggc 780 ggccaagact ttgttaagaa ggagattctt gaccttatga catgcgagcc gccccagcat 840 gccactattc cgctactacc tgatgaaata ttggccaagt gtcaagcgcg caatatacct 900 tccttaacgt acaatcagtt ggccgttata tacaagttaa tttggtacca ggatggctat 960 gagcagccat ctgaagagga tctcaggcgt ataatgagtc aacccgatga gaacgagagc 1020 caaacggacg tcagctttcg gcatataacc gagataacca tactcacggt ccagttgatt 1080 gttgagtttg ctaaaggtct accagcgttt acaaagatac cccaggagga ccagatcacg 1140 ttactaaagg cctgctcgtc ggaggtgatg atgctgcgta tggcacgacg ctatgaccac 1200 agctcggact caatattctt cgcgaataat agatcatata cgcgggattc ttacaaaatg 1260 gccggaatgg ctgataacat tgaagacctg ctgcatttct gccgccaaat gttctcgatg 1320 aaggtggaca acgtcgaata cgcgcttctc actgccattg tgatcttctc ggaccggccg 1380 ggcctggaga aggcccagct agtcgaagcg atccagagct actacatcga cacgctacgc 1440 atttatatac tcaaccgcca ctgcggcgac tcaatgagcc tcgtcttcta cgcaaagctg 1500 ctctcgatcc tcaccgagct gcgtacgctg ggcaaccaga acgccgagat gtgtttctca 1560 ctaaagctca aaaaccgcaa actgcccaag ttcctcgagg agatctggga cgttcatgcc 1620 atcccgccat cggtccagtc gcaccttcag attacccagg aggagaacga gcgtctcgag 1680 cgggctgagc gtatgcgggc atcggttggg ggcgccatta ccgccggcat tgattgcgac 1740 tctgcctcca cttcggcggc ggcagccgcg gcccagcatc agcctcagcc tcagccccag 1800 ccccaaccct cctccctgac ccagaacgat tcccagcacc agacacagcc gcagctacaa 1860 cctcagctac cacctcagct gcaaggtcaa ctgcaacccc agctccaacc acagcttcag 1920 acgcaactcc agccacagat tcaaccacag ccacagctcc ttcccgtctc cgctcccgtg 1980 cccgcctccg taaccgcacc tggttccttg tccgcggtca gtacgagcag cgaatacatg 2040 ggcggaagtg cggccatagg accaatcacg ccggcaacca ccagcagtat cacggctgcc 2100 gttaccgcta gctccaccac atcagcggta ccgatgggca acggagttgg agtcggtgtt 2160 ggggtgggcg gcaacgtcag catgtatgcg aacgcccaga cggcgatggc cttgatgggt 2220 gtagccctgc attcgcacca agagcagctt atcgggggag tggcggttaa gtcggagcac 2280 tcgacgactg catag 2295 20 764 PRT Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 20 Met Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15 Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 20 25 30 Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 35 40 45 His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 50 55 60 Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly Lys 65 70 75 80 Leu Leu Gly Thr Gly Ala Asp Thr Glu Asp Val Val Cys Cys Ser Met 85 90 95 Ser Tyr Thr Trp Thr Gly Ser Arg Arg Ile Ser Asn Ser Ile Ser Ser 100 105 110 Gly Arg Asp Asp Leu Ser Pro Ser Ser Ser Leu Asn Gly Tyr Ser Ala 115 120 125 Asn Glu Ser Cys Asp Ala Lys Lys Ser Lys Lys Gly Pro Ala Pro Arg 130 135 140 Val Gln Glu Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr 145 150 155 160 His Tyr Asn Ala Leu Thr Cys Gly Gly Cys Lys Gly Phe Phe Arg Arg 165 170 175 Ser Val Thr Lys Ser Ala Val Tyr Cys Cys Lys Phe Gly Arg Ala Cys 180 185 190 Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys 195 200 205 Lys Cys Leu Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Asn 210 215 220 Gln Cys Ala Met Lys Arg Arg Glu Glu Lys Ala Gln Lys Glu Lys Asp 225 230 235 240 Lys Met Thr Thr Ser Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu 245 250 255 Ala Ser Gly Gly Gly Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu 260 265 270 Met Thr Cys Glu Pro Pro Gln His Ala Thr Ile Pro Leu Leu Pro Asp 275 280 285 Glu Ile Leu Ala Lys Cys Gln Ala Arg Asn Ile Pro Ser Leu Thr Tyr 290 295 300 Asn Gln Leu Ala Val Ile Tyr Lys Leu Ile Trp Tyr Gln Asp Gly Tyr 305 310 315 320 Glu Gln Pro Ser Glu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro Asp 325 330 335 Glu Asn Glu Ser Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu Ile 340 345 350 Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu Pro 355 360 365 Ala Phe Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys Ala 370 375 380 Cys Ser Ser Glu Val Met Met Leu Arg Met Ala Arg Arg Tyr Asp His 385 390 395 400 Ser Ser Asp Ser Ile Phe Phe Ala Asn Asn Arg Ser Tyr Thr Arg Asp 405 410 415 Ser Tyr Lys Met Ala Gly Met Ala Asp Asn Ile Glu Asp Leu Leu His 420 425 430 Phe Cys Arg Gln Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr Ala 435 440 445 Leu Leu Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Lys 450 455 460 Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu Arg 465 470 475 480 Ile Tyr Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu Val Phe 485 490 495 Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg Thr Leu Gly Asn 500 505 510 Gln Asn Ala Glu Met Cys Phe Ser Leu Lys Leu Lys Asn Arg Lys Leu 515 520 525 Pro Lys Phe Leu Glu Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser 530 535 540 Val Gln Ser His Leu Gln Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu 545 550 555 560 Arg Ala Glu Arg Met Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly 565 570 575 Ile Asp Cys Asp Ser Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala Gln 580 585 590 His Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr Gln 595 600 605 Asn Asp Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln Leu Pro 610 615 620 Pro Gln Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln Pro Gln Leu Gln 625 630 635 640 Thr Gln Leu Gln Pro Gln Ile Gln Pro Gln Pro Gln Leu Leu Pro Val 645 650 655 Ser Ala Pro Val Pro Ala Ser Val Thr Ala Pro Gly Ser Leu Ser Ala 660 665 670 Val Ser Thr Ser Ser Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro 675 680 685 Ile Thr Pro Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser 690 695 700 Ser Thr Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly Val 705 710 715 720 Gly Val Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala Met 725 730 735 Ala Leu Met Gly Val Ala Leu His Ser His Gln Glu Gln Leu Ile Gly 740 745 750 Gly Val Ala Val Lys Ser Glu His Ser Thr Thr Ala 755 760 21 2301 DNA Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 21 atggcccccc cgaccgatgt cagcctgggg gacgaactcc acttagacgg cgaggacgtg 60 gcgatggcgc atgccgacgc gctagacgat ttcgatctgg acatgttggg ggacggggat 120 tccccaggtc cgggatttac cccccacgac tccgccccct acggcgctct ggatatggcc 180 gacttcgagt ttgagcagat gtttaccgat gcccttggaa ttgacgagta cggtgggaag 240 cttctaggta ccggcgccga cacggaagat gtcgtgtgct gctgatgatc aatgtcttat 300 acctggacag gctctagaag aatatcaaat tctatatctt caggtcgcga tgatctctcg 360 ccttcgagca gcttgaacgg atactcggcg aacgaaagct gcgatgcgaa gaagagcaag 420 aagggacctg cgccacgggt gcaagaggag ctgtgcctgg tttgcggcga cagggcctcc 480 ggctaccact acaacgccct cacctgtggg ggctgcaagg ggttctttcg acgcagcgtt 540 acgaagagcg ccgtctactg ctgcaagttc gggcgcgcct gcgaaatgga catgtacatg 600 aggcgaaagt gtcaggagtg ccgcctgaaa aagtgcctgg ccgtgggtat gcggccggaa 660 tgcgtcgtcc cggagaacca atgtgcgatg aagcggcgcg aagagaaggc ccagaaggag 720 aaggacaaaa tgaccacttc gccgagctct cagcatggcg gcaatggcag cttggcctct 780 ggtggcggcc aagactttgt taagaaggag attcttgacc ttatgacatg cgagccgccc 840 cagcatgcca ctattccgct actacctgat gaaatattgg ccaagtgtca agcgcgcaat 900 ataccttcct taacgtacaa tcagttggcc gttatataca agttaatttg gtaccaggat 960 ggctatgagc agccatctga agaggatctc aggcgtataa tgagtcaacc cgatgagaac 1020 gagagccaaa cggacgtcag ctttcggcat ataaccgaga taaccatact cacggtccag 1080 ttgattgttg agtttgctaa aggtctacca gcgtttacaa agatacccca ggaggaccag 1140 atcacgttac taaaggcctg ctcgtcggag gtgatgatgc tgcgtatggc acgacgctat 1200 gaccacagct cggactcaat attcttcgcg aataatagat catatacgcg ggattcttac 1260 aaaatggccg gaatggctga taacattgaa gacctgctgc atttctgccg ccaaatgttc 1320 tcgatgaagg tggacaacgt cgaatacgcg cttctcactg ccattgtgat cttctcggac 1380 cggccgggcc tggagaaggc ccagctagtc gaagcgatcc agagctacta catcgacacg 1440 ctacgcattt atatactcaa ccgccactgc ggcgactcaa tgagcctcgt cttctacgca 1500 aagctgctct cgatcctcac cgagctgcgt acgctgggca accagaacgc cgagatgtgt 1560 ttctcactaa agctcaaaaa ccgcaaactg cccaagttcc tcgaggagat ctgggacgtt 1620 catgccatcc cgccatcggt ccagtcgcac cttcagatta cccaggagga gaacgagcgt 1680 ctcgagcggg ctgagcgtat gcgggcatcg gttgggggcg ccattaccgc cggcattgat 1740 tgcgactctg cctccacttc ggcggcggca gccgcggccc agcatcagcc tcagcctcag 1800 ccccagcccc aaccctcctc cctgacccag aacgattccc agcaccagac acagccgcag 1860 ctacaacctc agctaccacc tcagctgcaa ggtcaactgc aaccccagct ccaaccacag 1920 cttcagacgc aactccagcc acagattcaa ccacagccac agctccttcc cgtctccgct 1980 cccgtgcccg cctccgtaac cgcacctggt tccttgtccg cggtcagtac gagcagcgaa 2040 tacatgggcg gaagtgcggc cataggacca atcacgccgg caaccaccag cagtatcacg 2100 gctgccgtta ccgctagctc caccacatca gcggtaccga tgggcaacgg agttggagtc 2160 ggtgttgggg tgggcggcaa cgtcagcatg tatgcgaacg cccagacggc gatggccttg 2220 atgggtgtag ccctgcattc gcaccaagag cagcttatcg ggggagtggc ggttaagtcg 2280 gagcactcga cgactgcata g 2301 22 94 PRT Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 22 Met Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp 1 5 10 15 Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 20 25 30 Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 35 40 45 His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 50 55 60 Glu Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly Lys 65 70 75 80 Leu Leu Gly Thr Gly Ala Asp Thr Glu Asp Val Val Cys Cys 85 90 23 5 PRT Drosophila 23 Glu Gly Cys Lys Gly 1 5 24 5 PRT Artificial Sequence Description of Artificial Sequenceman-made artificial sequence 24 Gly Ser Cys Lys Val 1 5

Claims (27)

We claim:
1. A fusion protein comprising:
a. a protease cleavage site;
b. a ligand binding domain; and
c. a DNA binding domain;
wherein the association of a ligand with said fusion protein mediates the binding of the fusion protein to a ligand-responsive element (“LRE”) operatively linked to a reporter gene; and
wherein the fusion protein also comprises an expression modulator domain or associates with a second protein having an expression modulator domain, wherein said expression modulator domain regulates the transcription of said reporter gene.
2. The fusion protein according to claim 1, wherein said protease cleavage site is recognized by HCV NS3 protease.
3. The fusion protein according to claim 1, wherein said protease cleavage site is recognized by HIV aspartyl protease.
4. The fusion protein according to claim 1, wherein said DNA binding domain is selected from a DNA binding domain of a steroid/thyroid superfamily receptor.
5. The fusion protein according to claim 1, wherein said DNA binding domain is from the ecdysone receptor and wherein said ecdysone receptor requires association with a protein binding partner to enable binding to DNA.
6. The fusion protein according to claim 5, wherein said DNA binding domain is modified such that the P-box region has the amino acid sequence Seq. ID. No. 24.
7. The fusion protein according to claim 1, wherein said expression modulator domain is the activation domain of the VP16 protein.
8. The fusion protein according to claim 1 having the sequence of SEQ ID NO: 18.
9. The fusion protein according to claim 1, wherein the amino acid sequence of the protease cleavage site is selected from the group consisting of Seq. ID. Nos. 1-10.
10. A DNA molecule encoding a fusion protein according to any one of claims 1 to 9.
11. The DNA molecule according to claim 6, wherein said DNA sequence is SEQ ID No. 17.
12. A vector comprising the DNA molecule according to claim 10 or 11.
13. A host cell transformed with a vector according to claim 12.
14. A method of producing a fusion protein according to any one of claims 1 to 9, comprising the steps of culturing a host cell according to claim 13 under conditions which cause expression of said fusion protein.
15. The host cell according to claim 13 further comprising a DNA molecule comprising:
a. an LRE which binds to said DNA binding domain of said fusion protein, wherein said binding is modulated by the presence of a ligand and is optionally modulated by the presence of a protein binding partner of said fusion protein;
b. a promoter which is modulated by said expression modulating domain of said fusion protein; and
c. a reporter gene, the expression of which is controlled by said promoter.
16. The host cell according to claim 15, wherein said promoter is a mammalian heat shock promoter.
17. The host cell according to claim 15, wherein said reporter gene is the luciferase gene.
18. The host cell according to claim 15, additionally comprising a DNA molecule which encodes a protein binding partner necessary for activating said DNA binding domain of said fusion protein.
19. A method for assaying protease activity in vitro comprising the steps of:
a. incubating a fusion protein according to any one of claims 1 to 5 in an in vitro transcription extract with a protease capable of cleaving said fusion protein at said protease cleavage site and a DNA molecule, wherein said DNA molecules comprise (i) a LRE which binds to the DNA binding domain of said fusion protein; (ii) a promoter which is modulated by the expression modulating domain of the fusion protein; and (iii) a reporter gene, the expression of which is controlled by the promoter, wherein said protease cleavage site of said fusion protein can be cleaved by said protease whose activity is being assayed;
b. adding to said incubation a ligand which associates with said fusion protein, wherein said ligand is required for said fusion protein to (i) bind to said LRE or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and
c. quantifying the gene product produced from said reporter gene.
20. A method for assaying protease activity in a cell comprising the steps of:
a. culturing a host cell according to any one of claims 15 to 18 under conditions which cause expression of said fusion protein and, if present, said protein binding partner, wherein said host cell expresses the protease to be assayed and wherein said protease cleavage site of said fusion protein can be cleaved by said protease;
b. adding to said host cell culture a ligand which associates with and regulates the activity of said fusion protein, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene;
c. quantifying the gene product produced from said reporter gene.
21. A method for determining the inhibitory activity of a compound against a protease comprising the steps of:
a. culturing in a first culture a host cell according to any one of claims 15 to 18 in the absence of said compound under conditions which cause expression of said fusion protein, wherein said host cell expresses the protease to be assayed and wherein said protease cleavage site of said fusion protein can be cleaved by said protease;
b. culturing in a second culture the host cell used in step a. in the presence of said compound under conditions which cause expression of said fusion protein;
c. adding to said first and to said second host cell cultures a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene which associates of said fusion protein;
d. comparing the amount of gene product produced from said reporter gene in said first host cell culture and said second host cell culture.
22. The method according to claim 21, comprising the additional steps of:
a. culturing in a third culture a host cell according to any one of claims 15 to 18 under conditions which cause expression of said fusion protein and wherein said host cell does not express a protease which recognizes the protease cleavage site in said fusion protein; and, as part of step d., comparing the amount of gene product produced from said reporter gene in said host cell culture with the amount of gene product produced from said reporter gene in said first and second host cell cultures.
23. A method of comparing the activity of two proteases which recognize the same cleavage site, said method comprising the steps of:
a. culturing a first host cell according to any one of claims 15 to 18 under conditions which cause expression of said fusion protein and, if present, said protein binding partner, wherein said first host cell expresses a first protease which is capable of cleaving said protease cleavage site of said fusion protein;
b. culturing a second host cell according to any one of claims 15 to 18 under conditions which cause expression of said fusion protein and, if present, said protein binding partner, wherein said second host cell expresses a second protease which is capable of cleaving said protease cleavage site of said fusion protein;
c. adding to said first and to said second host cell culture a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and
d. comparing the amount of gene product produced from said reporter gene in said first host cell culture and said second host cell culture.
24. The method according to claim 23, wherein said first and said second proteases are forms of HIV protease.
25. A kit for assaying protease activity comprising:
a. a DNA molecule according to claim 10 or 11 or a vector according to claim 12;
b. a DNA molecule comprising:
i. a LRE which is capable of being bound to said DNA binding domain of said fusion protein encoded by the DNA molecule of a.;
ii. a promoter which is capable of being modulated by said expression modulating domain of said fusion protein encoded by the DNA molecule of a.; and
iii. a reporter gene, the expression of which is controlled by said promoter;
c. a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE operatively linked to a reporter gene or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and
d. instructions for using said kit to assay protease activity.
26. The kit according to claim 25, wherein said DNA molecule of a. and said DNA molecule of b. are both present in a host cell.
27. The kit according to claim 26 further including a DNA molecule or a vector comprising a DNA molecule encoding a protein binding partner of said fusion protein.
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