# US 5,350,835 A · Cellular nucleic acid binding protein and uses thereof in regulating gene expression and in the treatment of aids > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,350,835 A - Document ID: US5350835A - Status: Granted - Kind: A - Application number: 07/788,266 - Filed: 1991-11-05 - Granted: 1994-09-27 - Earliest claimed: 1991-11-05 - Assignees: Board of Regents, The University of Texas - Inventors: Richard B. Gaynor; Foon K. Wu - Web page: https://patentgrep.com/patent/US5350835A ## CPC - C07K 14/005: from viruses - C07K 14/4702: {Regulators; Modulating activity} - C07K 16/18: against material from animals or humans - G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites} - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy - C07K 2319/00: Fusion polypeptide - C12N 2740/16322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - Y10S 435/974: Aids related test - Y10S 930/221: Retrovirus related, or human immunodeficiency virus related, or simian immunodeficiency virus related - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract The invention relates to a cellular protein which is specific and has high affinity for nucleic acid sequences characteristic of an intact TAR RNA loop sequence of the HIV LTR TAR region. The invention also relates to a between 1,000-10,000-fold purified, about 185 kD protein preparation isolated from a mammalian cell nuclear extract preparation, most specifically a HeLa cell extract. The about 185 kD protein is shown to regulate HIV viral gene expression by binding a TAR RNA region of an HIV LTR template, in the presence of a cofactor fraction (including at least a.about.100 kD cofactor), and a tat protein. The TRP-185, having a molecular weight of about 185 kD protein may also provide a research tool in the study of viral and cellular gene expression. A route for the development of immunodiagnostics for AIDS and related disorders may also be provided given the specific and high affinity of TRP-185 for HIV RNA. The 185 kD protein and related encoding amino acid and DNA sequences may also be used in therapeutic agents for AIDS and related disorders and, for the generation of specific antibodies for use in the diagnosis and study of the epidemiology of AIDS. ## Claims ### Claim 1 A nucleic acid binding element obtained from mammalian cells susceptible to infection with HIV comprising a TRP-185 cellular protein that binds with high affinity to a TAR bulge and loop region of an HIV RNA in the presence of a cofactor fraction having a molecular weight of about 85-100 kD by sucrose gradient analysis, said TRP-185 cellular protein having a molecular weight of about 175-190 kD by sucrose gradient analysis. ### Claim 2 (depends on 1) The nucleic acid binding element of claim 1 wherein said cellular protein is designated TRP-185. ### Claim 3 (depends on 2) The nucleic acid binding element of claim 2 wherein said cellular protein binds to a TAR region of an HIV mRNA with an affinity of about 3.times.10.sup.-10 M. ### Claim 4 (depends on 3) The nucleic acid binding element of claim 3 wherein said cellular protein increases transcription of wild type HIV LTR about 4-fold. ### Claim 5 (depends on 1) The nucleic acid binding element of claim 1 wherein the TAR region is a transactivating region. ### Claim 6 (depends on 1) The nucleic acid binding element of claim 1 wherein the TAR region is at least a 12 base pair segment of the TAR mRNA. ### Claim 7 (depends on 6) The nucleic acid binding element of claim 6 wherein the cellular protein binds a base pair segment between bases 23-34 of the TAR mRNA. ### Claim 8 (depends on 1) The nucleic acid binding element of claim 1 further defined having a molecular weight of about 185 kD. ### Claim 9 (depends on 1) The nucleic acid binding element of claim 1 prepared by a process comprising the steps of: obtaining a volume of mammalian cells susceptible to infection by HIV; preparing a nuclear extract from the mammalian cells; fractionating the nuclear extract and selecting fractions having HIV TAR binding activity; and isolating an element having a molecular weight of between 175-190 kD from the selected fractions to provide a nucleic acid binding element. ### Claim 10 (depends on 9) The nucleic acid binding element of claim 9 wherein preparing a nuclear extract of mammalian cells comprises: slicing a cell pellet of mammalian cells susceptible to infection with HIV with a cell homogenizer; isolating cell nuclei; lysing the nuclei to obtain a lysate; separating a supernatant from the lysate; and isolating a nuclear extract from the supernatant, said nuclear extract having a binding activity for a TAR region of a nucleic acid. ### Claim 11 (depends on 9) The nucleic acid binding element of claim 9 wherein the steps for fractionating the nuclear extract includes: chromatographing the nuclear extract on heparin agarose; eluting the chromatographed nuclear extract with potassium chloride to obtain fractions having TAR binding activity; preparing a dialyzate of the fractions having TAR binding activity; chromatographing the dialyzate on an anionic separation gel; precipitating TAR active binding fractions from the anionic separation gel with ammonium sulfate; applying the TAR active binding fractions to a molecular weight separation column; collecting TAR active binding fractions and applying a dialyzate thereof to a molecular weight separation column; collecting TAR active binding fractions and applying a dialyzate thereof to a anionic separation column; collecting TAR active binding fractions and applying a dialyzate thereof to a anionic separation column; collecting TAR active binding fractions and applying a dialyzate of selected washed fractions to a continuous sucrose gradient; and isolating a nucleic acid binding element having a molecular weight of between 175 kD-190 kD as determined by a continuous sucrose gradient and having TAR region binding activity. ### Claim 12 (depends on 1, 9, 10) The nucleic acid binding element of claims 1, 9 or 10 wherein the cellular protein is obtained from a HeLa cell nuclear extract. ### Claim 13 (depends on 11) The nucleic acid binding element of claim 11 wherein the cellular protein is designated TRP-185 and is obtained in a preparation from HeLa cells at an about 3,000-fold purification. ### Claim 14 A TRP-185 nucleic acid binding protein having a molecular weight of between about 175 kD-190 kD as determined by continuous sucrose gradient and having high binding affinity to a bulge and loop TAR region of HIV mRNA in the presence of a cofactor fraction, said cofactor fraction having a molecular weight of about 100 kD as determined by continuous sucrose gradient. ### Claim 15 (depends on 14) The TRP-185 nucleic acid binding protein of claim 14 wherein the co-factor fraction enhances the binding of the TRP-185 to the TAR region of HIV mRNA. ### Claim 16 A nucleic acid binding protein TRP-185 prepared by a process comprising the steps of: obtaining a volume of HeLa cells; preparing a nuclear extract; fractionating the nuclear extract; and isolating the nucleic acid binding protein comprising the TRP-185 protein from the nuclear extract fractions, said TRP-185 protein having a molecular weight of between about 175 kD-190 kD and also having binding affinity to a TAR bulge and loop region of HIV mRNA.