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WO1999011794A2 - Nouvelle kinase vrk1 - Google Patents

Nouvelle kinase vrk1 Download PDF

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Publication number
WO1999011794A2
WO1999011794A2 PCT/US1998/018524 US9818524W WO9911794A2 WO 1999011794 A2 WO1999011794 A2 WO 1999011794A2 US 9818524 W US9818524 W US 9818524W WO 9911794 A2 WO9911794 A2 WO 9911794A2
Authority
WO
WIPO (PCT)
Prior art keywords
nvrkl
sequence
sequences
kinase
polynucleotide sequence
Prior art date
Application number
PCT/US1998/018524
Other languages
English (en)
Other versions
WO1999011794A3 (fr
Inventor
Henry Yue
Preeti Lal
Neil C. Corley
Original Assignee
Incyte Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to AU93044/98A priority Critical patent/AU9304498A/en
Publication of WO1999011794A2 publication Critical patent/WO1999011794A2/fr
Publication of WO1999011794A3 publication Critical patent/WO1999011794A3/fr

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Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof.
  • the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
  • the invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • Microarray refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • “Fragments” are those nucleic acid sequences which are greater than 60 nucleotides than in length, and most preferably includes fragments that are at least 100 nucleotides or at least 1000 nucleotides, and at least 10,000 nucleotides in length.
  • substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • nucleotide sequences encoding NVRKl may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring NVRKl, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter NVRKl encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • natural, modified, or recombinant nucleic acid sequences encoding NVRKl may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between the NVRKl encoding sequence and the heterologous protein sequence, so that NVRKl may be cleaved and purified away from the heterologous moiety.
  • An insect system may also be used to express NVRKl.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding NVRKl may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of NVRKl will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which NVRKl may be expressed (Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6 to 10M are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding NVRKl. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding NVRKl, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; Bethesda, MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150: 1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci.
  • Antibodies to NVRKl may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding NVRKl or closely related molecules, may be used to identify nucleic acid sequences which encode NVRKl .
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding NVRKl, alleles, or related sequences.
  • oligonucleotides In order to produce oligonucleotides to a known sequence for a microarray, the gene of interest is examined using a computer algorithm which starts at the 5' or more preferably at the 3 'end of the nucleotide sequence.
  • the algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray.
  • the "pairs" will be identical, except for one nucleotide which preferably is located in the center of the sequence.
  • the second oligonucleotide in the pair serves as a control.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.
  • RNA or DNA from a biological sample is made into hybridization probes.
  • the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA).
  • aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray so that the probe sequences hybridize to complementary oligonucleotides of the microarray. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray.
  • the biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies on the sequences, mutations, variants, or polymorphisms among samples.
  • the frozen tissues were homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, NJ) in guanidinium isothiocyanate solution. Both lysates were extracted once with an equal volume acid phenol per Stratgene's RNA isolation protocol (Stratagene Inc., San Diego, CA). The RNA was extracted once with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in DEPC-treated water, and DNase treated at 37 °C for 25 minutes. The mRNA was then isolated using the Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, CA) and used to construct the cDNA libraries.
  • Qiagen Oligotex kit QIAGEN, Inc., Chatsworth, CA
  • sequences have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • threshold was set at 10 "25 for nucleotides and 10 "14 for peptides.
  • High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, PCR was performed using the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown,
  • NVRKl that is substantially purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • the amino acid sequence deduced from SEQ ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel et al. (supra), and others.
  • the oligopeptides are 15 residues in length, synthesized using an Applied Protocol

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une nouvelle kinase VRK1 (NVRK1) et des polynucléotides identifiant et codant la NVRK1. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des agonistes, des anticorps et des antagonistes. L'invention concerne, en outre, des méthodes de traitement de troubles associés à l'expression de la NVRK1.
PCT/US1998/018524 1997-09-04 1998-09-04 Nouvelle kinase vrk1 WO1999011794A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU93044/98A AU9304498A (en) 1997-09-04 1998-09-04 New vrk1 kinase

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92346997A 1997-09-04 1997-09-04
US08/923,469 1997-09-04

Publications (2)

Publication Number Publication Date
WO1999011794A2 true WO1999011794A2 (fr) 1999-03-11
WO1999011794A3 WO1999011794A3 (fr) 1999-07-08

Family

ID=25448731

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/018524 WO1999011794A2 (fr) 1997-09-04 1998-09-04 Nouvelle kinase vrk1

Country Status (2)

Country Link
AU (1) AU9304498A (fr)
WO (1) WO1999011794A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960938A1 (fr) * 1996-12-27 1999-12-01 Chugai Research Institute for Molecular Medicine Inc. Nouveau gene de serine-threonine kinase
WO2001011086A2 (fr) * 1999-08-11 2001-02-15 Eos Biotechnology, Inc. Nouvelles techniques de diagnostic de l'angiogenese, compositions et techniques de criblage pour modulateurs d'angiogenese
US6677437B1 (en) 1996-12-27 2004-01-13 Chugai Seiyaku Kabushiki Kaisha Serine-threonine kinase gene
WO2014160364A1 (fr) * 2013-03-13 2014-10-02 Dana-Farber Cancer Institute, Inc. Méthodes de traitement du cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029552A1 (fr) * 1996-12-27 1998-07-09 Chugai Research Institute For Molecular Medicine, Inc. Nouveau gene de serine-threonine kinase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029552A1 (fr) * 1996-12-27 1998-07-09 Chugai Research Institute For Molecular Medicine, Inc. Nouveau gene de serine-threonine kinase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NEZU J. ET AL.: "Identification of two novel human putative serine/threonine kinases VRK1 and VR2 with structural similarity to vaccinia virus B1R kinase" GENOMICS, vol. 45, no. 2, 15 October 1997, pages 327-331, XP002096180 *
NEZU J. ET AL.: "VRK1, COMPLETE CDS" EMBL DATABASE,1 May 1997, XP002096179 HEIDELBERG, DE *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960938A1 (fr) * 1996-12-27 1999-12-01 Chugai Research Institute for Molecular Medicine Inc. Nouveau gene de serine-threonine kinase
EP0960938A4 (fr) * 1996-12-27 2001-12-05 Chugai Res Inst Molecular Med Nouveau gene de serine-threonine kinase
US6677437B1 (en) 1996-12-27 2004-01-13 Chugai Seiyaku Kabushiki Kaisha Serine-threonine kinase gene
US7186536B2 (en) 1996-12-27 2007-03-06 Chugai Seiyaku Kabushiki Kaisha Serine-threonine kinase gene
WO2001011086A2 (fr) * 1999-08-11 2001-02-15 Eos Biotechnology, Inc. Nouvelles techniques de diagnostic de l'angiogenese, compositions et techniques de criblage pour modulateurs d'angiogenese
WO2001011086A3 (fr) * 1999-08-11 2002-03-07 Eos Biotechnology Inc Nouvelles techniques de diagnostic de l'angiogenese, compositions et techniques de criblage pour modulateurs d'angiogenese
WO2014160364A1 (fr) * 2013-03-13 2014-10-02 Dana-Farber Cancer Institute, Inc. Méthodes de traitement du cancer

Also Published As

Publication number Publication date
AU9304498A (en) 1999-03-22
WO1999011794A3 (fr) 1999-07-08

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