WO2000006714A1 - ACIDES NUCLEIQUES DDKh-3, POLYPEPTIDES, VECTEURS, CELLULES HOTES, PROCEDES ET UTILISATIONS - Google Patents

ACIDES NUCLEIQUES DDKh-3, POLYPEPTIDES, VECTEURS, CELLULES HOTES, PROCEDES ET UTILISATIONS Download PDF

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WO2000006714A1
WO2000006714A1 PCT/US1999/016963 US9916963W WO0006714A1 WO 2000006714 A1 WO2000006714 A1 WO 2000006714A1 US 9916963 W US9916963 W US 9916963W WO 0006714 A1 WO0006714 A1 WO 0006714A1
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polypeptide
ddkh
nucleic acid
isolated
present
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Brian Taylor Edmonds
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Eli Lilly and Co
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Eli Lilly and Co
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to compounds and compositions comprising novel human dickkopf-1 homolog 3 (DDKh-3) polypeptides and nucleic acids. More specifically, recombinant or isolated nucleic acid molecules are provided encoding human DDKh-3 polypeptides as well as vectors, host cells, antibodies and methods for producing and using different aspects of the invention.
  • DDKh-3 novel human dickkopf-1 homolog 3
  • the Spemann organizer in amphibian embryos is a tissue with potent head-inducing activity, the molecular nature of which is unresolved.
  • dickkopf-1 dkk-1
  • Dkk-1 a potent antagonist of nt signaling, suggesting that dkk genes encode a family of secreted Wnt inhibitors .
  • the present invention provides isolated nucleic acids and encoded DDKh-3 polypeptides, including fragments and specified variants, as well as DDKh-3 compositions, probes, vectors, host cells, antibodies, and methods, as described and enabled herein.
  • the present invention provides, in one aspect, isolated nucleic acid molecules comprising a polynucleotide encoding specific DDKh-3 polypeptides as fragments or specified variants comprising at least one domain thereof.
  • polypeptides are provided as non-limiting examples by the corresponding domains, fragments and/or variants as DDKh-3 polypeptides corresponding to at least five amino acid fragments of at least one of SEQ ID NOS:2, 3, respectively.
  • the present invention further provides recombinant vectors, comprising 1-40 of said isolated DDKh-3 nucleic acid molecules of the present invention, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such nucleic acid, vectors and/or host cells.
  • the present invention also provides methods of making or using such nucleic acids, vectors and/or host cells, such as but not limited to, using them for the production of DDKh-3 nucleic acids and/or polypeptides by known recombinant, synthetic and/or purification techniques, based on the teaching and guidance presented herein in combination with what is known in the art . *
  • the present invention also provides an isolated DDKh-3 polypeptide, comprising at least one fragment, domain or specified variant of at least 5-1,000 contiguous amino acids of at least one portion of at least one of SEQ ID NOS : 2 , 3.
  • the present invention also provides an isolated DDKh-3 polypeptide as described herein, comprising at least one amino acid sequence selected from at least one of SEQ ID NOS : 2 , 3.
  • the present invention also provides an isolated DDKh-3 polypeptide as described herein, wherein the polypeptide further comprises at least one specified mutation corresponding to portions or residues of SEQ ID NOS:2, 3.
  • the present invention also provides an isolated DDKh-3 polypeptide as described herein, wherein the polypeptide has at least one activity selected from Wnt antagonist (Glinka et al., Nature 391:357-362 (Jan. 1998);). A DDKh-3 polypeptide can thus be screened for a corresponding activity according to known methods.
  • the present invention also provides a composition comprising an isolated DDKh-3 nucleic acid and/or polypeptide as described herein and a carrier or diluent .
  • the carrier or diluent can optionally be pharmaceutically acceptable, according to known methods.
  • the present invention also provides an isolated nucleic acid probe as described herein, wherein the nucleic acid comprises a polynucleotide of at least 10 nucleotides, corresponding or complementary to at least 10 nucleotides of at least one of SEQ ID NO:l, or a consensus sequence thereof .
  • the present invention also provides a vector comprising an isolated DDKh-3 nucleic acid as described herein.
  • the present invention also provides a vector as described herein, wherein the vector is selected from a linear or circular, single or double stranded, DNA, RNA, or combination thereof, nucleic acid vector.
  • the present invention also provides a host cell, comprising a isolated DDKh-3 nucleic acid as described herein.
  • the present invention also provides a method for constructing a recombinant host cell that expresses a DDKh-3 polypeptide, comprising introducing into the host cell a DDKh-3 nucleic acid in replicatable form as described herein to provide the recombinant host cell.
  • the present invention also provides a recombinant host cell provided by a method as described herein.
  • the present invention also provides a method for expressing a DDKh-3 polypeptide in a recombinant host cell, comprising culturing a recombinant host cell as described herein under conditions wherein the DDKh-3 polypeptide is expressed in detectable or recoverable amounts .
  • the present invention also provides an isolated DDKh-3 polypeptide produced by a recombinant, synthetic and/or purification method as described herein and/or as known in the art .
  • the present invention also provides a DDKh-3 antibody or fragment, comprising a polyclonal and/or monoclonal antibody or fragment that specifically binds at least one epitope specific to an isolated DDKh-3 polypeptide as described herein.
  • the present invention also provides a method for providing a DDKh-3 antibody or fragment, comprising generating the antibody or fragment that binds at least one epitope that is specific to an isolated DDKh-3 polypeptide as described herein, the generating done by recombinant, synthetic and/or hybridoma techniques .
  • the present invention also provides a DDKh-3 antibody or fragment produced by a method as described herein.
  • the present invention also provides a method for identifying compounds that bind a DDKh-3 polypeptide, comprising a) admixing at least one isolated DDKh-3 polypeptide as described herein with a test compound or composition; and b) detecting at least one binding interaction between the polypeptide and the compound or composition.
  • the present invention provides isolated, recombinant and/or synthetic nucleic acid molecules comprising a polynucleotide encoding DDKh-3 polypeptides comprising specific fragments and specified variants, such polypeptides, and methods of making and using thereof.
  • a DDKh-3 polypeptide of the invention comprises at least one fragment domain, and/or specified variant as a portion or fragment of a DDKh-3 protein as described herein.
  • the present invention also provides at least one utility by providing isolated nucleic acid comprising polynucleotides of sufficient length and complementarity to a DDKh-3 nucleic acid to use as probes or amplification primers in the detection, quantitation, or isolation of gene transcripts.
  • isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for detecting mutations in the gene (e.g., substitutions, deletions, or additions), for monitoring upregulation of expression or changes in biological activity as described herein in screening assays of compounds, and/or for detection of any number of allelic variants (polymorphisms) of the gene.
  • the isolated nucleic acids of the present invention can also be used for recombinant expression of DDKh-3 polypeptides, or for use as immunogens in the preparation and/or screening of antibodies .
  • the isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more DDKh-3 genes, nucleic acids or genes in a host cell, or tissue. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation of at least one nucleic acid.
  • a "polynucleotide” comprises at least 5 - 10 nucleotides of a nucleic acid (RNA, DNA or combination thereof) , provided by any means, such as synthetic, recombinant isolation or purification method steps .
  • complementarity refers to the capacity of purine, pyrimidine, synthetic or modified nucleotides to associate by partial or complete complementarity through hydrogen or other bonding to form partial or complete double or triple stranded nucleic acid molecules .
  • the following base pairs occur by complete complementarity: (i) guanine (G) and cytosine (C) ; (ii) adenine (A) and thymine (T) ; and adenine (A) and uracil (U) .
  • Partial complementarity refers to association of two or more bases by one or more hydrogen bonds or attraction that is less than the complete complementarity as described above .
  • Partial or complete complementarity can occur between any two nucletides, including naturally occurring or modified bases, e.g., as listed in 37 CFR sec. 1.822. All such nucleotides are included in polynucleotides of the invention as described herein.
  • “Fragment” refers to a fragment, piece, portion, or sub-region of a nucleic acid or polypeptide molecule as disclosed herein, such that the fragment comprises 4 or more amino acids, or 10 or more nucleotides, that are contiguous in the referenced polypeptide or nucleic acid molecule. A fragment thereof may or may not retain biological activity.
  • a fragment of a polypeptide disclosed herein could be used as an antigen to raise a specific antibody against the referenced polypeptide molecule.
  • fusion protein denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain.
  • polypeptide also includes such fusion proteins .
  • "Host cell” refers to any eucaryotic, procaryotic, or fusion or other cell or pseudo cell or membrane containing construct that is suitable for propagating and/or expressing an isolated nucleic acid that is introduced into the host cell by any suitable means known in the art (e.g., but not limited to, transformation or transfection, or the like) .
  • the cell can be part of a tissue or organism, isolated in culture or in any other suitable form.
  • hybridization refers to a process in which a partially or completely single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. Hybridization can occur under conditions of low, moderate to high stringency, with high stringency preferred. The degree of hybridization depends upon, for example, the degree of homology, the stringency conditions, and the length of hybridizing strands .
  • isolated nucleic acid molecule is intended a nucleic acid molecule, DNA, RNA, or both which has been removed from its native or naturally occurring environment.
  • isolated nucleic acid molecules contained or generated in culture, a vector and/or a host cell are considered isolated for the purposes of the present invention.
  • isolated nucleic acid molecules include recombinant nucleic acid molecules maintained in heterologous host cells or purified (partially or substantially) nucleic acid molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the nucleic acid molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically, purified from or provided in cells containing such nucleic acids, where the nucleic acid in other than a naturally occurring form, quantitatively or qualitatively.
  • isolated used in reference to at least one polypeptide of the invention describes a state of isolation such that the peptide or polypeptide is not in a naturally occurring form and/or has been purified to remove at least some portion of cellular or non-cellular molecules with which the protein is naturally associated.
  • isolated may include the addition of other functional or structural polypeptides for a specific purpose, where the other peptide may occur naturally associated with at least one polypeptide of the present invention.
  • a “nucleic acid probe,” “oligonucleotide probe,” or “probe” as used herein comprises at least one detectably labeled or unlabeled nucleic acid which hybridizes under specified hybridization conditions with at least one other nucleic acid. This term also refers to a single or partially double stranded nucleic acid, oligonucleotide or polynucleotide that will associate with a complementary or partially complementary target nucleic acid to form at least a partially double-stranded molecule.
  • a nucleic acid probe may be an oligonucleotide or a nucleotide polymer.
  • a probe can optionally contain a detectable moiety which may be attached to the end(s) of the probe or be internal to the sequence of the probe, termed a “detectable probe” or “detectable nucleic acid probe.”
  • a “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule, e.g., using a amplification reaction, such as, but not limited to, a polymerase chain reaction (PCR) , as known in the art.
  • PCR polymerase chain reaction
  • promoter refers to a nucleic acid sequence that directs the initiation of transcription, for example, of DNA to RNA.
  • An inducible promoter is one that is regulatable by environmental signals, such as carbon source, heat , or metal ions , for example .
  • stringency refers to hybridization conditions for nucleic acids in solution. High stringency conditions disfavor non-homologous base pairing. Low stringency conditions have much less of this effect. Stringency may be altered, for example, by temperature and salt concentration, or other conditions, as well known in the art.
  • a non-limiting example of "high stringency” conditions includes, for example, (a) a temperature of about 42° C , a formamide concentration of about ⁇ 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65° C, or less, and a low salt (SSPE) concentration; (b) hybridization in 0.5 M NaHP04 , 7% sodium dodecyl sulfate (SDS) , 1 mM EDTA at 65°C (See, e.g., Ausubel et al . , ed. , Current Protocols in Molecular Biology, 1987-1998, Wiley Interscience, New York, at ⁇ 2.10.3).
  • SSC comprises a hybridization and wash solution.
  • a stock 2OX SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.
  • SSPE comprises a hybridization and wash solution.
  • a IX SSPE solution contains 180 mM NaCl, 9mM Na2HP04 , 0.9 mM NaH2P04 and 1 mM EDTA, pH 7.4.
  • vector refers to a nucleic acid compound used for introducing exogenous or endogenous nucleic acid into host cells .
  • a vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses, and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated nucleic acid molecule.
  • reaction conditions for particular enzymes are well known to one of ordinary skill in the art (New England Biolabs, Boston; Life Technologies, Rockville, Md.). Reaction conditions for particular enzymes are preferably carried out according to the manufacturer's recommendation.
  • nucleotide sequences identified by sequencing a nucleic acid molecule herein were identified using an automated nucleic acid sequencer, and all amino acid sequences of polypeptides encoded by nucleic acid molecules identified herein were identified by codon correspondence or by translation of a nucleic acid sequence identified as described herein or as known in the art. Therefore, as is well known in the art that for any nucleic acid sequence identified by this automated approach, any nucleotide sequence identified herein may contain some errors which are reproducibly correctable by resequencing based upon an available or a deposited vector or host cell containing the nucleic acid molecule using well-known methods .
  • the identified nucleotide sequence of a DDKh-3 nucleic acid of SEQ ID NO:l contains an open reading frame encoding a polypeptide of about 112 amino acid residues (SEQ ID NO:l).
  • Isolated nucleic acid molecules of the present invention include nucleic acid molecules comprising an open reading frame (ORF) shown in at least one SEQ ID NOS1; nucleic acid molecules comprising the coding sequence for a DDKh-3 polypeptide; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one DDKh-3 polypeptide as described and enabled herein.
  • ORF open reading frame
  • nucleic acid molecules comprising the coding sequence for a DDKh-3 polypeptide
  • the genetic code is well known in the art.
  • the invention provides isolated nucleic acid molecules encoding a DDKh-3 polypeptide having an amino acid sequence as encoded by the cDNA clone contained in the plasmid deposited as designated clone names and ATCC Deposit Nos., as follows: , respectively deposited as , deposited on .
  • nucleic acid molecules are provided encoding the mature DDKh-3 polypeptide or the full- length DDKh-3 polypeptide lacking the N-terminal methionine, such as of at least one of 2.
  • the invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in at least one of SEQ ID NO:l, or the nucleotide sequence of the DDKh-3 cDNA contained in at least one of the above-described deposited clones, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • isolated molecules particularly nucleic acid molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting transcription, translation and/or expression of the DDKh-3 gene in human tissue, for instance, by Northern blot analysis for mRNA detection.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having at least 10 nucleotides of a nucleotide sequence of a deposited cDNA or a nucleotide sequence shown in at least one of SEQ ID NO:l, and is intended fragments at least about 10 nt, at least about 15 nt, at least about 30 nt, and at least about 40 nt in length, which are useful, inter alia as diagnostic probes and primers as described herein.
  • fragments such as at least about 50, 100, 120, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, and/or 4000 nt in length, are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown at least one of SEQ ID NO:l.
  • a fragment at least 10 nt in length for example, is intended fragments which include 10 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:l, or consensus sequences thereof, as determined by methods known in the art .
  • nucleotide fragments are useful according to the present invention for screening DNA sequences that code for one or more fragments of a DDKh-3 polypeptide as described herein.
  • nucleic acid molecules of the present invention which comprise a nucleic acid encoding a DDKh-3 polypeptide can include, but are not limited to, those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non- coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA) ; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding a polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the
  • Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of a DDKh-3 polypeptide.
  • the invention provides a polynucleotide (either DNA or RNA) that comprises at least about 10 nucleotides (nt) , and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably at least about 30-2000 nt of a nucleic acid molecule described herein. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly (A) of a DDKh-3 polypeptide cDNA shown in at least one of SEQ ID NO:l
  • a complementary stretch of T (or U) resides would not be included in a probe of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone) .
  • the present invention also provides subsequences of full-length nucleic acids.
  • any number of subsequences can be obtained by reference to at least one of SEQ ID NO:l, and using primers which selectively amplify, under stringent conditions to: at least two sites to the polynucleotides of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it.
  • a variety of methods for obtaining 5' and/or 3' ends is well known in the art. See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in Frohman, M.
  • Primer sequences can be obtained by reference to a contiguous subsequence of a polynucleotide of the present invention.
  • Primers are chosen to selectively hybridize, under PCR amplification conditions, to a polynucleotide of the present invention in an amplification mixture comprising a genomic and/or cDNA library from the same species.
  • the primers are complementary to a subsequence of the amplicon they yield.
  • the primers will be constructed to anneal at their 5' terminal end's to the codon encoding the carboxy or amino terminal amino acid residue (or the complements thereof) of the polynucleotides of the present invention.
  • the primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50.
  • the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length.
  • a non-annealing sequence at the 5'end of the primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.
  • Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure. Antibody screening of peptide display libraries is well known in the art .
  • the displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 15 amino acids long.
  • several recombinant DNA methods have been described.
  • One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence.
  • the isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof , as well known in the art .
  • the nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention - excluding the polynucleotide sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention.
  • cloning and/or expression sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb.
  • Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.
  • RNA, cDNA, genomic DNA, or a hybrid thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
  • oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. While isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art. Nucleic Acid Screening and Isolation Methods
  • the cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms .
  • a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein.
  • Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms .
  • degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur.
  • the degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
  • the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through manipulation of the concentration of formamide within the range of 0% to 50%.
  • the degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.
  • the degree of complementarity will optimally be 100 percent; however, it should be understood that minor sequence variations in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.
  • RNA amplification includes, but are not limited to polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et al.; 4,795,699 and 4,921,794 to Tabor et al; 5,142,033 to Innis; 5,122,464 to Wilson et al .
  • PCR polymerase chain reaction
  • RNA mediated amplification which uses anti- sense RNA to the target sequence as a template for double stranded DNA synthesis (U.S. patent No. 5,130,238 to Malek et al, with the tradeneame NASBA) , the entire contents of which patents are herein entirely incorporated by reference.
  • PCR polymerase chain reaction
  • in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al., U.S. Patent No.
  • kits for genomic PCR amplification are known in the art. See, e.g., Advantage- GC Genomic PCR Kit (Clontech) .
  • the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.
  • the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al . , Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts.
  • Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • an automated synthesizer e.g., as described in Needham-VanDevanter et al., Nucleic Acids Res., 12: 6159-6168 (1984); and, the solid support method of U.S. Patent No. 4,458,066.
  • Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
  • the present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention.
  • a nucleic acid sequence coding for the desired polynucleotide of the present invention for example a cDNA or a genomic sequence encoding a full length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell.
  • heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter DDKh-3 content and/or composition in a desired tissue.
  • isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non- heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention.
  • endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution.
  • cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids.
  • Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065- 4076 describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences .
  • a report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785-789.
  • the present invention also relates to vectors which include isolated nucleic acid molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of DDKh-3 polypeptides or fragments thereof by recombinant techniques, as well known in the art. See, eg., Sambrook, et al . , 1989; Ausubel, et al . , 1987-1989, each entirely incorporated herein by reference.
  • the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host .
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells .
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include, e.g., dihydrofolate reductase or neomycin resistance for eucaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as
  • Drosophila S2 and Spodoptera Sf9 cells Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • Appropriate culture mediums and conditions for the above- described host cells are known in the art.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE- 9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • eucaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
  • the polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N- terminus of a polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a polypeptide to facilitate purification. Such regions can be removed prior to final preparation of a polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18. Expression of Proteins in Host Cells
  • nucleic acids of the present invention may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian.
  • a recombinantly engineered cell such as bacteria, yeast, insect, mammalian.
  • the cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
  • nucleic acid encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible) , followed by incorporation into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes .
  • Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention.
  • To obtain high level expression of a cloned gene it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • a strong promoter to direct transcription a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.
  • modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences .
  • Prokaryotic cells may be used as hosts for expression.
  • Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., Gene 22: 229-235 (1983); Mosbach, et al . , Nature 302: 543-545 (1983)).
  • yeast Synthesis of heterologous proteins in yeast is well known. Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast .
  • Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.
  • Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen) . Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • a protein of the present invention once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates .
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques .
  • Suitable animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992) .
  • Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus .
  • Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider, J. Embryol. Exp. Morphol . 27: 353-365 (1987).
  • polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene . Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45: 773-781 (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors.
  • a DDKh-3 polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eucaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention can be glycosylated or can be non- glycosylated.
  • polypeptides of the invention can also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
  • the present invention includes biologically active polypeptides of the present invention (i.e., enzymes).
  • biologically active polypeptides have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% - 100% of that of the native (non- synthetic) , endogenous polypeptide.
  • the substrate specificity (k cat /K is optionally substantially similar to the native (non-synthetic) , endogenous polypeptide.
  • the K is optionally substantially similar to the native (non-synthetic)
  • Sites that are critical for ligand-protein binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol . Biol. 224:899-904 (1992) and de Vos et al . Science 255:306-312 (1992) ) .
  • a DDKh-3 polypeptide can further comprise a polypeptide encoded by 1-3000 contiguous amino acids of SEQ ID NOS:2, 3
  • a DDKh-3 polypeptide further includes an amino acid sequence selected from one or more of SEQ ID NOS : 2 , 3.
  • Antigenic epitope- bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Antibodies included in this invention are useful in diagnostics, therapeutics or in diagnostic/therapeutic combinations.
  • the antibody or compound to be tested is labeled by any suitable method.
  • Competitive displacement of an antibody from an antibody-antigen complex by a test compound such that a test compound-antigen complex is formed provides a method for identifying compounds that bind HPLFP.
  • DDKh-3 nucleic acid can be introduced into a cell or genome of the animal embryo so as to be chromosomally incorporated and expressed according to known methods .
  • Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the a DDKh-3 polypeptide nucleic acid of the present invention such as animals produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic animal, are also intended to be within the scope of the present invention.
  • Chimeric non-human mammals having human cells or tissue engrafted therein are also encompassed by the present invention, which may be used for testing expression of at least one DDKh-3 polypeptide in human tissue and/or for testing the effectiveness of therapeutic and/or diagnostic agents associated with delivery vectors which preferentially bind to a DDKh-3 polypeptide of the present invention.
  • Methods for providing chimeric non-human mammals are provided, e.g, in U.S. serial Nos.
  • transgenic non-human mammals may be used for the production » of a transgenic non-human mammal of the present invention.
  • the various techniques described in Palmiter (1986) may also be used.
  • the animals carrying at least one DDKh-3 polypeptide nucleic acid can be used to test compounds or other treatment modalities which may prevent, suppress or cure a pathology using the a DDKh-3 polypeptide or DDKh-3 nucleic acid of the present invention.
  • Such transgenic animals will also serve as a model for testing of diagnostic methods for the same diseases .
  • Transgenic animals according to the present invention can also be used as a source of cells for cell culture .
  • Example 1 Expression and Purification of a DDKh-3 polypeptide in E. coli
  • the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., Chatsworth, CA) .
  • pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site (“RBS”), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri- acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN, Inc., and suitable single restriction enzyme cleavage sites.
  • a DNA fragment encoding a polypeptide can be inserted in such as way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide.
  • a polypeptide coding sequence can optionally be inserted such that translation of the six His codons is prevented and, therefore, a polypeptide is produced with no 6 X His tag.
  • the nucleic acid sequence encoding the desired portion of a DDKh-3 polypeptide lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers (based on the sequences presented, (e.g., as presented in at least one SEQ ID NO:l), which anneal to the amino terminal sequences of the desired portion of a DDKh-3 polypeptide and to sequences in the deposited construct 3 ' to the cDNA coding sequence . Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
  • the 5' and 3' primers have nucleotides corresponding or complementary to a portion of the coding sequence of a DDKh-3 e.g., as presented in at least one of SEQ ID NO:l, according to known method steps.
  • the point in a polypeptide coding sequence where the 5 ' primer begins can be varied to amplify a desired portion of the complete polypeptide shorter or longer than the mature form.
  • the amplified DDKh-3 nucleic acid fragments and the vector pQE60 are digested with appropriate restriction enzymes and the digested DNAs are then ligated together.
  • Insertion of the DDKh-3 DNA into the restricted pQE60 vector places a DDKh-3 polypeptide coding region including its associated stop codon downstream from the IPTG-inducible promoter and in-frame with an initiating AUG.
  • the associated stop codon prevents translation of the six histidine codons downstream of the insertion point .
  • the ligation mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook, et al.,1989; Ausubel, 1987-1998. E.
  • coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance (“Kanr") , is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing DDKh-3 polypeptide, is available commercially from QIAGEN, Inc. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 mg/microliters) and kanamycin (25 mg/microliters) .
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250.
  • the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
  • Isopropyl-b-D- thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor.
  • Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.
  • the cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCl, pH8.
  • the cell debris is removed by centrifugation, and the supernatant containing the DDKh-3 is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM NaCl .
  • a polypeptide can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH7.4, containing protease inhibitors.
  • the polypeptide is purified by ion exchange, hydrophobic interaction and size exclusion chromatography.
  • an affinity chromatography step such as an antibody column is used to obtain pure DDKh- 3 polypeptide.
  • the purified polypeptide is stored at 4°C or frozen at -80°C.
  • Example 2 Cloning and Expression of a DDKh-3 polypeptide in a Baculovirus Expression System
  • the plasmid shuttle vector pA2 GP is used to insert the cloned DNA encoding the mature polypeptide into a baculovirus to express a DDKh-3 polypeptide, using a baculovirus leader and standard methods as described in Summers et al . , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) .
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 polypeptide and convenient restriction sites such as BamHI, Xba I and Asp718.
  • the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
  • the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
  • the inserted genes are flanked on both sides by viral sequences for cell- mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide.
  • baculovirus vectors are used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
  • Such vectors are described, for instance, in Luckow et al . , Virology 170:31-39.
  • the cDNA sequence encoding the mature DDKh-3 polypeptide in the deposited or other clone, lacking the AUG initiation codon and the naturally associated nucleotide binding site, is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene .
  • Non- limiting examples include 5 ' and 3 ' primers having nucleotides corresponding or complementary to a portion of the coding sequence of a DDKh-3 polypeptide, e.g., as presented in at least one of SEQ ID NO:l, according to known method steps .
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit (e.g., "Geneclean,” BIO 101 Inc., La Jolla, Ca.) .
  • the fragment then is then digested with the appropriate restriction enzyme and again is purified on a 1% agarose gel. This fragment is designated herein "FI".
  • the plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This vector DNA is designated herein "VI”.
  • E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human DDKh-3 gene using the PCR method, in which one of the primers that is used to amplify the gene and the second primer is from well within the vector so that only those bacterial colonies containing the DDKh-3 gene fragment will show amplification of the DNA.
  • the sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac DDKh-3 .
  • plasmid pBacDDKh-3 Five mg of the plasmid pBacDDKh-3 is co-transfected with 1.0 mg of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987) . 1 mg of BaculoGoldTM virus DNA and 5 mg of the plasmid pBacDDKh-3 are mixed in a sterile well of a microtiter plate containing 50 microliters of serum-free Grace's medium (Life Technologies Inc., Rockville, MD) .
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, according to known methods. An agarose gel with "Blue Gal” (Life Technologies Inc., Rockville, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue- stained plaques . (A detailed description of a "plaque assay” of this type can also be found in the user ' s guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Rockville, MD, page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf) .
  • a micropipettor e.g., Eppendorf
  • the agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 microliters of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C.
  • the recombinant virus is called V-DDKh-3.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat - inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-DDKh-3 at a multiplicity of infection ("MOI") of about 2.
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, MD) .
  • SF900 II medium minus methionine and cysteine available from Life Technologies Inc., Rockville, MD
  • radiolabeled polypeptides 42 hours later, 5 mCi of 35S-methionine and 5 mCi 35S-cysteine (available from Amersham) are added.
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation.
  • polypeptides in the supernatant as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled) . Microsequencing of the amino acid sequence of the amino terminus of purified polypeptide can be used to determine the amino terminal sequence of the mature polypeptide and thus the cleavage point and length of the secretory signal peptide.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV) . However, cellular elements can also be used (e.g., the human actin promoter) .
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3. l/Hygro (+/-) (Invitrogen) , PSVL and PMSG (Pharmacia, Uppsala, Sweden) , pRSVcat (ATCC 37152) , pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) .
  • vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Ze
  • Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • the co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded polypeptide.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem J. 227:277-279 (1991); Bebbington et al . , Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome.
  • CHO Chinese hamster ovary
  • NSO cells are often used for the production of polypeptides.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al . ,
  • the expression plasmid, pDDKh-3 HA is made by cloning a cDNA encoding DDKh-3 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.) .
  • the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E.
  • coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eucaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • HA hemagglutinin fragment
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin polypeptide described by Wilson et al . , Cell 37:767-778 (1984).
  • the fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognizes the HA epitope.
  • pcDNAIII contains, in addition, the selectable neomycin marker.
  • a DNA fragment encoding the DDKh-3 is cloned into the polylinker region of the vector so that recombinant polypeptide expression is directed by the CMV promoter.
  • the plasmid construction strategy is as follows.
  • DDKh-3 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of DDKh-3 in E. coli.
  • suitable primers include those based on the coding sequences presented in at least one of SEQ ID NO:l, as they encode DDKh-3 polypeptides as described herein.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with suitable restriction enzyme (s) and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning
  • Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the DDKh-3-encoding fragment .
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989) .
  • Cells are incubated under conditions for expression of DDKh- 3 by the vector.
  • Expression of the DDKh-3-HA fusion polypeptide is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988) .
  • the cells are labeled by incubation in media containing 35S-cysteine for 8 hours.
  • the cells and the media are collected, and the cells are washed and lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al . cited above.
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody.
  • the precipitated polypeptides then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • Plasmid pC4 is used for the expression of DDKh- 3polypeptide.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146) .
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate .
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al . , Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al . , Cell 41:521-530 (1985) ) . Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes .
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • the plasmid contains the 3 ' intron and polyadenylation site of the rat preproinsulin gene.
  • Other high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the DDKh- 3 in a regulated way in mammalian cells (Gossen, M. , & Bujard, H. 1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551).
  • telomeres For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate .
  • the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art .
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the complete DDKh-3 polypeptide including its nucleotide binding site is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • Non-limiting examples include 5 ' and 3 ' primers having nucleotides corresponding or complementary to a portion of the coding sequence of a DDKh-3, e.g., as presented in at least one of SEQ ID NO:l, according to known method steps .
  • the amplified fragment is digested with suitable endonucleases and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
  • 5 mg of the expression plasmid pC4 is cotransfected with 0.5 mg of the plasmid pSV2-neo using lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/microliters G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/microliters of methotrexate plus 1 mg/microliters G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 microliters flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM) .
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM) . The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
  • Example 4 Tissue distribution of DDKh-3 mRNA expression
  • Northern blot analysis is carried out to examine DDKh-3 gene expression in human tissues, using methods described by, among others, Sambrook et al., cited above.
  • a cDNA probe containing the entire nucleotide sequence of a DDKh-3 polypeptide (SEQ ID N0:1) is labeled with 32P using the rediprime (TM) DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for DDKh-3 mRNA.
  • TM rediprime
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues

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Abstract

La présente invention concerne au moins un nouveau polypeptide DDKh-3 renfermant des acides nucléiques isolés qui codent pour au un polypeptide DDKh-3, des polypeptides DDKh-3, des vecteurs, des cellules hôtes, des transgéniques, des chimères, leurs procédés de fabrication et leurs utilisation, ainsi que des anticorps DDKh-3 et des procédés spécifiques.
PCT/US1999/016963 1998-07-31 1999-07-28 ACIDES NUCLEIQUES DDKh-3, POLYPEPTIDES, VECTEURS, CELLULES HOTES, PROCEDES ET UTILISATIONS Ceased WO2000006714A1 (fr)

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AU52335/99A AU5233599A (en) 1998-07-31 1999-07-28 Ddkh-3 nucleic acids, polypeptides, vectors, host cells, methods and uses thereof

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US9521798P 1998-07-31 1998-07-31
US9494798P 1998-07-31 1998-07-31
US9497698P 1998-07-31 1998-07-31
US60/094,947 1998-07-31
US60/094,976 1998-07-31
US60/095,217 1998-07-31

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7628989B2 (en) 2001-04-10 2009-12-08 Agensys, Inc. Methods of inducing an immune response
US8673306B2 (en) 2006-01-13 2014-03-18 Novartis Ag Compositions and methods of use for antibodies of dickkopf-1
WO2016004055A1 (fr) 2014-07-03 2016-01-07 Yale University Suppression de la formation de tumeurs par inhibition de dickkopf2 (dkk2)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GLINKA ET AL: "Dickkopf-1 is a Member of a New Family of Secreted Proteins and Functions in Head Induction", NATURE, vol. 391, 22 January 1998 (1998-01-22), pages 357 - 362, XP002921970 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7628989B2 (en) 2001-04-10 2009-12-08 Agensys, Inc. Methods of inducing an immune response
US7641905B2 (en) 2001-04-10 2010-01-05 Agensys, Inc. Methods of inducing an immune response
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
US8673306B2 (en) 2006-01-13 2014-03-18 Novartis Ag Compositions and methods of use for antibodies of dickkopf-1
US9296813B2 (en) 2006-01-13 2016-03-29 Novartis Ag Compositions and methods of use for antibodies of Dickkopf-1
WO2016004055A1 (fr) 2014-07-03 2016-01-07 Yale University Suppression de la formation de tumeurs par inhibition de dickkopf2 (dkk2)
EP3164194A4 (fr) * 2014-07-03 2018-05-09 Yale University Suppression de la formation de tumeurs par inhibition de dickkopf2 (dkk2)
US10398765B2 (en) 2014-07-03 2019-09-03 Yale University Dickkopf2 (Dkk2) inhibition suppresses tumor formation
EP3978524A1 (fr) * 2014-07-03 2022-04-06 Yale University Suppression de la formation de tumeurs par inhibition de dickkopf2 (dkk2)
US11497799B2 (en) 2014-07-03 2022-11-15 Yale University Dickkopf2 (Dkk2) inhibition suppresses tumor formation

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