WO2000005248A1 - Cellules hotes, vecteurs, polypeptides, acides nucleiques de gdf-3 humains et leur methodes d'utilisation - Google Patents

Cellules hotes, vecteurs, polypeptides, acides nucleiques de gdf-3 humains et leur methodes d'utilisation Download PDF

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WO2000005248A1
WO2000005248A1 PCT/US1999/016474 US9916474W WO0005248A1 WO 2000005248 A1 WO2000005248 A1 WO 2000005248A1 US 9916474 W US9916474 W US 9916474W WO 0005248 A1 WO0005248 A1 WO 0005248A1
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polypeptide
hgdf
nucleic acid
isolated
present
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James Robert Miller
Ho Yeong Song
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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
    • C07K14/475—Growth factors; Growth regulators
    • 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)

Definitions

  • the present invention relates to compounds and compositions comprising novel human GDF-3 homolog (hGDF-3) polypeptides and nucleic acids. More specifically, recombinant or isolated nucleic acid molecules are provided encoding human hGDF-3 polypeptides as well as vectors, host cells, antibodies and methods for producing and using different aspects of the invention. Also contemplated are methods for identifying compounds that bind said proteins, and methods for treating or preventing disorders involving aberrant cellular proliferation resulting from abnormal levels of GDF-3 expression. RELATED ART
  • the transforming growth factor-beta (TGF- ⁇ ) super- family includes a large and ever-increasing number of secreted growth and differentiation factors that play important roles in embryonic development, cellular proliferation, and tissue homeostasis.
  • the individual members display varying degrees of homology with each other and several subgroups within the super- family have been defined.
  • the family includes, for instance, the TGF- ⁇ s, the activins, the bone morphogenetic proteins (BMPs) , and the growth/differentiation factors (GDFs) among others (for review see Kingsley, Genes and Development 8:133-146, 1994).
  • the GDFs are a class of biological mediators that have been shown to play critical roles in the control of growth and differentiation processes including tissue repair and regeneration (McPherron, A. and Lee, S.(1993) J. Biol. Chem. 268(5) :3444-3449; McPherron, A. and Lee, S. (1997) Nature 387:83-90) . Recently, a cDNA encoding a novel variant of the human homolog to mouse GDF-3 (Lee, McPherron, W094/15965) was isolated. Like other members of the TGF- ⁇ super-family, GDFs are responsible for inducing diverse, tissue dependent, biological effects which are often very dramatic. Accordingly, there is a need to provide hGDF-3 polypeptides and nucleic acids, as well as methods of making and using thereof .
  • the present invention provides isolated nucleic acids and encoded hGDF-3 polypeptides, including fragments and specified variants, as well as hGDF-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 hGDF-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 hGDF-3 polypeptides corresponding to at least five amino acid fragments of SEQ ID NOS : 3 or 4.
  • the present invention further provides recombinant vectors, comprising 1-40 of said isolated hGDF-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 hGDF-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 hGDF-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 SEQ ID NOS : 3 or 4.
  • the present invention also provides an isolated hGDF-3 polypeptide as described herein, comprising at least one amino acid sequence selected from SEQ ID NOS : 3 or 4.
  • the present invention also provides an isolated hGDF-3 polypeptide as described herein, wherein the polypeptide further comprises at least one specified mutation corresponding to portions or residues of SEQ ID NOS: 3 or 4.
  • the present invention also provides an isolated hGDF-3 polypeptide as described herein, wherein the polypeptide has at least one activity selected from the following: stimulation of bone and/or cartilage formation, stimulation of wound healing processes, including ulcers of various origins and/or burns, increasing cellular proliferation, and/or inhibiting tumorigenesis .
  • a hGDF-3 polypeptide can thus be screened for a corresponding activity according to known methods .
  • the present invention also provides a composition
  • a composition comprising an isolated hGDF-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 NOS : 1 or 2 , or a consensus sequence thereof .
  • the present invention also provides a vector comprising an isolated hGDF-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 hGDF-3 nucleic acid as described herein.
  • the present invention also provides a method for constructing a recombinant host cell that expresses a hGDF-3 polypeptide, comprising introducing into the host cell a hGDF-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 hGDF-3 polypeptide in a recombinant host cell, comprising culturing a recombinant host cell as described herein under conditions wherein the hGDF-3 polypeptide is expressed in detectable or recoverable amounts.
  • the present invention also provides an isolated hGDF-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 hGDF-3 antibody or fragment, comprising a polyclonal and/or monoclonal antibody or fragment that specifically binds at least one epitope specific to an isolated hGDF-3 polypeptide as described herein.
  • the present invention also provides a method for providing a hGDF-3 antibody or fragment, comprising generating the antibody or fragment that binds at least one epitope that is specific to an isolated hGDF-3 polypeptide as described herein, the generating done by recombinant, synthetic and/or hybridoma techniques.
  • the present invention also provides a hGDF-3 antibody or fragment produced by a method as described herein.
  • the present invention also provides a screening system for discovering compounds that bind the hGDF-3 protein, said screening system comprising the steps of:
  • c) quantifying the binding of said compound to hGDF-3 protein by any suitable means.
  • Utilization of the screening system described above provides a means to determine compounds that may alter the biological function of hGDF-3. This screening method may be adapted to large-scale, automated procedures such as a
  • hGDF-3 is prepared as described herein, preferably using recombinant DNA technology.
  • a test compound is introduced into a reaction vessel containing the hGDF-3 protein or fragment thereof. Binding of hGDF-3 by a test compound is determined by any suitable means. For example, in one method radioactively- labeled or chemically-labeled test compound may be used. Binding of the protein by the compound is assessed, for example, by quantifying bound label versus unbound label using any suitable method. Binding of a test compound may also be carried out by a method disclosed in U.S.
  • Patent 5,585,277 which hereby is incorporated by reference.
  • binding of a test compound to a protein is assessed by monitoring the ratio of folded protein to unfolded protein, for example by monitoring sensitivity of said protein to a protease, or amenability to binding of said protein by a specific antibody against the folded state of the protein.
  • the foregoing screening methods are useful for identifying an agonist and/or antagonist of a hGDF-3 protein or related fragment thereof, perhaps as a lead to a pharmaceutical compound for treatment of cancer, for inhibiting tumor growth, for inhibiting and/or stimulating cellular proliferation, inflammation, bone formation, or hematopoiesis .
  • a compound that binds hGDF-3 is identified, for example, by combining a test compound with hGDF-3 under conditions that cause the protein to exist in a ratio of folded to unfolded states. If the test compound binds the folded state of the protein, the relative amount of folded protein will be higher than in the case of a test compound that does not bind the protein.
  • the ratio of protein in the folded versus unfolded state is easily determinable by, for example, susceptibility to digestion by a protease, or binding to a specific antibody, or binding to chaperonin protein, or binding to any suitable surface.
  • the proteins and peptides described herein are also useful therapeutic agents, for inhibiting cell proliferation in a patient in need thereof, for example, in the treatment of obesity, cancer, or as an adjunct therapy intended to protect hematopoietic stem cells during chemotherapy.
  • the present invention provides isolated, recombinant and/or synthetic nucleic acid molecules comprising a polynucleotide encoding hGDF-3 polypeptides comprising specific fragments and specified variants, such polypeptides, and methods of making and using thereof.
  • a hGDF-3 polypeptide of the invention comprises at least one fragment domain, and/or specified variant as a portion or fragment of a hGDF-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 hGDF-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 hGDF-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 hGDF-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.
  • the terms "complementary” or “complementarity” as used herein refer 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.
  • 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. For example, 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 .
  • recombinant 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.
  • amplification reaction such as, but not limited to, a polymerase chain reaction (PCR), as known in the art.
  • 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.
  • the term "vector” as used herein 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.
  • 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 .
  • Nucleotide sequences identified by automation are typically at least about 95% to at least about 99.999% identical to the actual nucleotide sequence of the sequenced nucleic acid molecule .
  • the actual sequence can be more precisely identified by other approaches including manual nucleic acid sequencing methods well known in the art .
  • a single insertion or deletion in a identified nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the identified amino acid sequence encoded by a identified nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced nucleic acid molecule, beginning at the point of such an insertion or deletion.
  • nucleic acid molecule of the present invention encoding a hGDF-3 polypeptide can be obtained using well-known cloning and/or screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • the identified nucleotide sequence of a hGDF-3 nucleic acid of SEQ ID NOS : 1 and 2 contain open reading frames encoding a polypeptides of about 364 amino acid residues (SEQ ID NOS: 3 or 4), and a identified molecular weight of about 36-42 kDa.
  • nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof.
  • the DNA can be triple-, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated nucleic acid molecules of the present invention include nucleic acid molecules comprising an open reading frame (ORF) as shown in SEQ ID NOS: 1 or 2; nucleic acid molecules comprising the coding sequence for a hGDF-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 hGDF-3 polypeptide as described and enabled herein.
  • ORF open reading frame
  • the invention provides isolated nucleic acid molecules encoding a hGDF-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 hGDF-3 polypeptide or the full- length hGDF-3 polypeptide lacking the N-terminal methionine .
  • the invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown as in SEQ ID NOS : 1 or 2, or the nucleotide sequence of the hGDF-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.
  • Such isolated molecules are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting transcription, translation and/or expression of the hGDF-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 SEQ ID NOS : 1 or 2, 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 in SEQ ID NOS:l or 2.
  • 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 NOS : 1 or 2, or consensus sequences thereof, as determined by methods known in the art .
  • Such nucleotide fragments are useful according to the present invention for screening DNA sequences that code for one or more fragments of a hGDF-3 polypeptide as described herein.
  • nucleic acid molecules of the present invention which comprise a nucleic acid encoding a hGDF-3 polypeptide can include, but are not limited to, those encoding the amino acid sequence of the mature polypeptide; those encoding the mature polypeptide and the signal polypeptide; those encoding the aforementioned coding sequences along with additional, non-coding sequences.
  • additional, non-coding sequences may include, but are 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) ; and the aforementioned sequences with other coding sequences which provide additional functionalities.
  • sequence encoding a polypeptide can be fused to a marker sequence or a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the hGDF-3 polypeptides of the present invention.
  • 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 hGDF-3 polypeptide cDNA shown in SEQ ID NOS : 1 or 2) , or to 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 SEQ ID NOS : 1 or 2, 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.
  • the present invention provides hGDF-3 polynucleotides having the sequence of the hGDF-3 gene, nuclear transcript, cDNA, or complementary sequences and/or subsequences thereof .
  • 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.
  • the amplification primers may optionally be elongated in the 3' direction with additional contiguous nucleotides from the polynucleotide sequences, such as at least one of SEQ ID NOS: 1 or 2 , from which they are derived.
  • the number of nucleotides by which the primers can be elongated is selected from the group of integers consisting of from at least 1 to at least 25.
  • the primers can be elongated with an additional 1, 5, 10, or 15 nucleotides.
  • a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence.
  • the amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra.
  • the resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention.
  • the present invention provides isolated nucleic acids comprising hGDF-3 polynucleotides, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide as discussed, supra.
  • the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
  • polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
  • the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a dicot or monocot nucleic acid library.
  • the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full- length sequences, and more preferably at least 95% full- length sequences.
  • the cDNA libraries can be normalized to increase the representation of rare sequences.
  • Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences .
  • Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
  • Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences .
  • the polynucleotides of this embodiment will share an epitope with a polypeptide encoded by the polynucleotides described above.
  • these polynucleotides encode a first polypeptide which elicits production of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide described above.
  • the polynucleotides of this embodiment embrace nucleic acid sequences which can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.
  • 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 present invention provides isolated nucleic acids comprising hGDF-3 polynucleotides, wherein the polynucleotides are complementary to the polynucleotides described herein, above.
  • complementary sequences base-pair throughout the entirety of their length with such polynucleotides (i.e., have 100% sequence identity over their entire length) .
  • Complementary bases associate through hydrogen bonding in double stranded nucleic acids .
  • the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
  • 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.
  • 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.
  • 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, or 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.
  • nucleic acids encoding portions of FBPs according to the present invention allows for advantages over known amplification primers, due to the increase in sensitivity, selectivity and/or rate of amplification.
  • Known methods of DNA or RNA amplification include, 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. 4,683,202 (1987) ; and, PCR Protocols A Guide to Methods and
  • 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.
  • 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.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • 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 hGDF-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.
  • a polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable characteristics.
  • Another method of suppression is sense suppression.
  • Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes .
  • 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 hGDF-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.
  • 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. 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; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • 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
  • 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.
  • 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.
  • prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used.
  • Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al . , Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al . , Nucleic Acids Res.
  • DNA vectors transfected in E. coli are also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol .
  • the vector is selected to allow introduction into the appropriate host cell .
  • Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA.
  • 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)).
  • eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art .
  • polypeptides of the present invention can be expressed in these eukaryotic systems.
  • 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.
  • 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.
  • sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin.
  • Mammalian cells are illustrative of cell cultures useful for the production of these peptides.
  • Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter) , an enhancer (Queen et al . , Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • Other 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 Autographica californica nuclear polyhedrosis virus "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 hGDF-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.
  • hGDF-3 POLYPEPTIDE FRAGMENTS AND VARIANTS The invention further provides isolated hGDF-3 polypeptides having fragments or specified variants of the amino acid sequence encoded by the deposited cDNAs, or the amino acid sequence in SEQ ID NOS : 3 or 4.
  • the isolated proteins of the present invention comprise a polypeptide having at least 5 - 10 amino acids encoded by any one of the polynucleotides of the present invention as discussed more fully, supra, or polypeptides which are conservatively modified variants thereof.
  • Exemplary polypeptide sequences are provided in SEQ ID NOS : 3 and 4.
  • the proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in a full- length hGDF-3 polypeptide.
  • this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length.
  • the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5.
  • 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 ⁇ /K is optionally substantially similar to the native (non-synthetic) , endogenous polypeptide.
  • the K is optionally substantially similar to the native (non-synthetic) , endogenous polypeptide.
  • polypeptides of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention encoded by a polynucleotide of the present invention as described, supra.
  • Exemplary polypeptides include those which are full-length, such as those disclosed in SEQ ID NOS: 3 and 4.
  • the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide .
  • Immunoassays for determining binding are well known to those of skill in the art.
  • a preferred immunoassay is a competitive immunoassay as discussed, infra.
  • the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.
  • a hGDF-3 polypeptide of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions for any given hGDF-3 polypeptide will not be more than 20, 10, 5, or 3 , such as 1-20 or any range or value therein, as specified herein.
  • Amino acids in a hGDF-3 polypeptide of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity. 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 hGDF-3 polypeptide can further comprise a polypeptide encoded by 1-364 contiguous amino acids of SEQ ID NOS : 3 or 4.
  • a hGDF-3 polypeptide further includes an amino acid sequence selected from one or more of SEQ ID NOS : 3 or 4.
  • Non-limiting mutants that can enhance or maintain at least one of the listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation corresponding to at least one substitution selected from the group consisting of 53E, 55S, 129V, 157R, 182S, 209N, 213R, 213G, 221N, and 307R of SEQ ID NOS: 3 or 4.
  • Antigenic/Epitope comprising hGDF-3 Peptides or Polypeptides
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention according to methods well known in the art. See, e.g., Colligan, et al, . ed., Current Protocols in Immunology, Greene Publishing, N.Y. (1993- 1998) , Ausubel, supra , entirely incorporated herein by reference .
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein.
  • An "immunogenic epitope" can be defined as a part of a polypeptide that elicits an antibody response when the whole polypeptide is the immunogen.
  • an antigenic epitope a region of a polypeptide molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a polypeptide generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc . Natl . Acad . Sci . USA 81:3998-4002 (1983).
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a polypeptide, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact polypeptides (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson, et al., Cell 37:767-778 (1984) at 777.
  • 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.
  • the epitope-bearing peptides and polypeptides of the invention can be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid- phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl . Acad. Sci . USA 82:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS) " process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986) .
  • SMPS Simultaneous Multiple Peptide Synthesis
  • hGDF-3 polypeptides of the present invention and the epitope- bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG) , resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4- polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al . , Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric hGDF-3 polypeptide or polypeptide fragment alone (Fountoulakis et al . , J. Biochem 270:3958-3964 (1995)).
  • the polypeptides of this invention and fragments thereof may be used in the production of antibodies .
  • the term "antibody” as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab', Fab2 ' , and Fv fragments), and modified versions thereof, as well known in the art (e.g., chimeric, humanized, recombinant, veneered, resurfaced or CDR-grafted) such antibodies are capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived.
  • the instant invention also encompasses single chain polypeptide binding molecules.
  • Single chain antibodies and libraries thereof are yet another variety of genetically engineered antibody technology that is well known in the art. (See, e.g. R.E. Bird, et al . , Science 242:423-426 (1988); PCT Publication Nos. WO 88/01649, WO 90/14430, and WO 91/10737.
  • Single chain antibody technology involves covalently joining the binding regions of heavy and light chains to generate a single polypeptide chain. The binding specificity of the intact antibody molecule is thereby reproduced on a single polypeptide chain.
  • Antibodies included in this invention are useful in diagnostics, therapeutics or in diagnostic/therapeutic combinations .
  • polypeptides of this invention or suitable fragments thereof can be used to generate polyclonal or monoclonal antibodies, and various inter-species hybrids, or humanized antibodies, or antibody fragments, or single-chain antibodies.
  • the techniques for producing antibodies are well known to skilled artisans. (See, e.g., Colligan supra ; Monoclonal Antibodies: Principles & Applications Ed. J.R.Birch & E.S. Lennox, Wiley-Liss, 1995.
  • a polypeptide used as an immunogen may be modified or administered in an adjuvant, by subcutaneous or intraperitoneal injection into, for example, a mouse or a rabbit.
  • spleen cells from immunized animals are removed, fused with myeloma or other suitable known cells, and allowed to become monoclonal antibody producing hybridoma cells in the manner known to the skilled artisan.
  • Hybridomas that secrete a desired antibody molecule can be screened by a variety of well known methods, for example ELISA assay, Western blot analysis, or radioimmunoassay (Lutz et al . Exp. Cell Res. 175, 109-124 (1988); Monoclonal Antibodies: Principles & Applications Ed. J.R.Birch & E.S. Lennox, Wiley-Liss, 1995; Colligan, supra) .
  • labeled antibodies are desirable.
  • Procedures for labeling antibody molecules are widely known, including for example, the use of radioisotopes, affinity labels, such as biotin or avidin, enzymatic labels, for example horseradish peroxidase, and fluorescent labels, such as FITC or rhodamine (See, e.g., Colligan, supra).
  • Labeled antibodies are useful for a variety of diagnostic applications.
  • the present invention relates to the use of labeled antibodies to detect the presence of a hGDF-3 polypeptide.
  • the antibodies could be used in a screen to identify potential modulators of a hGDF-3 polypeptide.
  • 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.
  • the present invention is also directed to a transgenic non-human eukaryotic animal (preferably a rodent, such as a mouse) the germ cells and somatic cells of which contain genomic DNA according to the present invention which codes for a hGDF-3 polypeptide.
  • a transgenic non-human eukaryotic animal preferably a rodent, such as a mouse
  • the germ cells and somatic cells of which contain genomic DNA according to the present invention which codes for a hGDF-3 polypeptide At least one hGDF-3 nucleic acid can be introduced into the animal to be made transgenic, or an ancestor of the animal, at an embryonic stage, preferably the 1-1000 cell or oocyte, stage, and preferably not later than about the 64-cell stage.
  • transgene, " as used herein, means a gene which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of at least one hGDF-3 polypeptide in the transgenic animal .
  • hGDF-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 hGDF-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. These chimeras may be used for testing expression of at least one hGDF-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 hGDF-3 polypeptide of the present invention.
  • Methods for providing chimeric non-human mammals are provided, e.g, in U.S. serial Nos.
  • the animals carrying at least one hGDF-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 hGDF-3 polypeptide or hGDF-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 hGDF-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 hGDF-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 NOS : 1 or 2), which anneal to the amino terminal sequences of the desired portion of a hGDF-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 1 and 3' sequences, respectively.
  • the 5' and 3' primers have nucleotides corresponding or complementary to a portion of the coding sequence of a hGDF-3 e.g., as presented in SEQ ID NOS : 1 or 2 , according to known method steps .
  • SEQ ID NOS : 1 or 2 SEQ ID NOS : 1 or 2 .
  • 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 hGDF-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 hGDF-3 DNA into the restricted pQE60 vector places a hGDF-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.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing hGDF-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 ⁇ g/ml) and kanamycin (25 ⁇ g/ml) .
  • 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.
  • hGDF-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 can be purified by ion exchange, hydrophobic interaction, and size exclusion chromatography.
  • an affinity chromatography step such as an antibody column is used to obtain pure hGDF-3 polypeptide.
  • the purified polypeptide is stored at 4°C or frozen at -80°C.
  • Example 2 Cloning and expression of a hGDF-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 hGDF-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 Asp7l8.
  • 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 .
  • viral sequences for cell- mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide .
  • baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941 and pAcIMl, , 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 hGDF-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 hGDF-3, e.g., as presented in SEQ ID NOS : 1 or 2 , 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.
  • 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”.
  • Fragment FI and the dephosphorylated plasmid VI are ligated together with T4 DNA ligase.
  • 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 GDF-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 hGDF-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 hGDF-3 .
  • plasmid pBachGDF-3 Five ⁇ g of the plasmid pBachGDF-3 is co-transfected with 1.0 ⁇ g 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 ⁇ g of BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBachGDF-3 are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Rockville, MD) .
  • 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 ⁇ l 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-hGDF-3.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat - inactivated FBS .
  • the cells are infected with the recombinant baculovirus V-hGDF-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) . If radiolabeled polypeptides are desired, 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.
  • Example 3 Cloning and expression of hGDF-3 in mammalian cells
  • 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.
  • 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.
  • 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 QCl-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.
  • 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. Chinese hamster ovary (CHO) and 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 . , Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al . , Cell 41:521-530 (1985)).
  • LTR Rous Sarcoma Virus
  • CMV-enhancer Boshart, et al . , Cell 41:521-530 (1985)
  • Multiple cloning sites e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest .
  • the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • the expression plasmid, phGDF-3 HA is made by cloning a cDNA encoding hGDF-3 into the expression vector pcDNAl/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.) .
  • the expression vector pcDNAl/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.
  • an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells
  • an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells
  • 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 hGDF-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.
  • the hGDF-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 hGDF-3 in E. coli.
  • suitable primers include those based on the coding sequences presented in at least one of SEQ ID NOS : 1 or 2 , as they encode hGDF-3 polypeptides as described herein.
  • the PCR amplified DNA fragment and the vector, pcDNAl/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 Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the hGDF-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 hGDF- 3 by the vector.
  • hGDF-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 .
  • the vector pC4 is used for the expression of hGDF-
  • 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 .
  • the amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F. W. et al., J Biol.
  • 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
  • plasmid Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites 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 hGDF- 3 in a regulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl . Acad. Sci . USA 89: 5547-5551).
  • 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 hGDF-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 hGDF-3, e.g., as presented in at least one of SEQ ID NOS : 1 or 2 , 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 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g 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 ⁇ g/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 ⁇ g/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml 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 hGDF-3 mRNA expression
  • Northern blot analysis is carried out to examine hGDF-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 hGDF-3 polypeptide (SEQ ID NOS : 1 or 2) 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 hGDF-3 mRNA.
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues

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Abstract

L'invention concerne au moins un nouveau polypeptide de hGDF-3, comprenant des acides nucléiques isolés codant au moins un polypeptide de hGDF-3, des vecteurs, des cellules hôtes, des organismes transgéniques, des chimères, des polypeptides de hGDF-3, des méthodes de fabrication et d'utilisation de ceux-ci, ainsi que des anticorps spécifiques à hGDF-3 et des méthodes.
PCT/US1999/016474 1998-07-24 1999-07-21 Cellules hotes, vecteurs, polypeptides, acides nucleiques de gdf-3 humains et leur methodes d'utilisation Ceased WO2000005248A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097090A1 (fr) * 2001-05-31 2002-12-05 Sumitomo Pharmaceuticals Co., Ltd. Genes presentant une expression specifique aux cellules es
EP1432726A4 (fr) * 2001-09-07 2005-05-25 Curagen Corp Polypeptides therapeutiques, acides nucleiques codant ces derniers et methodes d'utilisation correspondantes
WO2022119418A1 (fr) * 2020-12-04 2022-06-09 주식회사 프리모리스 Procédé d'isolement et de culture de cellules souches de sang de cordon exprimant gdf-3 à un niveau élevé, et utilisation de gdf -3

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994015965A1 (fr) * 1993-01-12 1994-07-21 Johns Hopkins University School Of Medicine Facteur-3 de croissance et de differenciation

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WO1994015965A1 (fr) * 1993-01-12 1994-07-21 Johns Hopkins University School Of Medicine Facteur-3 de croissance et de differenciation

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Title
CARICASOLE A.A.D. ET AL: "Human growth-differentiation factor-3 (hGDF-3): developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours", ONCOGENE, vol. 16, January 1998 (1998-01-01), pages 95 - 103, XP002922407 *
MCPHERRON A.C. ET AL: "GDF-3 and GDF-9: Two New Members of the Transforming Growth Factor-beta Superfamily Containing a Novel Pattern of Cysteines", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 5, 15 February 1993 (1993-02-15), pages 3444 - 3449, XP002922402 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097090A1 (fr) * 2001-05-31 2002-12-05 Sumitomo Pharmaceuticals Co., Ltd. Genes presentant une expression specifique aux cellules es
US7250255B2 (en) 2001-05-31 2007-07-31 Shinya Yamanaka Genes with ES cell-specific expression
US8158766B2 (en) 2001-05-31 2012-04-17 Shinya Yamanaka Genes with ES cell-specific expression
US8597895B2 (en) 2001-05-31 2013-12-03 Shinya Yamanaka Genes with ES cell-specific expression
EP1432726A4 (fr) * 2001-09-07 2005-05-25 Curagen Corp Polypeptides therapeutiques, acides nucleiques codant ces derniers et methodes d'utilisation correspondantes
WO2022119418A1 (fr) * 2020-12-04 2022-06-09 주식회사 프리모리스 Procédé d'isolement et de culture de cellules souches de sang de cordon exprimant gdf-3 à un niveau élevé, et utilisation de gdf -3

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