WO2001004307A1 - Proteine alpha-27 - Google Patents

Proteine alpha-27 Download PDF

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WO2001004307A1
WO2001004307A1 PCT/US2000/018849 US0018849W WO0104307A1 WO 2001004307 A1 WO2001004307 A1 WO 2001004307A1 US 0018849 W US0018849 W US 0018849W WO 0104307 A1 WO0104307 A1 WO 0104307A1
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leu
arg
gly
ala
val
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PCT/US2000/018849
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Darrell C. Conklin
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Zymogenetics, Inc.
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Priority to AU59279/00A priority Critical patent/AU5927900A/en
Publication of WO2001004307A1 publication Critical patent/WO2001004307A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • RNA Ribonucleic acid
  • Ribonucleic acid is often isolated in the process of discovering new genes and proteins, and making cDNA libraries.
  • the isolated RNA can be destroyed if it is contaminated with ribonuclease, an enzyme that destroys RNA.
  • proteins that can be used to inhibit ribonucleases.
  • the present invention addresses this need by providing for novel polypeptides and related compositions and methods that can be used to inhibit ribonucleases.
  • the present invention provides an isolated polynucleotide encoding a mammalian cytokine termed 'alpha protein-27', hereinafter referred to as "Zalpha27".
  • Zalpha27 is defined by SEQ ID NOs 1 and 2 and has a molecular weight of about 17,054 Daltons (D).
  • an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding Zalpha27 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.
  • a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein polypeptide encoded by the DNA segment.
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion of the chimeric polypeptide consists essentially of (a) a Zalpha27 polypeptide as shown in SEQ ID NO: 2 (b) allelic variants of SEQ ID NO:2; and (c) protein polypeptides that are at least 80% identical to (a) or (b).
  • the second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag.
  • the affinity tag is an immunoglobulin F c polypeptide.
  • the invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
  • an antibody that specifically binds to a Zalpha27 polypeptide as disclosed above, and also an anti-idiotypic antibody that neutralizes the antibody to a Zalpha27 polypeptide.
  • An additional embodiment of the present invention relates to a peptide or polypeptide that has the amino acid sequence of an epitope-bearing portion of a Zalpha27 polypeptide having an amino acid sequence described above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalpha27 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention.
  • Examples of epitope-bearing polypeptides of the present invention are SEQ ID NOs: 4 - 9. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A, Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991), glutathione S transferase, Smith and Johnson, Gene 67:31 (1988), Glu-Glu affinity tag, Grussenmeyer et al, Proc. Natl.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • Angiogenic denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of ⁇ l ⁇ " M " l .
  • polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGCTTAGCTT-3' are 5'- TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5'.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:71 '4-78 (1985).
  • an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a- globin, b-globin, and myoglobin are paralogs of each other.
  • polynucleotide is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucieotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucieotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb”). Where the context allows, the latter two terms may describe polynucieotides that are single-stranded or double-stranded.
  • double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • a bioactive molecule i.e., a ligand
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention provides novel cytokine polypep tides/proteins.
  • the novel cytokine termed “alpha helical protein-32" hereinafter referred to as "Zalpha27” was discovered and identified to be a cytokine by the presence of polypeptide and polynucleotide features characteristic of four-helix -bundle cytokines (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth hormone).
  • Analysis of the amino acid sequence shown in SEQ ID NO:2 indicates a signal sequence which extends from the methionine at position 1 to and including amino acid residue 25.
  • the mature sequence extends from amino acid residue 26, a glutamine, to an including amino acid residue 170, a phenylalanine.
  • the mature Zalpha27 polypeptide is also represented by the amino acid sequence of SEQ ID NO:3 which has an unglycosylated molecular weight of approximately 16,578 Daltons (D).
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the Zalpha27 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.
  • Polynucieotides generally a cDNA sequence, of the present invention encode the described polypeptides herein.
  • a cDNA sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows.
  • Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;
  • Cysteine (Cys) is encoded by TGC or TGT;
  • Aspartic acid is encoded by GAC or GAT;
  • Glutamic acid is encoded by GAA or GAG
  • Phenylalanine is encoded by TTC or TTT;
  • Glycine is encoded by GGA, GGC, GGG or GGT;
  • Histidine is encoded by CAC or CAT;
  • Isoleucine (lie) is encoded by ATA, ATC or ATT;
  • Lysine is encoded by AAA, or AAG;
  • Leucine is encoded by TTA, TTG, CTA, CTC, CTG or CTT;
  • Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA, CCC, CCG or CCT; Glutamine (Gin) is encoded by CAA or CAG; Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;
  • Serine is encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
  • Valine (Val) is encoded by GTA, GTC, GTG or GTT;
  • Trp Tryptophan
  • Tyrosine (Tyr) is encoded by TAC or TAT.
  • mRNA messenger RNA
  • T thymine nucleotide
  • U uracil nucleotide
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucieotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the isolated polynucieotides will hybridize to similar sized regions of SEQ ID NO: 1, or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucieotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of Zalpha27 RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc. Natl. Acad. Sci. USA 77:5201 (1980) and are discussed below.
  • Total RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al., Biochemistry 78:52-94 (1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl.
  • cDNA Complementary DNA
  • genomic DNA can be isolated.
  • Polynucieotides encoding Zalpha27 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding Zalpha27 can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to Zalpha27, receptor fragments, or other specific binding partners.
  • the polynucieotides of the present invention can also be synthesized using DNA synthesizers.
  • DNA synthesizers Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • the present invention further provides counterpart polypeptides and polynucieotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zalpha27 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zalpha27 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zalpha27 as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A Zalpha27 -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to Zalpha27 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ ID NO: 1 represents a single allele of human Zalpha27 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the Zalpha27 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated Zalpha27 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO:2 and their orthologs.
  • substantially homologous is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence and the amino acid sequence of a putative variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Natl Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:AAA (1970); Sellers, SIAM J. Appl Math. 26:787 (1974), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from four to six.
  • the present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:2.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins [Henikoff and Henikoff, Proc. Nat 'I Acad. Sci. USA 89:10915 (1992)]. Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
  • the language "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0,1,2, or 3.
  • Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1,2 or 3), while more preferred conservative substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • the present invention claims those polypeptides which are at least 90%, preferably 95% and most preferably 99% identical to SEQ ID NO: 3 and which are able to stimulate antibody production in a mammal, and said antibodies are able to bind the native sequence of SEQ ID NO:2.
  • Variant Zalpha27 polypeptides or substantially homologous Zalpha27 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from 20 to 30 amino acid residues that comprise a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ ID NO:4.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the Zalpha27 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites. Table 2
  • Aromatic phenylalanine tryptophan tyrosine
  • the present invention further provides a variety of other polypeptide fusions [and related multimeric proteins comprising one or more polypeptide fusions].
  • a Zalpha27 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin-Zalpha27 polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric Zalpha27 analogs].
  • Auxiliary domains can be fused to Zalpha27 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a Zalpha27 polypeptide or protein could be targeted to a predetermined cell type by fusing a Zalpha27 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a Zalpha27 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34 :l-9 (1996).
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, rrans-3-methylproline, 2,4-methanoproline, c 5-4-hydroxyproline, trans-A- hydroxyproline, N-methylglycine, ⁇ //o-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, terf-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • desired non-naturally occurring amino acid(s) e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476 (1994).
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site- directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zalpha27 amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al, Proc. Natl. Acad. Sci. USA 88:4498-502 (1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al, Proc. Natl. Acad. Sci. USA 88:4498-502 (1991).
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 277:4699-708, 1996.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312 (1992); Smith et al, J. Mol. Biol. 224:899-904 (1992); Wlodaver et al, FEBS Lett. 309:59-64 (1992).
  • variants of the disclosed Zalpha27 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc. Natl. Acad. Sci. USA 97:10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the Zalpha27 polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a Zalpha27 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of Zalpha27, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the Zalpha27 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5,143,830).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al, Cell 14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al, EMBO J.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al, J. Gen. Virol. 36:59 (1977) and Chinese hamster ovary (e.g. CHO- Kl; ATCC No. CCL 61) cell lines.
  • COS-1 ATCC No. CRL 1650
  • COS-7 ATCC No. CRL 1651
  • BHK ATCC No. CRL 1632
  • BHK 570 ATCC No. CRL 10314
  • 293 ATCC No. CRL 1573
  • Chinese hamster ovary e.g. CHO- Kl; ATCC No. CCL 61
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • hygromycin resistance multi-drug resistance
  • puromycin acetyltransferase can also be used.
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 77:47 (1987). Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
  • DNA encoding the Zalpha27 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods.
  • the first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the Zalpha27 flanked by AcNPV sequences.
  • Suitable insect cells e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a Zalpha27 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences. See, King, L.A.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow, V.A, et al., J Virol 67:4566 (1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zalpha27 polypeptide into a baculovirus genome maintained in E.
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case Zalpha27.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Peer, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen Virol 77:971 (1990); Bonning, B.C.
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed that replace the native Zalpha27 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native Zalpha27 secretory signal sequence.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zalpha27 polypeptide, for example, a Glu- Glu epitope tag, Grussenmeyer, T. et al, Proc Natl Acad Sci. 82:7952 (1985).
  • a transfer vector containing Zalpha27 is transformed into E. coli, and screened for bacmids that contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
  • Recombinant virus that expresses Zalpha27 is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall army worm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994).
  • host cells typically a cell line derived from the fall army worm, Spodoptera frugiperda.
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells.
  • Suitable media are Sf900 UTM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • the recombinant virus-infected cells typically produce the recombinant Zalpha27 polypeptide at 12-72 hours post-infection and secrete it with varying efficiency into the medium.
  • the culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant).
  • the supernatant containing the Zalpha27 polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the Zalpha27 polypeptide from the supernatant can be achieved using methods described herein.
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311;
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al, U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
  • Transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 o ⁇ AUG2).
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P.
  • methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli,
  • Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art; see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a Zalpha27 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient, which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25 °C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
  • Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a Zalpha27 polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • a region of a protein to which an antibody can bind is defined as an "antigenic epitope”.
  • an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J.G. et al. Science 279:660-666 (1983).
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • epitope-bearing polypeptides of the present invention are the following polypeptides: a polypeptide containing the sequence extending from amino acid residue 8, a proline, through amino acid residue 68, and arginine of SEQ ID NO: 2, also represented by SEQ ID NO: 2, also represented by SEQ ID NO: 2, also represented by SEQ ID NO: 2, also represented by SEQ ID NO: 2, also represented by SEQ ID NO: 2, also represented by SEQ ID NO:
  • 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.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein.
  • the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred.
  • All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides defined by SEQ ID NOs: 4-9.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zalphal polypeptide described herein.
  • Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
  • Immunogenic epitope-bearing peptides can be identified using standard methods [see, for example, Geysen et al, supra. See also U.S. Patent No. 4,708,781 (1987) further describes how to identify a peptide bearing an immunogenic epitope of a desired protein.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant Zalpha27 polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, MontgomeryviUe, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl-Sepharose FF Pharmacia
  • Toyopearl butyl 650 Toso Haas, MontgomeryviUe, PA
  • Octyl-Sepharose Pharmacia
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3: 1 (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. Methods in Enzymol, Vol. 782, "Guide to Protein Purification", M. Lieber, (ed.),page 529-539 (Acad. Press, San Diego, 1990).
  • an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
  • polypeptide fusions, or hybrid Zalpha27 proteins are constructed using regions or domains of the inventive Zalpha27, Sambrook et al, ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511 (1994). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest.
  • Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between Zalpha27 of the present invention with the functionally equivalent domain(s) from another family member. Such domains include, but are not limited to, the secretory signal sequence, conserved, and significant domains or regions in this family. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Antagonists are useful as research reagents for characterizing sites of ligand-receptor interaction.
  • Inhibitors of Zalpha27 activity include anti-Zalpha27 antibodies and soluble Zalpha27 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
  • Zalpha27 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of Zalpha27. In addition to those assays disclosed herein, samples can be tested for inhibition of Zalpha27 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of Zalpha27- dependent cellular responses. For example, Zalpha27 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a Zalpha27-stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a Zalpha27-DNA response element operably linked to a gene encoding a protein that can be assayed, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE), Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273 (1990) and serum response elements (SRE) (Shaw et al. Cell
  • Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063 (1988) and Habener, Molec. Endocrinol 4 (8): 1087 (1990). Hormone response elements are reviewed in Beato, Cell 56:335 (1989).
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of Zalpha27 on the target cells as evidenced by a decrease in Zalpha27 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block Zalpha27 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding.
  • compounds or other samples can be tested for direct blocking of Zalpha27 binding to receptor using Zalpha27 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like).
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • a Zalpha27 polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two constant region domains and lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify the ligand.
  • the chimeras are bound to a support via the F c region and used in an ELISA format.
  • a Zalpha27 ligand-binding polypeptide can also be used for purification of ligand.
  • the polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross- linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross- linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to disrupt ligand-receptor binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229 (1991) and Cunningham and Wells, J. Mol. Biol. 234:554 (1993).
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell.
  • a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity, Scatchard, Ann. NY Acad. Sci. 51: 660 (1949) and calorimetric assays, Cunningham et al., Science 253:545 (1991); Cunningham et al., Science 245:821 (1991).
  • Zalpha27 polypeptides can also be used to prepare antibodies that specifically bind to Zalpha27 epitopes, peptides or polypeptides.
  • the Zalpha27 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • Suitable antigens would be the Zalpha27 polypeptides encoded by SEQ ID NOs: 4 - 9.
  • Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a Zalpha27 polypeptide or a fragment thereof.
  • the immunogenicity of a Zalpha27 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zalpha27 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to Zalpha27 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zalpha27 protein or peptide).
  • Genes encoding polypeptides having potential Zalpha27 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • These random peptide display libraries can be used to screen for peptides that interact with a known target, which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al, US Patent NO. 4,946,778; Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the Zalpha27 sequences disclosed herein to identify proteins which bind to Zalpha27.
  • binding proteins which interact with Zalpha27 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as Zalpha27 "antagonists" to block Zalpha27 binding and signal transduction in vitro and in vivo. Antibodies are determined to be specifically binding if: 1.
  • antibodies herein specifically bind if they bind to a Zalpha27 polypeptide, peptide or epitope with a binding affinity (K a ) of 10 M " or
  • binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis.
  • antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides.
  • Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zalpha27 but not known related polypeptides using a standard Western blot analysis (Ausubel et al, ibid.).
  • Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family (e.g. IL-16), Zalpha27 polypeptides, and non-human Zalpha27.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides.
  • antibodies raised to Zalpha27 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zalpha27 will flow through the matrix under the proper buffer conditions.
  • Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.) (Raven Press, 1993); Getzoff et al., Adv. in Immunol.
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Zalpha27 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant Zalpha27 protein or polypeptide.
  • Antibodies to Zalpha27 may be used for tagging cells that express Zalpha27 and for isolating Zalpha27 by affinity purification.
  • Zalpha27 can inhibit the digestion of RNA by ribonucleases.
  • concentration of Zalpha27 should be about 100 ⁇ M in solution to inhibit the action of ribonucleases.
  • Polynucieotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of Zalpha27 can be used as standards or as "unknowns" for testing purposes.
  • Zalpha27 polynucieotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein Zalpha27 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucieotides; determining mRNA and DNA localization of Zalpha27 polynucieotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucieotides and polypeptides by nucleic acid hybridization.
  • Zalpha27 polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed Zalpha27 polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo.
  • native and tagged protein i.e., receptor binding, signal transduction, proliferation, and differentiation
  • Zalpha27 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the locations of the disulfide bonds, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the locations of the disulfide bonds, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • a kit containing the Zalpha27 can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of Zalpha27 would be unique unto itself.
  • the antibodies which bind specifically to Zalpha27 can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify Zalpha27, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies.
  • the Zalpha27 gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum.
  • Such educational kits containing the Zalpha27 gene, polypeptide, or antibody are considered within the scope of the present invention. The invention is further illustrated by the following non-limiting examples.
  • RNA extracted from each one of the cell lines was reversed transcribed in the following manner.
  • the first strand cDNA reaction contained 10 ⁇ l of twice poly d(T)-selected poly (A)+ mRNA from 562, Daudi, HL-60, MOLT-4 or Raji Cells (Clontech, Palo Alto, CA) at a concentration of 1.0 mg/ml, and 2 ⁇ l of 20 pmole/ ⁇ l first strand primer SEQ ID NO: 10 (GTC TGG GTT CGC TAC TCG AGG CGG CCG CTA TTT TTT TTT TTT TTT TTT TTT TTT TTT) containing an Xho I restriction site. The mixture was heated at 70°C for 3.0 minutes and cooled by chilling on ice.
  • First strand cDNA synthesis was initiated by the addition of 8 ⁇ l of first strand buffer (5x SUPERSCRIPTTM buffer; Life Technologies, Gaithersburg, MD), 4.0 ⁇ l of 100 mM dithiothreitol, and 3.0 ⁇ l of a deoxynucleotide triphosphate (dNTP) solution containing 10 mM each of dTTP, dATP, dGTP and 5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, NJ) to the RNA-primer mixture.
  • dNTP deoxynucleotide triphosphate
  • 32p- ⁇ dCTP to a 5 ⁇ l aliquot from one of the reaction mixtures to label the reaction for analysis.
  • the reactions were incubated at 37°C for 10 minutes, 45°C for 50 minutes, then incubated at 50°C for 10 minutes.
  • Unincorporated 32p . r ⁇ dCTP in the labeled reaction was removed by chromatography on a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA).
  • the unincorporated nucleotides and primers in the unlabeled first strand reactions were removed by chromatography on 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA).
  • the length of labeled first strand cDNA was determined by agarose gel electrophoresis.
  • the second strand reaction contained 135 ⁇ l of the unlabeled first strand cDNA, 40 ⁇ l of 5x polymerase I buffer (125 mM Tris: HC1, pH 7.5, 500 mM KC1, 25 mM MgCl 2 , 50mM (NH 4 ) 2SO4)), 2.5 ⁇ l of 100 mM dithiothreitol, 5.0 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ⁇ l of 5 mM ⁇ -NAD, 2.5 ⁇ l of 10 U/ ⁇ l E. coli DNA ligase (New England Biolabs; Beverly, MA), 7 ⁇ l of 10 U/ ⁇ l E.
  • 5x polymerase I buffer 125 mM Tris: HC1, pH 7.5, 500 mM KC1, 25 mM MgCl 2 , 50mM (NH 4 ) 2SO4
  • Eco RI adapters were ligated onto the 5' ends of the cDNA described above to enable cloning into an expression vector.
  • a 12.0 ⁇ l aliquot of cDNA (-2.0 ⁇ g) and 4 ⁇ l of 69 pmole/ ⁇ l of Eco RI adapter (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) were mixed with 2.5 ⁇ l lOx ligase buffer (660 mM Tris-HCl pH 7.5, 100 mM MgCl 2 ), 2.5 ⁇ l of 10 mM ATP, 3.0 ⁇ l 0.1 M DTT and 1 ⁇ l of 15 U/ ⁇ l T4 DNA ligase (Promega Corp., Madison, WI).
  • the reaction was incubated in a 0° to 22° C temperature gradient for 48 hours.
  • the reaction was terminated by the adding 65 ⁇ l H 2 O and 10 ⁇ l 10X H buffer (Boehringer Mannheim, Indianapolis, IN) and incubating the mixture at 70° C for 20 minutes.
  • the cDNA was digested with Xho I, resulting in a cDNA having a 5' Eco RI cohesive end and a 3' Xho I cohesive end.
  • the Xho I restriction site at the 3' end of the cDNA had been previously introduced.
  • Restriction enzyme digestion was carried out in a reaction mixture by the addition of 1.0 ⁇ l of 40 U/ ⁇ l Xho I (Boehringer Mannheim, Indianapolis, IN). Digestion was carried out at 37°C for one hour. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400 pore size gel filtration column (Clontech Laboratories, Palo Alto, CA).
  • the cDNA was ethanol precipitated, washed with 70% ethanol, air dried and resuspended in 13.5 ⁇ l water, 2 ⁇ l of 10X kinase buffer (660 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 ), 0.5 ⁇ l 0.1 M DTT, 3 ⁇ l 10 mM ATP, 1.0 ⁇ l T4 polynucleotide kinase (10 U/ ⁇ l, Life Technologies, Gaithersburg, MD). Following incubation at 37° C for 30 minutes, the cDNA was ethanol precipitated in the presence of 2.5 M Ammonium Acetate, and electrophoresed on a 0.8% low melt agarose gel.
  • 10X kinase buffer 660 mM Tris-HCl, pH 7.5, 100 mM MgCl 2
  • 0.5 ⁇ l 0.1 M DTT 0.5 ⁇ l 0.1 M DTT
  • 3 ⁇ l 10 mM ATP 3 ⁇ l 10
  • the contaminating adapters and cDNA 2 kb or less in length were excised and discarded.
  • the electrodes were reversed, and the cDNA was electrophoresed until the greater than 2 kb length cDNAs were concentrated near the lane origin.
  • the areas of the gel containing the concentrated cDNAs were excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined.
  • An aliquot of water approximately three times the volume of the gel slice (300 ⁇ l) and 35 ⁇ l lOx ⁇ -agarose I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65°C for 15 minutes.
  • the K562G library was created to enrich for receptor-encoding cDNAs, which are usually encoded by long cDNAs. Since long cDNAs normally constitute only a small fraction of the total cDNA species of the cell, the K562G library would also be enriched for cDNAs of low abundant mRNAs.
  • the resulting cDNA library were subjected to large scale sequencing to identify novel express sequence tags (ESTs). Sequencing was done using an ABI 377 sequencer using either the T3 or the reverse primer.
  • Zalpha27 was discovered by the cDNA library of Example 1 with Expressed Sequence Tag of SEQ ID NO:3 the a clone was discovered and sequenced resulting in SEQ ID NOs: 1 and 2.

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Abstract

La présente invention concerne des molécules polynucléotidiques et polypeptidiques d'une protéine alpha-27 (Zalpha27) mammalienne. Les polypeptides et les polynucléotides codant pour ces polypeptides sont hormonaux et peuvent s'utiliser pour réguler le fonctionnement du système immunitaire. La présente invention concerne également des anticorps contre les polypeptides Zalpha27.
PCT/US2000/018849 1999-07-13 2000-07-11 Proteine alpha-27 WO2001004307A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7569681B2 (en) * 2002-04-30 2009-08-04 The University Of North Carolina At Chapel Hill Caterpiller gene family

Citations (2)

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WO1990012881A1 (fr) * 1989-04-24 1990-11-01 Promega Corporation Inhibiteur de ribonuclease placentaire humain de recombinaison et son procede de production
WO1998006845A1 (fr) * 1996-08-16 1998-02-19 Life Technologies, Inc. Inhibiteurs de ribonuclease mammaliens et utilisation desdits inhibiteurs

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1990012881A1 (fr) * 1989-04-24 1990-11-01 Promega Corporation Inhibiteur de ribonuclease placentaire humain de recombinaison et son procede de production
WO1998006845A1 (fr) * 1996-08-16 1998-02-19 Life Technologies, Inc. Inhibiteurs de ribonuclease mammaliens et utilisation desdits inhibiteurs

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DATABASE EMBL EMBL; 18 April 1997 (1997-04-18), EVANS G A ET AL.: "Homo sapiens chromosome 11 clone pDJ393o15, *** sequencing in progress ***, 8 unordered pieces", XP002152367 *
DATABASE EMBL EMBL; 9 May 1996 (1996-05-09), MACKE J ET AL.: "59c7 Human retina cDNA Tsp509I-cleaved sublibrary Homo sapiens cDNA not directional, mRNA sequence", XP002152368 *
DATABASE EMBLNEW EMBL; 29 September 2000 (2000-09-29), SUGANO S ET AL.: "Homo sapiens cDNA: FLJ21478, clone COL05012", XP002152369 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7569681B2 (en) * 2002-04-30 2009-08-04 The University Of North Carolina At Chapel Hill Caterpiller gene family
US7923545B2 (en) 2002-04-30 2011-04-12 The University Of North Carolina At Chapel Hill Caterpiller gene family

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