WO2001018205A1 - Polypeptide secrete zalpha30 - Google Patents

Polypeptide secrete zalpha30 Download PDF

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Publication number
WO2001018205A1
WO2001018205A1 PCT/US2000/024326 US0024326W WO0118205A1 WO 2001018205 A1 WO2001018205 A1 WO 2001018205A1 US 0024326 W US0024326 W US 0024326W WO 0118205 A1 WO0118205 A1 WO 0118205A1
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seq
residues
zalpha30
polypeptide
cells
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PCT/US2000/024326
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English (en)
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Darrell C. Conklin
Zeren Gao
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Zymogenetics, Inc.
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Priority to AU71134/00A priority Critical patent/AU7113400A/en
Publication of WO2001018205A1 publication Critical patent/WO2001018205A1/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

Definitions

  • Multicellular organisms contain a vast number and variety of proteins that are produced in one cell or organ and act on another cell or organ. These proteins are secreted by the cells in which they are synthesized. Secreted proteins may be embedded in the membrane of the synthesizing cell, or may be exported into the surrounding milieu. Such exported proteins may be transported, for example by the bloodstream, to act at on a distant target cell.
  • proteins that are produced by one organ and act on a different site are medically important. For example, insulin is administered for the treatment of diabetes, and coagulation factors are used in the treatment of hemophilia.
  • Other secreted proteins that have been developed for therapeutic use in humans include tissue- type plasminogen activator, platelet derived growth factor, erythropoietin, and human growth hormone.
  • the pituitary gland is an endocrine gland that secretes a number of different hormones which are carried by the bloodstream to act on distant target cells in the body.
  • the pituitary gland is intimately connected with the hypothalamus, and together these organs form the most complex and dominant portion of the endocrine system.
  • the known hormones of the hypothalamus-pituitary complex regulate the function of the thyroid, adrenal, and reproductive glands, and also control somatic growth, lactation, milk secretion, and water metabolism.
  • the present invention provides novel polypeptides, polynucleotides encoding them, methods of making them, and methods of using them.
  • an isolated polypeptide comprising at least fifteen contiguous amino acid residues of SEQ ID NO:2, SEQ ID NO:10, or SEQ ID NO:12.
  • the isolated polypeptide is from 15 to 1500 amino acid residues in length.
  • the at least fifteen contiguous amino acid residues of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, a polyhistidine tag, and a peptide as shown in SEQ ID NO:5.
  • the isolated polypeptide comprises residues 1-25, 42-56, or 171-202 of SEQ ID NO:2 or SEQ ID NO: 12.
  • the isolated polypeptide comprises residues 75-80, 106-111, 107-112, 187-192, or 290-295 of SEQ ID NO:2; residues 268-273, 427-432, 75-80, 641-646, or 639-644 of SEQ ID NO:10; or residues 601-606, 691-696, 517-522, 425-430, or 72-77 of SEQ ID NO: 12.
  • the isolated polypeptide comprises 71-263, 42-263, 71-236, 42-236, or 26-295 of SEQ ID NO:2; residues 26-697 or 304-693 of SEQ ID NO:10; or residues 71-235, 42-235, 26-700, or 302-690 of SEQ ID NO: 12.
  • the isolated polypeptide comprises at least 30 contiguous residues of SEQ ID NO:2, SEQ ID NO:10, or SEQ ID NO: 12.
  • the isolated polypeptide comprises an amino acid sequence motif as shown in SEQ ID NO:3.
  • an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising at least 20 contiguous residues of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12; and a transcription terminator.
  • the polypeptide comprises residues 71- 263, 42-263, 71-236, 42-236, or 26-295 of SEQ ID NO:2; residues 304-693 or 26-697 of SEQ ID NO: 10; or residues 71-236, 42-236, 302-690, or 26-700 of SEQ ID NO: 12.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment.
  • the secretory signal sequence encodes residues 1-25 of SEQ ID NO:2 or SEQ ID NO: 12.
  • the DNA segment encodes residues 1-25 of SEQ ID NO:2 or SEQ ID NO: 12 and the DNA segment is operably linked to a second DNA segment encoding an additional polypeptide.
  • a method of making a polypeptide comprising the steps of culruring a cell into which has been introduced an expression vector as disclosed above under conditions whereby the DNA segment is expressed and the polypeptide is produced, and recovering the produced polypeptide.
  • the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, and the polypeptide is secreted by the cell and recovered from a medium in which the cell is cultured.
  • an antibody that specifically binds to a polypeptide comprising residues 26-295 of SEQ ID NO:2, residues 26-697 of SEQ ID NO: 10, or residues 26-700 of SEQ ID NO: 12.
  • a method of detecting, in a test sample, the presence of an antagonist of zalpha30 activity comprising the steps of (a) culruring a cell that is responsive to zalpha30; (b) exposing the cell to a zalpha30 polypeptide in the presence and absence of a test sample; (c) comparing levels of response to the zalpha30 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and (d) determining from the comparison the presence of an antagonist of zalpha30 activity in the test sample.
  • Fig. 1 is a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
  • Fig. 2 is an alignment of human (SEQ ID NO: 10) and mouse (SEQ ID NO: 12) zalpha30 proteins.
  • Fig. 3 is a partial restriction map of a portion of the polynucleotide of SEQ ID NO: 1.
  • Fig. 4 is a partial restriction map of a portion of the polynucleotide of
  • Fig. 5 is a partial restriction map of a portion of the polynucleotide of SEQ ID NO:l l.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification 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.
  • DNAs encoding affinity tags and other reagents are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, MA; Eastman Kodak, New Haven, CT).
  • 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.
  • a "complement" of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • corresponding to when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.
  • 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.
  • 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 316:774-78, 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 or protein can be prepared substantially free of other polypeptides or proteins, particularly those of animal origin.
  • the polypeptides and proteins can be prepared in a highly purified form, i.e. greater than 95% pure or greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide or protein in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • “Operably linked” means that two or more entities are joined together such that they function in concert for their intended purposes.
  • DNA segments the phrase indicates, for example, that coding sequences are joined in the correct reading frame, and transcription initiates in the promoter and proceeds through the coding segment(s) to the terminator.
  • "operably linked” includes both covalently (e.g., by disulfide bonding) and non-covalently (e.g., by hydrogen bonding, hydrophobic interactions, or salt-bridge interactions) linked sequences, wherein the desired function(s) ofthe sequences are retained.
  • 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.
  • a "polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • bp base pairs
  • nt nucleotides
  • kb kilobases
  • 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.
  • a “secretory signal sequence” is 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.
  • a “segment” is a portion of a larger molecule (e.g., polynucleotide or polypeptide) having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5' to the 3' direction, encodes the sequence of amino acids ofthe specified polypeptide.
  • the present invention provides novel polypeptides and proteins.
  • This novel protein termed "zalpha30" was initially identified by querying a database of human pituitary cDNA sequences for a secretory signal sequence encoding a signal peptide characterized by a hydrophobic core, a stop codon upstream of the predicted initiation Met codon, and an alpha helical region.
  • a full-length human zalpha30 cDNA is shown in SEQ ID NO: 1.
  • Northern blot analysis indicated a corresponding message of approximately 3.0 kb.
  • the sequence of SEQ ID NO:l consists of 2305 bp, and does not including a poly(A) tail that was present at the 3' end of the original cDNA.
  • the sequence includes an open reading frame of 888 base pairs (including the TAA stop codon).
  • SEQ ID NO:9 and SEQ ID NO: 10 This longer protein comprises 697 amino acid residues, and diverges from SEQ ID NO:2 following residue 255.
  • a mouse sequence corresponding to the longer human sequence is shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • An alignment of SEQ ID NO: 10 and SEQ ID NO: 12 is shown in Fig. 2.
  • structural features of zalpha30 include a predicted secretory signal peptide comprising residues 1 through 25 and an alpha helical region comprising residues 42 through 56.
  • the zalpha30 polypeptide also contains the cysteine-rich motif cxcx ⁇ 6 ⁇ cx ⁇ 3 ⁇ cccx ⁇ 3 ⁇ cx ⁇ l l ⁇ c (SEQ ID NO:3), wherein c is cysteine, x is any residue except cysteine, and ⁇ y ⁇ indicates the number of x residues, at residues 171 - 202.
  • SEQ ID NO:2 includes eight additional cysteine residues at positions 71, 105, 118, 133, 219, 236, 256, and 263.
  • the calculated molecular weight of the peptide backbone ofthe 295-residue precursor is approximately 30 kD.
  • the longer form of human zalpha30 (SEQ ID NO: 10) and the mouse zalpha30 shown in SEQ ID NO: 12 each include a predicted secretory signal peptide, alpha helical region, and cysteine-rich domain as disclosed above. While not wishing to be bound by theory, residues 304-693 of SEQ ID NO: 10 and residues 302-690 of SEQ ID NO: 12 may form an additional domain comprising 12 cysteine residues.
  • the zalpha30 protein was found to be most highly expressed in endocrine and neuroendocrine tissues, including thyroid, trachea, adrenal, and pituitary, with somewhat lower expression in prostate, testis, and ovary. Expression was also detected in kidney and pancreas. This tissue distribution suggests that zalpha30 has an endocrine function. Zalpha30 is thus predicted to modulate growth, development, reproduction, cell homeostasis, energy production, and/or behavior.
  • Polypeptides of the present invention comprise at least 15 contiguous amino acid residues of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12.
  • the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12, up to the entire predicted mature polypeptide (residues 26 to 295 of SEQ ID NO:2, residues 26-697 of SEQ ID NO: 10, or residues 26-700 of SEQ ID NO: 12) or the primary translation product (residues 1 to 295 of SEQ ID NO:2, residues 1 to 697 of SEQ ID NO: 10, or residues 1-700 of SEQ ID NO: 12).
  • polypeptides of the present invention also include those comprising residues 71-263 or residues 42-263 of SEQ ID NO:2; residues 71-236 or residues 42-236 of SEQ ID NO:2; and residues 71-235 or residues 42-235 of SEQ ID NO:12.
  • the polypeptides ofthe present invention comprise the secretory peptide of zalpha30 (residues 1-25 of SEQ ID NO:2 or SEQ ID NO: 12), and optionally residues 26-x of SEQ ID NO:2, wherein x is a peptide bond or an integer from 27 to 295, inclusive; residues 26-y of SEQ ID NO: 10, wherein y is a peptide bond or an integer from 27 to 697, inclusive; or residues 26 to z of SEQ ID NO: 12, wherein z is an integer from 27 to 700, inclusive.
  • these polypeptides can further comprise additional, non-zalpha30, polypeptide sequence(s).
  • polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO:2.
  • An "epitope” is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984.
  • Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length.
  • 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 et al., Science 219:660-666, 1983.
  • Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells or tissue samples.
  • Antibodies to linear epitopes are also useful for detecting fragments of zalpha30, such as might occur in body fluids or cell culture media.
  • Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zalpha30 protein.
  • Antigenic, epitope-bearing polypeptides contain a sequence of at least six, often at least nine, often from 15 to about 30 contiguous amino acid residues of a zalpha30 protein (e.g., SEQ ID NO:2).
  • Polypeptides comprising a larger portion of a zalpha30 protein, i.e. from 30 to 50 residues up to the entire sequence, are included.
  • amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided.
  • Such regions include residues 75-80, 106-111, 107-112, 187-192, and 290- 295 of SEQ ID NO:2; residues 268-273, 427-432, 75-80, 641-646, and 639-644 of SEQ ID NO:10; and residues 601-606, 691-696, 517-522, 425-430, and 72-77 of SEQ ID NO:12.
  • zalpha30 polypeptides are prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO:2.
  • These changes can be of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, an amino or carboxyl- terminal cysteine residue to facilitate subsequent linking to maleimide-activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination.
  • Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zalpha30 polypeptide and the affinity tag.
  • exemplary cleavage sites include thrombin cleavage sites and factor Xa cleavage sites.
  • a zalpha30 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.
  • Exemplary dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin-zalpha30 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zalpha30 analogs.
  • a zalpha30 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multi-functional molecule.
  • One or more helices of a zalpha30 polypeptide can be joined to another cytokine to enhance or otherwise modify its biological properties.
  • Auxiliary domains can be fused to zalpha30 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
  • a zalpha30 polypeptide or protein can be targeted to a predetermined cell type by fusing a zalpha30 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • a zalpha30 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 34:1-9, 1996.
  • Polypeptide fusions of the present invention will generally contain not more than about 2,000 amino acid residues, often not more than about 1,800 amino acid residues, not more than about 1,500 amino acid residues, not more than about 1,200 residues, or not more than about 1,000 residues, and will in many cases be considerably smaller.
  • a zalpha30 polypeptide of 672 residues can be fused to E. coli ?-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol.
  • a 10-residue spacer and a 4-residue factor Xa cleavage site to yield a polypeptide of 1,707 residues.
  • a zalpha30 polypeptide of 270 residues can be fused to E. coli ff-galactosidase, a 10-residue spacer, and a 4-residue factor Xa cleavage site to yield a polypeptide of 1,305 residues.
  • residues 26-295 of SEQ ID NO:2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
  • the present invention provides fusions comprising a secretory peptide of zalpha30 (e.g., residues 1 through 25 of SEQ ID NO:2).
  • a zalpha30 secretory peptide can be used to direct the secretion of other proteins of interest by joining a polynucleotide sequence encoding it to the 5' end of a sequence encoding a protein of interest.
  • SEQ ID NO:2 comprising residues 1-25 is fused to a second polypeptide by joining their respective DNA sequences in the correct reading frame.
  • the zalpha30 secretory peptide alone is sufficient to direct the secretion of a second polypeptide to which it is fused
  • portions of the mature polypeptide sequence of zalpha30, up to the entire zalpha30 sequence may be included in the fusion.
  • a segment of zalpha30 comprising an epitope can be fused to a second polypeptide, and the resulting fusion polypeptide can be purified by immunoaffinity chromatography using an antibody specific for the zalpha30 epitope.
  • a cleavage site such as a factor Xa cleavage site, may be provided between the zalpha30 sequence and the second polypeptide to allow separation ofthe sequences by proteolysis.
  • the polypeptides of the present invention comprise at least 15 contiguous residues of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO:12.
  • polypeptides may further comprise additional residues as shown in SEQ ID NO:
  • SEQ ID NO:2 SEQ ID NO: 10, or SEQ ID NO: 12; a variant of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12; or another protein as disclosed herein.
  • variants of SEQ ID NO:2, SEQ ID NO: 10 SEQ ID NO: 10; SEQ ID NO: 12; or another protein as disclosed herein.
  • SEQ ID NO: 10 or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical to the corresponding region of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12 will generally be at least 90% and commonly at least 95% identical
  • Percent sequence identity is determined by conventional methods. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
  • 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 three to six, most preferably three, with other parameters set as default.
  • 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 one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • the proteins of the present invention can also comprise non-naturally occuring amino acid residues.
  • Non-naturally occuring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, c s-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ tV ⁇ -threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • 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 occuring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • the non-naturally occuring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.
  • Naturally occuring amino acid residues can be converted to non-naturally occuring 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).
  • Amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can identify specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing, and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376,
  • a hydrophilicity profile of SEQ ID NO:2 is shown in Fig. 1.
  • Hydrophilicity profiles of SEQ ID NO: 10 and SEQ ID NO: 12 can be readily generated by those skilled in the art using the methods disclosed by Hopp, Methods Enzymol. 178:571-585, 1989. Those skilled in the art will recognize that hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zalpha30 polypeptide, so as not to disrupt the overall profile.
  • Essential amino acids in the polypeptides ofthe present invention can be identified experimentally 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 88:4498-4502, 1991).
  • site- directed mutagenesis or alanine-scanning mutagenesis
  • 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.
  • Variants of the disclosed zalpha30 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751, 1994. Briefly, variant genes are generated by in vitro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes 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.
  • the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zalpha30 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • expressing the complete zalpha30 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved.
  • Differential processing of individual chains may result in heterogeneity of expressed polypeptides.
  • Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zalpha30 variant polypeptides.
  • Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format.
  • Mutagenized DNA molecules that encode active zalpha30 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 present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zalpha30 polypeptides disclosed above.
  • Representative DNA sequences encoding the human and mouse zalpha30 proteins are shown in SEQ ID NO:l, SEQ ID NO:9, and SEQ ID NO:l 1.
  • SEQ ID NO:4 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha30 polypeptide of SEQ ID NO:2.
  • SEQ ID NO: 13 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha30 polypeptide of SEQ ID NO: 10.
  • SEQ ID NO: 14 is a degenerate DNA sequence that encompasses all DNAs that encode the zalpha30 polypeptide of SEQ ID NO: 12.
  • Those skilled in the art will recognize that these degenerate sequences also provides all RNA sequences encoding the corresponding proteins by substituting U for T.
  • zalpha30 polypeptide-encoding polynucleotides comprising nucleotides 1-885 or nucleotides 76-885 of SEQ ID NO:4, nucleotides 1-2091 or nucleotides 76-2091 of SEQ ID NO:13, nucleotides 1-2100 or nucleotides 76-2100 of SEQ ID NO: 14, and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO:4, SEQ ID NO: 13, and SEQ ID NO: 14 encoding other zalpha30 polypeptides disclosed herein.
  • Table 2 sets forth the one-letter codes used within SEQ ID NOS:4, 13, and 14 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter. “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NOS:4, 13, and 14, encompassing all possible codons for a given amino acid are set forth in Table 3, below.
  • Gap - One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 10, or SEQ ID NO: 12. Variant sequences can be readily tested for functionality as described herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO:l l, 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 polynucleotides of the present invention include DNA and RNA.
  • RNA is isolated from a tissue or cell that produces large amounts of zalpha30 RNA, such as pituitary tissue.
  • Other source tissues include heart, kidney, pancreas, prostate, testis, ovary, stomach, thyroid, spinal cord, trachea, and adrenal gland.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
  • cDNA Complementary DNA
  • genomic DNA can be isolated.
  • Polynucleotides encoding zalpha30 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • Full-length clones encoding zalpha30 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 ofthe sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to zalpha30, receptor fragments, or other specific binding partners.
  • Zalpha30 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a zalpha30 gene.
  • Promoter elements from a zalpha30 gene can thus be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zalpha30 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670.
  • an endogenous zalpha30 gene in a cell is altered by introducing into the zalpha30 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a zalpha30 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zalpha30 locus, whereby the sequences within the construct become operably linked with the endogenous zalpha30 coding sequence.
  • an endogenous zalpha30 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • sequences disclosed herein represent single alleles of human and mouse zalpha30. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs").
  • zalpha30 polypeptides from other mammalian species, including porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
  • Orthologs of human and mouse zalpha30 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 zalpha30 as disclosed above.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a zalpha30-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 sequence.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zalpha30 sequence disclosed herein.
  • 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 zalpha30 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • any zalpha30 polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 3 and 4, above.
  • those of skill in the art can use standard software to devise zalpha30 variants based upon the nucleotide and amino acid sequences described herein.
  • the present invention thus provides a computer-readable medium encoded with a data structure that provides at least one ofthe following sequences: SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID
  • Suitable forms of computer-readable media include magnetic media and optically-readable media.
  • Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIPTM disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD- rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • compact discs e.g., CD-read only memory (ROM), CD- rewritable (RW), and CD-recordable
  • DVD digital versatile/video discs
  • the zalpha30 polypeptides ofthe present invention can be produced according to conventional techniques using cells into which have been introduced an expression vector encoding the polypeptide.
  • cells into which have been introduced an expression vector include both cells that have been directly manipulated by the introduction of exogenous DNA molecules and progeny thereof that contain the introduced DNA.
  • 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.
  • a DNA sequence encoding a zalpha30 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 ofthe 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 a zalpha30, or may be derived from another secreted protein (e.g., t-PA; see, U.S. Patent No. 5,641,655) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zalpha30 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly sythesized 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 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). As discussed above, a zalpha30 secretory signal sequence can be used to direct the secretion of non-zalpha30 polypeptides.
  • Cultured mammalian cells can be used as hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell J4:725, 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
  • promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Expression vectors for use in mammalian cells include pZP-1 and pZP-9, which have been deposited with the American Type Culture Collection, Manassas, VA USA under accession numbers 98669 and 98668, respectively, and derivatives thereof.
  • 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.”
  • An exemplary 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.
  • An exemplary amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • the adenovirus system can also be used for protein production in vitro.
  • the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adeno viral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector-infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol.
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non- secreted proteins can also be effectively obtained.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) according to methods known in the art.
  • recombinant baculovirus is produced through the use of a transposon-based system described by Luckow et al. (J. Virol. 67:4566-4579, 1993). This system, which utilizes transfer vectors, is commercially available in kit form (Bac-to-BacTM kit; Life Technologies, Rockville, MD).
  • the transfer vector (e.g., pFastBaclTM; Life Technologies) contains a Tn7 transposon to move the DNA encoding the protein of interest into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 11:911-916, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.
  • transfer vectors can include an in-frame fusion with DNA encoding a polypeptide extension or affinity tag as disclosed above.
  • a transfer vector containing a zalpha30-encoding sequence is transformed into E. coli host cells, and the cells are screened for bacmids which 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, such as Sf9 cells.
  • Recombinant virus that expresses zalpha30 protein 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 armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., High FiveTM cells; Invitrogen, Carlsbad, CA). See, for example, U.S. Patent No. 5,300,435. Serum-free media are used to grow and maintain the cells. Suitable media formulations are known in the art and can be obtained from commercial suppliers.
  • 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
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11 :47-58, 1987.
  • 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; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
  • 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.
  • 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.). When expressing a zalpha30 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.
  • 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.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • Zalpha30 polypeptides can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is particularly advantageous for the preparation of smaller polypeptides.
  • polypeptides and proteins of the present invention can be purified to >80% purity, to >90% purity, to >95% purity, or to a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the purified polypeptide or protein is substantially free of other polypeptides or proteins, particularly those of animal origin.
  • Zalpha30 proteins are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity
  • Proteins comprising a polyhistidine affinity tag are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag can be purified by immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid.
  • Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
  • zalpha30 proteins can be prepared glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Target cells for use in zalpha30 activity assays include, without limitation, vascular cells (especially endothelial cells and smooth muscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells (including hepatocytes, fenestrated endothelial cells, Kupffer cells, and Ito cells), fibroblasts (including human dermal fibroblasts and lung fibroblasts), neurite cells (including astrocytes, glial cells, dendritic cells, and PC- 12 cells), fetal lung cells, articular synoviocytes, pericytes, chondrocytes, osteoblasts, adipocytes, and prostate epithelial cells. Endothelial cells and hematopoietic cells are derived from a common ancestral cell, the hemangioblast (Ch
  • Zalpha30 activity can be measured with a silicon-based biosensor microphysiometer that measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary such device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-1912, 1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth.
  • the microphysiometer By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zalpha30 proteins, their agonists, and antagonists.
  • assays measuring cell proliferation or differentiation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:149-113, 1985; Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al., Analytical Biochem. 119:1-1, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J.
  • Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63- 75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989). Effects of zalpha30 proteins on tumor cell growth and metastasis can be analyzed using the Lewis lung carcinoma model, for example as described by Cao et al., J. Exp. Med. 182:2069-2077, 1995. Activity of zalpha30 proteins on cells of neural origin can be analyzed using assays that measure effects on neurite growth as disclosed below.
  • Zalpha30 activity may also be detected using assays designed to measure zalpha30-induced production of one or more additional growth factors or other macromolecules.
  • assays include those for determining the presence of hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF ⁇ ), interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF), angiogenin, and other macromolecules produced by the liver.
  • Suitable assays include mitogenesis assays using target cells responsive to the macromolecule of interest, receptor-binding assays, competition binding assays, immunological assays (e.g., ELISA), and other formats known in the art.
  • Metalloprotease secretion is measured from treated primary human dermal fibroblasts, synoviocytes and chondrocytes.
  • the relative levels of collagenase, gelatinase and stromalysin produced in response to culturing in the presence of a zalpha30 protein is measured using zymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106, 1990).
  • Procollagen/collagen synthesis by dermal fibroblasts and chondrocytes in response to a test protein is measured using 3 H-proline incorporation into nascent secreted collagen.
  • 3 H-labeled collagen is visualized by SDS-PAGE followed by autoradiography (Unemori and Amento, J. Biol.
  • GAG Glycosaminoglycan secretion from dermal fibroblasts and chondrocytes is measured using a 1,9- dimethylmethylene blue dye binding assay (Farndale et al., Biochim. Biophys. Ada 883:173-177, 1986). Collagen and GAG assays are also carried out in the presence of IL-l ⁇ or TGF- ⁇ to examine the ability of zalpha30 protein to modify the established responses to these cytokines.
  • Monocyte activation assays are carried out (1) to look for the ability of zalpha30 proteins to further stimulate monocyte activation, and (2) to examine the ability of zalpha30 proteins to modulate attachment-induced or endo toxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987).
  • IL-l ⁇ and TNF ⁇ levels produced in response to activation are measured by ELISA (Biosource, Inc. Camarillo, CA).
  • Monocyte/macrophage cells by virtue of CD14 (LPS receptor), areakily sensitive to endotoxin, and proteins with moderate levels of endotoxin- like activity will activate these cells.
  • zalpha30 proteins can be measured by culturing target cells in the presence and absence of zalpha30 and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like. Suitable assays are known in the art.
  • Hematopoietic activity of zalpha30 proteins can be assayed on various hematopoietic cells in culture.
  • Such assays include primary bone marrow colony assays and later stage lineage-restricted colony assays, which are known in the art (e.g., Holly et al., WIPO Publication WO 95/21920).
  • Marrow cells plated on a suitable semi- solid medium e.g., 50% methylcellulose containing 15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic mix
  • Known hematopoietic factors are used as controls. Mitogenic activity of zalpha30 polypeptides on hematopoietic cell lines can be measured as disclosed above.
  • Cell migration is assayed essentially as disclosed by Kahler et al. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997).
  • a protein is considered to be chemotactic if it induces migration of cells from an area of low protein concentration to an area of high protein concentration.
  • a typical assay is performed using modified Boyden chambers with a polystryrene membrane separating the two chambers (Transwell; Corning Costar Corp.). The test sample, diluted in medium containing 1% BSA, is added to the lower chamber of a 24-well plate containing Transwells. Cells are then placed on the Transwell insert that has been pretreated with 0.2% gelatin. Cell migration is measured after 4 hours of incubation at 37°C.
  • Non- migrating cells are wiped off the top of the Transwell membrane, and cells attached to the lower face of the membrane are fixed and stained with 0.1 % crystal violet. Stained cells are then extracted with 10% acetic acid and absorbance is measured at 600 nm. Migration is then calculated from a standard calibration curve. Cell migration can also be measured using the matrigel method of Grant et al. ("Angiogenesis as a component of epithelial-mesenchymal interactions" in Goldberg and Rosen, Epithelial- Mesenchymal Interaction in Cancer, Birkhauser Verlag, 1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997).
  • Zalpha30 proteins can be assayed for the ability to modulate axon guidance and growth.
  • Suitable assays that detect changes in neuron growth patterns include, for example, those disclosed in Hastings, WIPO Publication WO 97/29189 and Walter et al., Development 101 :685-96, 1987.
  • Assays to measure the effects on neuron growth are well known in the art.
  • the C assay e.g., Raper and Kapfhammer, Neuron 4:21-9, 1990 and Luo et al., Cell 75:217-27, 1993
  • Conditioned media from cells expressing a zalpha30 protein, or aggregates of such cells can by placed in a gel matrix near suitable neural cells, such as dorsal root ganglia (DRG) or sympathetic ganglia explants, which have been co-cultured with nerve growth factor.
  • DRG dorsal root ganglia
  • SVG sympathetic ganglia
  • zalpha30-induced changes in neuron growth can be measured (as disclosed by, for example, Messersmith et al., Neuron 14:949-59, 1995 and Puschel et al., Neuron 14:941-8, 1995).
  • Neurite outgrowth can be measured using neuronal cell suspensions grown in the presence of molecules of the present invention. See, for example, O'Shea et al., Neuron 7:231-7, 1991 and DeFreitas et al., Neuron 15:333-43, 1995.
  • Cell adhesion activity is assayed essentially as disclosed by LaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter plates are coated with the test protein, non-specific sites are blocked with BSA, and cells (such as smooth muscle cells, leukocytes, or endothelial cells) are plated at a density of approximately 10 4 - 10 s cells/well. The wells are incubated at 37°C (typically for about 60 minutes), then non-adherent cells are removed by gentle washing. Adhered cells are quantitated by conventional methods (e.g., by staining with crystal violet, lysing the cells, and determining the optical density of the lysate). Control wells are coated with a known adhesive protein, such as fibronectin or vitronectin.
  • a known adhesive protein such as fibronectin or vitronectin.
  • Assays for angiogenic activity are also known in the art.
  • the effect of zalpha30 proteins on primordial endothelial cells in angiogenesis can be assayed in the chick chorioallantoic membrane angiogenesis assay (Leung, Science 246:1306-1309, 1989; Ferrara, Arm. NY Acad. Sci. 752:246-256, 1995). Briefly, a small window is cut into the shell of an eight-day old fertilized egg, and a test substance is applied to the chorioallantoic membrane. After 72 hours, the membrane is examined for neovascularization.
  • Suitable assays include microinjection of early stage quail (Coturnix coturnix japonic ⁇ ) embryos as disclosed by Drake et al. (Proc. Natl. Acad. Sci. USA 92:7657-7661, 1995); the rodent model of corneal neovascularization disclosed by Muthukkaruppan and Auerbach (Science 205:1416-1418, 1979), wherein a test substance is inserted into a pocket in the cornea of an inbred mouse; and the hampster cheek pouch assay (H ⁇ ckel et al., Arch. Surg. 128:423-429, 1993).
  • Induction of vascular permeability is measured in assays designed to detect leakage of protein from the vasculature of a test animal (e.g., mouse or guinea pig) after administration of a test compound (Miles and Miles, J. Physiol. U8:228-257, 1952; Feng et al., J. Exp. Med. 183:1981-1986, 1996).
  • In vitro assays for angiogenic activity include the tridimensional collagen gel matrix model (Pepper et al. Biochem. Biophys. Res. Comm. 189:824-831, 1992 and Ferrara et al., Ann. NY Acad. Sci.
  • Angiogenesis assays can be carried out in the presence and absence of vascular endothelial growth factor (VEGF) to assess possible combinatorial effects.
  • VEGF vascular endothelial growth factor
  • VEGF can be used as a control within in vivo assays.
  • Receptor activation can be detected in target cells by: (1) measurement of adenylate cyclase activity (Salomon et al., Anal. Biochem. 58:541-48, 1974; Alvarez and Daniels, Anal. Biochem. 187:98-103, 1990); (2) measurement of change in intracellular cAMP levels using conventional radioimmunoassay methods (Steiner et al, J. Biol. Chem. 247: 1106- 13, 1972; Harper and Brooker, J. Cyc. Nucl. Res. 1:207- 18, 1975); or (3) through use of a cAMP scintillation proximity assay (SPA) method (such as available from Amersham Corp., Arlington Heights, IL).
  • SPA cAMP scintillation proximity assay
  • the assays disclosed above can be modified by those skilled in the art to detect the presence of agonists and antagonists of zalpha30 activity.
  • Expression of zalpha30 polynucleotides in animals provides models for further study of the biological effects of overproduction or inhibition of protein activity in vivo.
  • Zalpha30-encoding polynucleotides and antisense polynucleotides can be introduced into test animals, such as mice, using viral vectors or naked DNA, or transgenic animals can be produced.
  • test animals such as mice
  • viral vectors or naked DNA or transgenic animals can be produced.
  • One in vivo approach for assaying proteins of the present invention utilizes viral delivery systems.
  • Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997.
  • the adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (e.g., the human 293 cell line).
  • the host cell e.g., the human 293 cell line.
  • the host's tissue e.g., liver
  • the host's tissue will express and process (and, if a signal sequence is present, secrete) the heterologous protein.
  • Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid. The transformed cells are then re-implanted in the body. Naked DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963- 967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.
  • Transgenic mice engineered to express a zalpha30 gene, and mice that exhibit a complete absence of zalpha30 gene function, referred to as "knockout mice” (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-742, 1993). These mice can be employed to study the zalpha30 gene and the protein encoded thereby in an in vivo system. Transgenic mice are particularly useful for investigating the role of zalpha30 proteins in early development in that they allow the identification of developmental abnormalities or blocks resulting from the over- or underexpression of a specific factor. See also, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997. Promoters for transgenic expression include, for example, promoters from metallothionein and albumin genes.
  • Antisense methodology can be used to inhibit zalpha30 gene transcription to examine the effects of such inhibition in vivo.
  • Polynucleotides that are complementary to a segment of a zalpha30-encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:l
  • Such antisense oligonucleotides can also be used to inhibit expression of zalpha30 polypeptide-encoding genes in cell culture.
  • Zalpha30 polypeptides can be used to prepare a variety of inhibitors of zalpha30 activity ("zalpha30 antagonists").
  • Zalpha30 antagonists include antibodies that specifically bind to zalpha30 polypeptides, soluble zalpha30 receptors, inactive receptor-binding fragments of zalpha30 polypeptides, and other peptidic and non- peptidic agents (including ribozymes and small molecule antagonists). Such antagonists can be used to block the effects of zalpha30 on cells or tissues.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies.
  • 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.
  • Antibodies are defined to be specifically binding if they bind to a zalpha30 polypeptide or protein with an affinity at least 10- fold greater than the binding affinity to control (non-zalpha30) polypeptide or protein.
  • the affinity of a monoclonal antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, Ann. NY Acad. Sci. 5 _: 660-672, 1949). Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, J. G.
  • polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
  • the immunogenicity of a zalpha30 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 a zalpha30 polypeptide 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.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to zalpha30 polypeptides, and selection of antibody display libraries in phage or similar vectors (e.g., through the use of immobilized or labeled zalpha30 polypeptide).
  • Human antibodies can be produced in transgenic, non- human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zalpha30 polypeptides. 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, radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
  • concurrent immunoelectrophoresis radio-immunoassays, radio- immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays.
  • Antibodies to zalpha30 may be used for affinity purification of zalpha30, polypeptide fragments thereof, and fusion proteins containing a zalpha30 segment; within diagnostic assays for determining circulating levels of the protein; for detecting or quantitating soluble zalpha30 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vitro and in vivo.
  • Antibodies to zalpha30 may also be used for tagging cells that express zalpha30; for affinity purification of zalpha30 polypeptides and proteins; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies.
  • Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targetting of those compounds to cells expressing receptors for zalpha30. For certain applications, including in vitro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies of the present invention may also be directly or indirectly conjugated to drugs (both peptidic and non-peptidic), toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications (e.g., inhibition of cell proliferation) as disclosed in more detail below. See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.
  • the proteins of the present invention can be used in a variety of diagnostic applications.
  • zalpha30 proteins can be used as standards in assays of circulating or excreted protein levels, wherein abnormal levels are indicative of disease, including acute (e.g., infections) and chronic (e.g., genetic abnormalities) diseases.
  • Assays useful in this regard include antibody-based assay methods (e.g., ELISA, RIA) using labeled anti-zalpha30 antibodies or labeled second antibodies.
  • Antibodies can be labeled with radioisotopes, enzymes, fluorophores, and the like according to methods known in the art.
  • zalpha30 proteins include imaging and in vivo localization, including imaging of cells expressing a cell-surface receptor for zalpha30 and detection abnormal expression of zalpha30 receptor using a labeled zalpha30 protein.
  • Suitable labels in this regard include radioisotopes, radio-opaque substances, paramagnetic compounds, electron-dense elements that scatter X-rays anomolously, and others known in the art.
  • Zalpha30-encoding polynucleotide molecules and polynucleotide molecules complementary thereto can also be used within diagnostic applications.
  • the zalpha30 gene, a probe comprising zalpha30 DNA or RNA, or a subsequence thereof can be used to determine the presence of mutations in human chromosome 12 at or near the zalpha30 locus at position 12q24.
  • Detectable chromosomal aberrations at the zalpha30 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and rearrangements.
  • Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
  • PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region ofthe gene is targetted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more.
  • Probes will generally comprise a polynucleotide linked to a signal-generating moiety such as a radionucleotide.
  • these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (c) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
  • Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof.
  • Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymo ⁇ hism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A.J.
  • Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion.
  • RNA-RNA hybrid reaction product
  • PCR assays a patient genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
  • Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-38, 1991).
  • SSCP single strand conformational polymorphism
  • Zalpha30 proteins can be used to treat conditions associated with a lack of zalpha30 activity, for example metabolic disorders resulting in under-production of zalpha30 protein or reduced protein activity.
  • the proteins can also be used to deliver cytotoxic agents to cells expressing a cell-surface zalpha30 receptor (e.g., tumor cells).
  • Cytotoxic agents useful in this regard include bacterial and plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188, and yttrium-90.
  • Proteins can also be conjugated to cytotoxic drugs such as adriamycin and the like.
  • cytotoxic drugs such as adriamycin and the like.
  • the cytotoxic molecule can be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the zalpha30 protein portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • zalpha30-cytokine fusion proteins may be used for enhancing in vitro cytotoxicity (for instance, that mediated by monoclonal antibodies against tumor targets) and for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers). See, generally, Hornick et al., Blood 89:4437-4447, 1997).
  • cytokines are toxic if administered systemically.
  • Such fusion proteins enable targeting of a cytokine to a desired site of action, such as a cell having binding sites for zalpha30, thereby providing an elevated local concentration of cytokine.
  • Suitable cytokines for this pu ⁇ ose include, for example, interleukin-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intra-arterially or intraductally, or may be introduced locally at the intended site of action.
  • Polynucleotides encoding zalpha30 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zalpha30 activity. If a mammal has a mutated or absent zalpha30 gene, a zalpha30 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zalpha30 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as he ⁇ es simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective he ⁇ es simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest.
  • HSV1 defective he ⁇ es simplex virus 1
  • a zalpha30 gene can be introduced in a retroviral vector as described, for example, by Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol.
  • the vector can be introduced by liposome-mediated transfection ("lipofection").
  • lipofection Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988).
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages, including molecular targeting of liposomes to specific cells. Directing transfection to particular cell types is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the pu ⁇ ose of targeting. Peptidic and non-peptidic molecules can be coupled to liposomes chemically.
  • cells are removed from the body, a vector is introduced into the cells as a naked DNA plasmid, and the transformed cells are re-implanted into the body as disclosed above.
  • Antisense methodology can be used to inhibit zalpha30 gene transcription in a patient as generally disclosed above.
  • zalpha30 proteins can be used as molecular weight standards or as reagents in assays for determining circulating levels of the protein, such as in the diagnosis of disorders characterized by over- or underproduction of zalpha30 protein or in the analysis of cell phenotype.
  • Antibody-based assays as disclosed above are exemplary.
  • kits for courses related to genetics, molecular biology, protein chemistry, antibody production and analysis, and the like.
  • kits will optionally contain one or more of an instruction sheet, a sheet of specifications, one or more standards or controls, and additional reagents.
  • Kits may also contain various combinations of zalpha30 polynucleotides, polypeptides, and antibodies.
  • molecules of zalpha30 can be used as standards or as ''unknowns" for testing pu ⁇ oses.
  • zalpha30 polynucleotides can be used to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein the zalpha30 gene is to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides (which cleavage sites can be readily determined by those skilled in the art; partial restriction maps of SEQ ID NOS:l, 9, and 11 are shown in Figs.
  • Zalpha30 polypeptides can be used in teaching preparation of antibodies, identification of proteins by Western blotting, protein purification, determination of the mass of expressed zalpha30 polypeptides as a ratio to total protein expressed, identification of peptide cleavage sites, coupling of 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
  • Zalpha30 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; and 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; and nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
  • a kit containing a zalpha30 polypeptide can be given to the student to analyze in order to develop or test the student's skills. Since the amino acid sequence and other properties of the polypeptide would be known by the instructor, the instructor would then know whether or not the student has correctly analyzed the polypeptide.
  • Antibodies that specifically bind to zalpha30 polypeptides can be used, for example, as teaching aids to instruct students how to prepare affinity chromatography columns to purify zalpha30 polypeptides and how to perform immunological assays and histological analysis.
  • Anti-zalpha30 antibodies can also be used as tools in the cloning and sequencing of a polynucleotide that encodes an antibody and are thus useful for teaching a student how to design humanized antibodies. Such kits are considered within the scope ofthe present invention.
  • Zalpha30 proteins can also be used to identify inhibitors of their activity. Test compounds are added to the assays disclosed above to identify compounds that inhibit the activity of zalpha30 protein. In addition to those assays disclosed above, samples can be tested for inhibition of zalpha30 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zalpha30- dependent cellular responses. For example, zalpha30-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zalpha30-stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a zalpha30-activated serum response element (SRE) operably linked to a gene encoding an assayable protein, such as luciferase.
  • SRE serum response element
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zalpha30 on the target cells as evidenced by a decrease in zalpha30 stimulation of reporter gene expression.
  • Assays of this type will detect compounds that directly block zalpha30 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 zalpha30 binding to receptor using zalpha30 tagged with a detectable label (e.g., I25 I, biotin, horseradish peroxidase, FITC, and the like).
  • a detectable label e.g., I25 I, biotin, horseradish peroxidase, FITC, and the like.
  • the ability of a test sample to inhibit the binding of labeled zalpha30 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • Zalpha30-encoding polynucleotides and polynucleotides complementary thereto can be used within the laboratory field, for example in chromosomal analysis and for subtracting out known sequences in preparing libraries.
  • zalpha30 proteins, antibodies, or other antagonists are formulated for topical or parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • pharmaceutical formulations will include a zalpha30 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
  • Zalpha30 will typically be used in a concentration of about 10 to 100 ⁇ g/ml of total volume, although concentrations in the range of 1 ng/ml to 1000 ⁇ g/ml may be used, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc.
  • a therapeutically effective amount of zalpha30 is an amount sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in hematopoietic or immune function, a significant reduction in morbidity, or a significantly increased histological score.
  • the invention is further illustrated by the following non-limiting examples.
  • Example 1 RNA extracted from cells of pituitary gland was purchased from
  • the first strand cDNA reaction mixture contained 10 ⁇ l of human pituitary twice poly d(T)-selected poly(A) + mRNA (Clontech Laboratories, Inc., Palo Alto, CA) at a concentration of 1.0 mg/ml, and 2 ⁇ l of 20 pmole/ ⁇ l first strand primer ZC6191 (SEQ ID NO:6), containing an Xho I restriction site.
  • the mixture was heated at 70°C for 2.0 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 ⁇ l of 100 mM dithiothreitol, and 2 ⁇ 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.
  • the reaction mixture was incubated at 37°C for 2 minutes, followed by the addition of 10 ⁇ l of 200 U/ ⁇ l RNase H " reverse transcriptase (Superscript II® 5 Life Technologies).
  • the length of labeled first strand cDNA was determined by agarose gel electrophoresis.
  • the second strand reaction mixture contained 100 ⁇ l of the unlabeled first strand cDNA, 30 ⁇ l of 5x polymerase I buffer (125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl 2 , 50mM (NH 4 ) 2 SO 4 ), 2.0 ⁇ l of 100 mM dithiothreitol, 3.0 ⁇ l of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 ⁇ l of 5 mM ⁇ - NAD, 2.0 ⁇ l of 10 U/ ⁇ l E.
  • 5x polymerase I buffer 125 mM Tris: HCl, pH 7.5, 500 mM KCl, 25 mM MgCl 2 , 50mM (NH 4 ) 2 SO 4
  • reaction mixtures were incubated at 16°C for two hours, then 1 ⁇ l of a 10 mM dNTP solution and 5.0 ⁇ l T4 DNA polymerase (10 U/ ⁇ l, Boehringer Mannheim, Indianapolis, IN) were added, and the mnixtures were incubated for an additional 10 minutes at 16°C. Uninco ⁇ orated 32 P- ⁇ dCTP in the labeled reaction was removed by chromatography through a 400 pore size gel filtration column before analysis by agarose gel electrophoresis.
  • the reaction was terminated by the addition of 10.0 ⁇ l 0.5 M EDTA and extraction with phenol/chloroform and chloroform followed by ethanol precipitation in the presence of 3.0 M Na acetate and 2 ⁇ l of a dye- labeled carrier (Pellet PaintTM Co-Precipitant; Novagen, Madison, Wl).
  • the yield of cDNA was estimated to be approximately 2 ⁇ g from starting mRNA template of 10 ⁇ g.
  • Eco RI adapters were ligated onto the 5' ends of the cDNA described above to enable cloning into an expression vector.
  • 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 ofthe 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). Digestion was carried out at 37°C for 45 minutes. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400 pore size gel filtration column.
  • 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 MgClj), 0.5 ⁇ l 0.1 M DTT, 2 ⁇ l 10 mM ATP, 2 ⁇ l T4 polynucleotide kinase (10 U/ ⁇ l, Life Technologies). 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 MgClj
  • 0.5 ⁇ l 0.1 M DTT 2 ⁇ l 10 mM ATP
  • 2 ⁇ l T4 polynucleotide kinase 10 U/ ⁇ l,
  • the contaminating adapters and cDNA below 0.6 Kb in length were excised from the gel.
  • the electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin.
  • the area of the gel containing the concentrated cDNA was 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 ⁇ -agarase I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65°C for 15 minutes.
  • the cDNA was cloned into the Eco RI and Xho I sites of a phagemid vector (pBluescript® SK(+); Stratagene, La Jolla, CA), which was then electroporated into E. coli host cells (Electromax DH10BTM cells; obtained from Life Technologies, Inc., Gaithersburg, MD). Bacterial colonies containing known sequences were identified and eliminated from sequence analysis by reiterative cycles of probe hybridization to high-density colony filter arrays (Genome Systems).
  • cDNAs of known genes were pooled in groups of 50 - 100 inserts and were labeled with 32 P using a commercially available labeling kit (MegaprimeTM DNA labeling system; Amersham, Arlington Heights, IL). Colonies that did not hybridize to the probe mixture were selected for sequencing. Sequencing was done using an ABI 377 sequencer using either the T3 or the reverse primer. The resulting data were analyzed, and a database of partial sequences (ESTs) was prepared. The pituitary EST database was analyzed using a quasi-threading method to identify assemblies of contiguous sequences that comprised a predicted secretory signal, an alpha-helical region, and an in- frame stop codon upstream of the predicted intiation Met.
  • ESTs partial sequences
  • a cDNA clone (designated LPIF20653910) corresponding to one such assembly was recovered from the library and sequenced. Analysis indicated that the clone contained a full-length cDNA encoding a secreted polypeptide of 295 amino acid residues (SEQ ID NO:l and SEQ ID NO:2).
  • tissue distribution was performed by the Northern blotting technique using Human Human Multiple Tissue (MTNI,II,and III) and Master Dot blots obtained from Clontech Laboratories, Inc.
  • a probe was obtained by restriction digest of the LPIF2065391 clone with EcoRI and PvuII to remove a 640 bp fragment of the insert from the vector.
  • the reaction mixture was electrophoresed on a 2% agarose gel, and the fragment was excised and purified using commercially available gel purification reagents and protocol (QIAEX® II gel extraction kit; Qiagen, Valencia, CA).
  • the purified DNA was radioactively labeled with 32 P using a commercially available kit (RediprimeTM II random-prime labeling system; Amersham Co ⁇ .).
  • the probe was purified using a commercially available size exclusion column (NucTrap® column; Stratagene, La Jolla, CA).
  • a commercially available hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories, Inc.) was used for hybridization and prehybridization.
  • the final hybridization solution contained 8 ml ExpressHybTM solution, 80 ⁇ l sheared salmon sperm DNA (10 mg/ml; 5 Prime-3 Prime, Boulder, CO), 48 ⁇ l human Cot-1 DNA (1 mg/ml; Life Technologies) and labeled probe (1.2 x 10 6 CPM). Hybridization took place overnight at 55°C. After hybridization the blots were washed in 2X SSC, 0.1 % SDS at room temperature; then in in 2X SSC, 0.1% SDS at 65°C, followed by a 0.1 X SSC, 0.1% SDS wash at 65°C. The blots were exposed to autoradiographic film overnight and developed.
  • Tissue Northern signals were observed at ⁇ 3 kb in most tissues. The strongest signals were observed in prostate, testis, thyroid, and trachea. Moderate signals were observed in heart, kidney, pancreas, ovary, stomach, spinal cord, and adrenal gland. Lesser signal intensity was observed in brain, placenta, lung, liver, skeletal muscle, spleen, thymus, small intestine, colon, and lymph node. Dot blot signals were observed in all tissues, including fetal tissues with a strong signal observed in pituitary gland.
  • Zalpha30 was mapped to human chromosome 12 using the commercially available version of the Stanford G3 Radiation Hybrid Mapping Panel (Research Genetics, Inc., Huntsville, AL). This panel contains PCRable DNAs from each of 83 radiation hybrid clones ofthe whole human genome, plus two control DNAs (the RM donor and the A3 recipient).
  • a publicly available WWW server http://shgc- www.stanford.edu) allows chromosomal localization of markers.
  • Each of the 85 reaction mixtures consisted of 2 ⁇ l buffer (10X KlenTaq PCR reaction buffer, Clontech Laboratories, Inc., Palo Alto, CA), 1.6 ⁇ l dNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, CA), 1 ⁇ l sense primer ZC22,745 (SEQ ID NO: 7), 1 ⁇ l antisense primer ZC22,746 (SEQ ID NO:8), 2 ⁇ l of a density increasing agent and tracking dye (RediLoad, Research Genetics, Inc., Huntsville, AL), 0.4 ⁇ l of a commercially available DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, obtained from Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x ⁇ l ddH 2 O for a total volume of 20 ⁇ l.
  • 2 ⁇ l buffer 10X KlenTaq PCR reaction buffer, Clon
  • the mixtures were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 5 minute denaturation at 94°C; 35 cycles of 45 seconds denaturation at 94°C, 45 seconds annealing at 62°C, and 1 minute 15 seconds extension at 72°C; followed by a final extension of 7 minutes at 72°C.
  • the reaction products were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
  • the human zalpha30 sequence was used to search a public database of mouse sequence data. A clone was identified that encoded a 700-residue open reading frame. The DNA sequence and encoded amino acid sequence are shown in SEQ ID NO: 11 and SEQ ID NO: 12.
  • Example 5 The mouse Zalpha30 gene was mapped to chromosome 5 using the commercially available mouse T31 whole genome radiation hybrid (WGRH) panel (Research Genetics, Inc.). This panel contains DNA from each of 100 radiation hybrid clones, plus two control DNAs (the 129aa donor and the A23 recipient).
  • WGRH whole genome radiation hybrid
  • a publicly available WWW server http://www.genome.wi.mit.edu cgi-bin/mouse_rh rhmap- auto/rhmapper.cgi
  • WICGR Whitehead Institute/MIT Center for Genome Research's
  • 102 20- ⁇ l PCR reaction mixtures were prepared essentially as disclosed in Example 3 using sense primer ZC27,703 (SEQ ID NO: 15), antisense primer ZC27,704 (SEQ ID NO: 16), and 0.4 ⁇ l of a commercially available DNA polymerase/antibody mix (50X AdvantageTM KlenTaq Polymerase Mix, obtained from Clontech Laboratories, Inc., Palo Alto, CA).
  • the reactions run with an initial 5-minute denaturation at 94°C; 35 cycles of 45 seconds at 94°C, 45 seconds at 56°C, and 1 minute at 72°C; followed by 7 minutes at 72°C.
  • reaction products were separated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown, NJ) and visualized by staining with ethidium bromide.
  • the mouse zalpha30 gene mapped 19.72 cR_3000 distal of the framework marker D5Mit65 on the mouse chromosome 5 WICGR radiation hybrid map.
  • Proximal and distal framework markers were D5Mitl73 and D5Mit426, respectively.
  • the human zalpha30 sequence was used to search a public database of human sequence data. Two sequences were identified that encoded an extended carboxyl terminus of the protein. The longer protein appeared to be an alternatively spliced form that conesponded to the mouse zalpha30 of SEQ ID NO: 12. The longer human DNA and protein sequences are shown in SEQ ID NO:9 and SEQ ID NO: 10. An alignment ofthe mouse protein and longer human protein is shown in Fig. 2.
  • An expression plasmid containing all or part of a polynucleotide encoding zalpha30 is constructed via homologous recombination.
  • a fragment of zalpha30 cDNA is isolated by PCR using the polynucleotide sequence of SEQ ID NO: 1 with flanking regions at the 5' and 3' ends conesponding to the vector sequences flanking the zalpha30 insertion point.
  • the primers for PCR each include from 5' to 3' end: 40 bp of flanking sequence from the vector and 17 bp corresponding to the amino and carboxyl termini from the open reading frame of zalpha30.
  • Plamid pZMP6 is a mammalian expression vector containing an expression cassette having the cytomegalovirus immediate early promoter, multiple restriction sites for insertion of coding sequences, a stop codon, and a human growth hormone terminator; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae. It was constructed from pZP9 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No.
  • yeast genetic elements taken from pRS316 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, under Accession No. 77145), an internal ribosome entry site (IRES) element from poliovirus, and the extracellular domain of CD8 truncated at the C-terminal end ofthe transmembrane domain.
  • IRS internal ribosome entry site
  • yeast/DNA mixtures are electropulsed using power supply (BioRad Laboratories, Hercules, CA) settings of 0.75 kV (5 kV/cm), co ohms, 25 ⁇ F.
  • power supply BioRad Laboratories, Hercules, CA
  • To each cuvette is added 600 ⁇ l of 1.2 M sorbitol, and the yeast is plated in two 300- ⁇ l aliquots onto two URA-D plates and incubated at 30°C.
  • the Ura + yeast transformants from a single plate are resuspended in 1 ml H2O and spun briefly to pellet the yeast cells.
  • the cell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
  • lysis buffer 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA.
  • Five hundred microliters of the lysis mixture is added to an Eppendorf tube containing 300 ⁇ l acid-washed glass beads and 200 ⁇ l phenol- chloroform, vortexed for 1 minute intervals two or three times, and spun for 5 minutes in an Eppendorf centrifuge at maximum speed.
  • E. coli host cells (Electromax DH10BTM cells; obtained from Life Technologies, Inc., Gaithersburg, MD) is done with 0.5-2 ml yeast DNA prep and 40 ⁇ l of cells. The cells are electropulsed at 1.7 kV, 25 ⁇ F, and 400 ohms.
  • the inserts of positive clones are subjected to sequence analysis. Larger scale plasmid DNA is isolated using a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen, Valencia, CA) according to manufacturer's instructions. The correct construct is designated pZMP6/zalpha30.
  • Example 8 CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,
  • the cells are then transfected with the plasmid zalpha30/pZMP6 by liposome-mediated transfection using a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium- trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in membrane- filetered water (LipofectamineTM Reagent, Life Technologies), in serum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L- glutamine and 1% sodium pyruvate).
  • SF serum free
  • Zalpha30/pZMP6 is diluted into 15-ml tubes to a total final volume of 640 ⁇ l with SF media.
  • 35 ⁇ l of LipofectamineTM is mixed with 605 ⁇ l of SF medium. The resulting mixture is added to the DNA mixture and allowed to incubate approximately 30 minutes at room temperature.
  • Five ml of SF media is added to the DNA: LipofectamineTM mixture.
  • the cells are rinsed once with 5 ml of SF media, aspirated, and the DNA:LipofectamineTM mixture is added. The cells are incubated at 37°C for five hours, then 6.4 ml of Ham's F 12/10% FBS, 1% PSN media is added to each plate.
  • the plates are incubated at 37°C overnight, and the DNA:LipofectamineTM mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 3 post-transfection, the cells are split into T-175 flasks in growth medium.
  • the cells are stained with FITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, CA) followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec).
  • the CD8-positive cells are separated using commercially available columns (mini-MACS columns; Miltenyi Biotec) according to the manufacturer's directions and put into DMEM Ham's F12/5% FBS without nucleosides but with 50 nM methotrexate (selection medium).
  • Cells are plated for subcloning at a density of 0.5, 1 and 5 cells per well in 96-well dishes in selection medium and allowed to grow out for approximately two weeks. The wells are checked for evaporation of medium and brought back to 200 ⁇ l per well as necessary during this process. When a large percentage of the colonies in the plate are near confluency, 100 ⁇ l of medium is collected from each well for analysis by dot blot, and the cells are fed with fresh selection medium. The supernatant is applied to a nitrocellulose filter in a dot blot apparatus, and the filter is treated at 100°C in a vacuum oven to denature the protein.
  • the filter is incubated in 625 mM Tris- glycine, pH 9.1, 5mM ⁇ -mercaptoethanol, at 65°C, 10 minutes, then in 2.5% non-fat dry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4°C on a rotating shaker.
  • the filter is incubated with the antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer for 1 hour at room temperature on a rotating shaker.
  • the filter is then washed three times at room temperature in PBS plus 0.01% Tween 20, 15 minutes per wash.
  • the filter is developed with chemiluminescence reagents (ECLTM direct labelling kit;
  • Full-length zalpha30 protein is produced in BHK cells transfected with pZMP6/zalpha30 (Example 7).
  • BHK 570 cells (ATCC CRL-10314) are plated in 10- cm tissue culture dishes and allowed to grow to approximately 50 to 70% confluence overnight at 37°C, 5% CO 2 , in DMEM/FBS media (DMEM, Gibco/BRL High Glucose;
  • the cells are then transfected with pZMP6/zalpha30 by liposome-mediated transfection (using LipofectamineTM; Life Technologies), in serum free (SF) media (DMEM supplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).
  • SF serum free
  • the plasmid is diluted into 15-ml tubes to a total final volume of 640 ⁇ l with SF media.
  • 35 ⁇ l of the lipid mixture is mixed with 605 ⁇ l of SF medium, and the resulting mixture is allowed to incubate approximately 30 minutes at room temperature. Five milliliters of SF media is then added to the DNA:lipid mixture. The cells are rinsed once with 5 ml of SF media, aspirated, and the DNA:lipid mixture is added. The cells are incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media is added to each plate. The plates are incubated at 37°C overnight, and the DNA:lipid mixture is replaced with fresh 5% FBS/DMEM media the next day.
  • the cells are split into T-162 flasks in selection medium (DMEM + 5% FBS, 1% L-Gln, 1% NaPyr, 1 ⁇ M methotrexate). Approximately 10 days post-transfection, two 150-mm culture dishes of methotrexate-resistant colonies from each transfection are trypsinized, and the cells are pooled and plated into a T-162 flask and transfened to large-scale culture.
  • selection medium DMEM + 5% FBS, 1% L-Gln, 1% NaPyr, 1 ⁇ M methotrexate.
  • Example 10 For construction of adenovirus vectors, the protein coding region of human zalpha30 is amplified by PCR using primers that add Pmel and Ascl restriction sites at the 5' and 3' termini respectively. Amplification is performed with a full-length zalpha30 cDNA template in a PCR reaction as follows: one cycle at 95°C for 5 minutes; followed by 15 cycles at 95°C for 1 min., 61°C for 1 min., and 72°C for 1.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak. The PCR reaction product is loaded onto a 1.2% low-melting-temperature agarose gel in TAE buffer (0.04 M Tris- acetate, 0.001 M EDTA).
  • the zalpha30 PCR product is excised from the gel and purified using a commercially available kit comprising a silica gel mambrane spin column (QIAquickTM PCR Purification Kit and gel cleanup kit; Qiagen, Inc.) as per kit instructions.
  • the PCR product is then digested with Pmel and Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 ml TE (Tris/EDTA pH 8).
  • the zalpha30 fragment is then ligated into the Pmel- Ascl sites of the transgenic vector pTG12-8 and transformed into E. coli DH10BTM competent cells by electroporation.
  • Vector pTG12-8 was derived from p2999B4 (Palmiter et al., Mol. Cell Biol. 13:5266- 5275, 1993) by insertion of a rat insulin II intron (ca. 200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I site.
  • the vector comprises a mouse metallothionein (MT-1) promoter (ca. 750 bp) and human growth hormone (hGH) untranslated region and polyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5' flanking sequence and 7 kb of MT-1 3' flanking sequence.
  • MT-1 mouse metallothionein
  • hGH human growth hormone
  • the cDNA is inserted between the insulin II and hGH sequences.
  • Clones containing zalpha30 are identified by plasmid DNA miniprep followed by digestion with Pmel and Ascl. A positive clone is sequenced to insure that there were no deletions or other anomalies in the construct.
  • DNA is prepared using a commercially available kit (Maxi Kit, Qiagen, Inc.), and the zalpha30 cDNA is released from the pTG12-8 vector using Pmel and Ascl enzymes.
  • the cDNA is isolated on a 1 % low melting temperature agarose gel and excised from the gel. The gel slice is melted at 70°C, and the DNA is extracted twice with an equal volume of Tris-buffered phenol, precipitated with ⁇ tOH, and resuspended in 10 ⁇ l H 2 O.
  • the zalpha30 cDNA is cloned into the ⁇ coRV-AscI sites of a modified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).
  • This construct contains the green fluorescent protein (GFP) marker gene.
  • GFP green fluorescent protein
  • the CMV promoter driving GFP expression is replaced with the SV40 promoter, and the SV40 polyadenylation signal is replaced with the human growth hormone polyadenylation signal.
  • the native polylinker is replaced with Fsel, ⁇ coRV, and Ascl sites.
  • This modified form of pAdTrack-CMV is named pZyTrack.
  • Ligation is performed using a commercially available DNA ligation and screening kit (Fast-LinkTM kit; Epicentre Technologies, Madison, Wl). Clones containing zalpha30 are identified by digestion of mini prep DNA with Fsel and Ascl. In order to linearize the plasmid, approximately 5 ⁇ g of the resulting pZyTrack zalpha30 plasmid is digested with Pmel. Approximately 1 ⁇ g of the linearized plasmid is cotransformed with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli BJ5183 cells (He et al., ibid.).
  • the co-transformation is done using a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25 ⁇ Fa.
  • the entire co-transformation mixture is plated on 4 LB plates containing 25 ⁇ g/ml kanamycin. The smallest colonies are picked and expanded in LB/kanamycin, and recombinant adenovirus DNA is identified by standard DNA miniprep procedures.
  • the recombinant adenovirus miniprep DNA is transformed into E. coli DH10BTM competent cells, and DNA is prepared using a Maxi Kit (Qiagen, Inc.) aaccording to kit instructions.
  • recombinant adenoviral DNA is digested with Pad enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30U of Pad.
  • the digested DNA is extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol.
  • the DNA pellet is resuspended in lO ⁇ l distilled water.
  • a T25 flask of QBI-293A cells Quantantum Biotechnologies, Inc. Montreal, Qc. Canada, inoculated the day before and grown to 60-70% confluence, is transfected with the Pad digested DNA.
  • the Pa -digested DNA is diluted up to a total volume of 50 ⁇ l with sterile HBS (150mM NaCl, 20mM HEPES).
  • HBS 150mM NaCl, 20mM HEPES
  • 20 ⁇ l of lmg/ml N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP) (Boehringer Mannheim, Indianapolis, IN) is diluted to a total volume of 100 ⁇ l with HBS.
  • the DNA is added to the DOTAP, mixed gently by pipeting up and down, and left at room temperature for 15 minutes.
  • the media is removed from the 293 A cells and washed with 5 ml serum-free minimum essential medium (MEM) alpha containing ImM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and
  • MEM serum-free minimum essential medium
  • the cells Seven days after transfection of 293A cells with the recombinant adenoviral DNA, the cells express the GFP protein and start to form foci (viral "plaques"). The crude viral lysate is collected using a cell scraper to collect all of the
  • the lysate is transfened to a 50-ml conical tube.
  • the crude lysate is amplified (Primary (1°) amplification) to obtain a working "stock" of zalpha30 rAdV lysate.
  • Ten 10cm plates of nearly confluent (80- 90%)) 293 A cells are set up 20 hours previously, 200 ml of crude rAdV lysate is added to each 10-cm plate, and the cells are monitored for 48 to 72 hours for CPE (cytopathic effect) under the white light microscope and expression of GFP under the fluorescent microscope.
  • CPE cytopathic effect
  • Twenty 15-cm tissue culture dishes of 293 A cells are prepared so that the cells are 80- 90%) confluent. All but 20 ml of 5% MEM media is removed, and each dish is inoculated with 300-500 ml of the 1° amplified rAdv lysate. After 48 hours the 293A cells are lysed from virus production, the lysate is collected into 250-ml polypropylene centrifuge bottles, and the rAdV is purified.
  • NP-40 detergent is added to a final concentration of 0.5% to the bottles of crude lysate in order to lyse all cells.
  • Bottles are placed on a rotating platform for 10 minutes agitating as fast as possible without the bottles falling over.
  • the debris is pelleted by centrifugation at 20,000 X G for 15 minutes.
  • the supernatant is transfened to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 M NaCl solution is added.
  • the bottles are shaken overnight on ice.
  • the bottles are centrifuged at 20,000 X G for 15 minutes, and the supernatant is discarded into a bleach solution.
  • the white, virus/PEG precipitate from 2 bottles is resuspended in 2.5 ml PBS.
  • the resulting virus solution is placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G in the microcentrifuge for 10 minutes to remove any additional cell debris.
  • the supernatant from the 2-ml microcentrifuge tubes is transfened into a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCl.
  • the solution is transferred to 3.2-ml, polycarbonate, thick-walled centrifuge tubes and spun at 348,000 X G for 3-4 hours at 25°C.
  • the virus forms a white band.
  • the virus band is collected.
  • a commercially available ion-exchange columns e.g., PD-10 columns prepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway, NJ
  • the column is equilibrated with 20 ml of PBS.
  • the virus is loaded and allowed to run into the column.
  • 5 ml of PBS is added to the column, and fractions of 8-10 drops are collected.
  • the optical densities of 1 :50 dilutions of each fraction are determined at 260 nm on a spectrophotometer. Peak fractions are pooled, and the optical density (OD) of a 1 :25 dilution is determined. OD is converted to virus
  • glycerol is added to the purified virus to a final concentration of 15%, mixed gently but effectively, and stored in aliquots at -80°C.

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Abstract

L'invention concerne des polypeptides secrétés, des matériels et des méthodes permettant leur production ainsi que leur méthode d'utilisation. Les polypeptides contiennent au moins neuf restes d'acides aminés contigus de SEQ ID NO:2, SEQ ID NO:10 ou SEQ ID NO:12, et ils peuvent être préparés sous la forme de fusions polypeptidiques contenant des séquences hétérologues, telles que des étiquettes d'affinité. Les polypeptides et les polynucléotides les codant peuvent être utilisés dans diverses applications thérapeutiques, diagnostiques et de recherche.
PCT/US2000/024326 1999-09-07 2000-09-05 Polypeptide secrete zalpha30 WO2001018205A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046757A2 (fr) * 1997-04-15 1998-10-22 Genetics Institute, Inc. Proteines secretees et polynucleotides codant ces proteines
WO1999029720A2 (fr) * 1997-12-10 1999-06-17 Zymogenetics, Inc. Proteine 1 mammalienne a helice alpha

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046757A2 (fr) * 1997-04-15 1998-10-22 Genetics Institute, Inc. Proteines secretees et polynucleotides codant ces proteines
WO1999029720A2 (fr) * 1997-12-10 1999-06-17 Zymogenetics, Inc. Proteine 1 mammalienne a helice alpha

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL 4 July 1999 (1999-07-04), ROBERT STRAUSBERG: "oq52h10.y5 NCI_CGAP_Kid5 Homo sapiens cDNA clone", XP002153546 *

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