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WO1998041627A1 - Polypeptides secretes presentant une homologie avec les proteines de la glande cementaire du xenopus - Google Patents

Polypeptides secretes presentant une homologie avec les proteines de la glande cementaire du xenopus Download PDF

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Publication number
WO1998041627A1
WO1998041627A1 PCT/US1998/005251 US9805251W WO9841627A1 WO 1998041627 A1 WO1998041627 A1 WO 1998041627A1 US 9805251 W US9805251 W US 9805251W WO 9841627 A1 WO9841627 A1 WO 9841627A1
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Prior art keywords
seq
polypeptide
zsiglo
sequence
amino acid
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PCT/US1998/005251
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English (en)
Inventor
Paul O. Sheppard
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Zymogenetics, Inc.
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Priority to AU67622/98A priority Critical patent/AU6762298A/en
Publication of WO1998041627A1 publication Critical patent/WO1998041627A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • Proteins expressed in secretory tissues may be implicated in secretory function or in development or repair of such tissues.
  • Such exposed tissues include the throat, the mouth, the lungs and the like.
  • Other secretory tissues include the prostate, the intestines and the like.
  • Expression of such proteins may serve protective functions for secretory tissue and/or exposed tissue, acting, for example, as an anti-microbial agent or as a mucous- modulating agent, such as a mucous-clearing or a mucous- degrading agent. Inappropriate expression of such proteins involved in secretory function may cause or connote improper mucous composition or secreted amount.
  • proteins or agonists or antagonists thereof are therefore sought to study, detect, prevent and treat secretory tissue disorders and/or exposed tissue maladies. More specifically, moieties which are components of mucous, modulators of mucous secretion or mucous degradation factors are sought.
  • anti-microbial protective agents may be directly acting or indirectly acting. Such agents, operating via membrane association or pore forming mechanisms of action, directly attach to the offending microbe. Anti-microbial agents can also act via an enzymatic mechanism, breaking down microbial protective substances or the cell wall/membrane thereof. Antimicrobial agents capable of inhibiting microorganism growth are also sought.
  • An example of a microbial - associated condition with mucous involvement in humans is the diminution of the defensive properties of the gastroduodenal mucosa by Helicobacter pylori , potentially resulting in ulcer formation. See, for example, Beligotskii et al . , Klin. Khir. 8_: 3-6, 1994.
  • the cement gland is an ectodermal organ in the head of frog embryos anterior to neural tissue.
  • Two proteins, believed to be secreted by the cement gland, have been discovered and designated XLU82110_1 and XLU76752_1.
  • the sequence of XLU82110_1 was published by direct submission without accompanying data.
  • the amphibian cement gland appears to be involved in anterior/posterior axis formation and may play other roles in amphibian embryogenesis, such as in neural development.
  • Otte et al . Nature 334 : 618- 20, 1988, have shown a correlation between neural induction and protein kinase C activation.
  • the cement gland is a mucous- secreting organ, which attaches the embryo to a solid support before swimming and feeding begin and provides sensory signals to the embryo to stop moving once such attachment is made. In this manner, the embryo ceases to move, thereby drawing less attention from potential predators. Before feeding begins, the cement gland is undergoes apoptosis. Proteins secreted by the cement gland may also be involved in preparing the substrate for attachment and/or protecting the embryo from microbial attack.
  • proteins secreted by the cement gland may have anti-microbial activity and/or be involved in adhesion, differentiation or neural development.
  • Mammalian homologs of such proteins may be useful for anti-microbial applications and/or mucous-modulating functions.
  • homologs or antagonists or agonists thereof are expected to be useful in circumstances where enhancement (homolog or agonist) or inhibition (antagonist) of adhesion is desired. For example, inhibition of microbial pathogen-cell adhesion and pathological tissue adhesions is desired.
  • the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 21-175 of SEQ ID NO : 2. Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 21-175 of SEQ ID NO : 2. Within another embodiment the polypeptide further comprises a cysteine residue corresponding to amino acid residue 81 of SEQ ID NO: 2. Within another embodiment the polypeptide further comprises a copper binding site corresponding to amino acid residues 74-78 of SEQ ID N0:2. Within another embodiment the polypeptide comprises residues 26-175 of SEQ ID NO: 2. Within another embodiment the polypeptide comprises residues 21-175 of SEQ ID NO:2.
  • polypeptide comprises residues 1-175 of SEQ ID NO : 2.
  • polypeptide is at least 1 kb in length.
  • polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides , enzymes and fluorophores .
  • moiety is an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region.
  • the invention provides a DNA construct encoding a polypeptide fusion, said fusion comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-20 of SEQ ID NO : 2 , wherein said secretory signal sequence is operably linked to an additional polypeptide.
  • the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcriptional terminator.
  • the DNA segment further encodes a secretory signal sequence operably linked to said polypeptide.
  • the DNA segment encodes the secretory signal sequence having the amino acid sequence of residues 1-20 of SEQ ID NO: 2.
  • a cultured cell into which has been introduced the expression vector described above, wherein the cell expresses the polypeptide encoded by the DNA segment.
  • the invention provides a method of producing a protein comprising: culturing a cell into which has been introduced an expression vector as described above whereby the cell expresses the protein encoded by the DNA segment; and recovering the expressed protein.
  • a pharmaceutical composition comprising a polypeptide as described above in combination with a pharmaceutically acceptable vehicle.
  • An antibody that specifically binds to an epitope of a polypeptide as described above.
  • a binding protein that specifically binds to an epitope of a polypeptide as described above.
  • the invention provides an isolated polynucleotide encoding a polypeptide as described above.
  • the polynucleotide is selected from the group consisting of, a) a sequence of nucleotides from nucleotide 138 to nucleotide 587 of SEQ ID NO:l; b) a sequence of nucleotides from nucleotide 123 to nucleotide 587 of SEQ ID NO : 2 ; c) a sequence of nucleotides from nucleotide 63 to nucleotide 587 of SEQ ID NO : 2 ; d) allelic variants of a) , b) , or c) ; and e) nucleotide sequences complementary to a) , b) , c) or d) .
  • polynucleotide is from 742 to 881 nucleotides in length.
  • an isolated polynucleotide comprising nucleotide 1 to nucleotide 525 of SEQ ID NO: 14.
  • the polynucleotide is DNA.
  • an oligonucleotide probe or primer comprising 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 14 or a sequence complementary to SEQ ID NO: 14.
  • the invention provides method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient .
  • the invention provides a method for detecting zsiglO polypeptides comprising: exposing a polypeptide containing sample to an antibody attached to a solid support, wherein said antibody binds to an epitope of a zsiglO polypeptide; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody- polypeptide to a second antibody directed to a second epitope of a zsiglO polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Figure 1 schematically depicts the zsiglO polypeptide structure, with "M” indicating the initial methionine residue; “Signal” indicating a secretory peptide through amino acid residue 20; and the solid bar encompassing amino acid residue 21 to residue 175 indicating a polypeptide homologous to a Xenopus secreted protein, with “ ⁇ ” and “ ⁇ ” indicating three alpha helical and five beta sheet structural domains, “ ⁇ Cu” indicating a putative copper binding site at amino acid residues 74-78 and "C” indicating a free cysteine residue at position 81
  • Figure 2 illustrates a multiple alignment of two Xenopus laevis secreted proteins (XLU821 (SEQ ID NO : 3 ) , which corresponds to an abbreviation of XLU82110_1 in published literature and XLU767 (SEQ ID NO:4), which corresponds to XLU76752_1) and a zsiglO polypeptide (SEQ ID NO: 2) of the present invention.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A
  • allelic variant denotes 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 and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 9 M "1 .
  • complements of polynucleotide molecules denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3 ' .
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5 ' - ATGGCTTAGCTT-3 ' are 5 ' -TAGCTTgagtct-3 ' and 3'- gtcgacTACCGA-5 ' .
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
  • expression vector denotes 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 may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide molecule, 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).
  • the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the protein in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure.
  • DNA segments denotes that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • the term "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.
  • polynucleotide denotes 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 vi tro, or prepared from a combination of natural and synthetic molecules.
  • bp nucleotide
  • nt nucleotides
  • kb polynucleotides that are single-stranded or double-stranded.
  • base pairs base pairs
  • 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.
  • a "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 denotes a portion of a gene containing DNA sequences that provide for the binding of
  • RNA polymerase and initiation of transcription.
  • Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • Membrane-bound receptors are characterized by a multi- domain structure comprising an extracellular ligand- binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
  • receptors also exhibit a multi-domain structure, including an amino-terminal , transactivating domain, a DNA binding domain and a ligand binding domain.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor) .
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence.
  • splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention is based in part upon the discovery of a novel DNA sequence that encodes a polypeptide having homology to two secreted proteins found in Xenopus laevis (SEQ ID NO: 3; XLU82110_1, with Met is at position 1 and SEQ ID NO: 4; XLU76752_1, with Met also at position 1) .
  • the protein of the present invention appears to be a soluble protein formed of alpha helical and beta sheet structures (designated " ⁇ " and " ⁇ " in Fig. 1) .
  • the zsiglO polypeptide is therefore characterized by a mixed alpha helix-beta sheet structure.
  • the zsiglO polypeptide of the present invention has a free cysteine residue at position 81 of SEQ ID NO: 2 (designated “C” in Fig. 1) and a putative copper binding site at amino acids 74-78 of SEQ ID NO: 2 (designated “ ⁇ Cu” in Fig. 1) .
  • This copper binding motif most closely matches the cytochrome C oxidase subunit I copper B binding site.
  • the presence of a free cysteine may indicate that the zsiglO polypeptide forms homodimers or heterodimers via disulfide bond formation. More specifically, the cysteine residue may constitute part of an intra-chain disulfide in active form.
  • ZsiglO polypeptide homodimers and zsiglO polypeptide-containing proteinaceous heterodimers are also contemplated by the present invention.
  • the free cysteine may be an essential moiety necessary for catalytic activity.
  • the zsiglO polypeptides of the present invention also preferably incorporate six potential protein kinase C phosphorylation sites, at amino acids 24, 68, 114, 136, 142 and 146 of SEQ ID NO: 2. Such putative sites of phosphorylation may indicate that the zsiglO polypeptides of the present invention are involved in neural induction, since a correlation between protein kinase C activity and induction has been noted. See Otte et al . referenced above.
  • the zsiglO polypeptides of the present invention preferably incorporate one potential casein kinase II phosphorylation site at amino acid 57 of SEQ ID NO: 2.
  • Such a phosphorylation site may impact zsiglO polypeptide in vivo half-life or localization, protein-protein interaction or function.
  • Analysis of the tissue distribution of the mRNA corresponding to this novel DNA by both Northern blot and Dot blot showed that expression was highest in lung, prostate, small intestine, colon, trachea and stomach, followed by apparent but decreased expression levels in uterus, pancreas and kidney.
  • Two transcript sizes were observed, one at approximately 1 kb and one at approximately 2 kb.
  • the 1 kb message was detected in much higher abundance than the 2 kb message, with the 1 kb message expressed at least about 50 times higher in most tissues except trachea where the expression appeared to be approximately 25 times higher.
  • the polynucleotide sequence in SEQ ID NO : 1 appears to correspond to the 1 kb message.
  • the polypeptide encoded by that polynucleotide sequence has been designated zsiglO.
  • novel zsiglO polypeptide-encoding polynucleotides of the present invention were initially identified by querying an EST database for secretory signal sequences characterized by an upstream methionine start site, a hydrophobic region of approximately 13 amino acids and a cleavage site (SEQ ID NO: 5, wherein cleavage occurs between the alanine and arginine amino acid residues) in an effort to select for secreted proteins.
  • Polypeptides corresponding to ESTs meeting those search criteria were compared to known sequences to identify secreted proteins having homology to known ligands.
  • a single EST sequence was discovered and predicted to be a secreted protein. Full length sequencing thereof allowed discovery of a homolog relationship to two secreted proteins found in Xenopus laevis (XLU82110_1 and
  • the full sequence of the zsiglO polypeptide was obtained from a single clone believed to contain it, wherein the clone was obtained from a small intestine tissue library.
  • Other libraries that might also be searched for such clones include colon, ovary, prostate, stomach, fetal liver and/or spleen, small intestine, trachea, lung, fetal lung and the like.
  • the full length nucleotide sequence encoding zsiglO polypeptide is described in SEQ ID N0:1, and its deduced amino acid sequence is described in SEQ ID NO: 2.
  • aligned positions 83-87 (amino acids 74-78 of the polypeptide of SEQ ID NO: 2) showed one conservative amino acid substitution (valine for isoleucine) at position 84 (74 in SEQ ID NO:2) .
  • the aligned polypeptides appear to share a putative copper binding site.
  • Fig. 2 shows that the aligned proteins share a free cysteine at aligned position 90 (position 81 of SEQ ID NO: 2) .
  • XLU82110_1 (SEQ ID NO: 3) and XLU76752_1 (SEQ ID NO : 4 ) appear to share the mixed alpha helix/beta sheet structure characteristic of zsiglO polypeptides.
  • SEQ ID NO:l Analysis of the DNA encoding a zsiglO polypeptide (SEQ ID NO:l) revealed an open reading frame encoding 175 amino acids (SEQ ID NO: 2) comprising a signal peptide of 20 amino acid residues (residue 1 to residue 20 of SEQ ID NO: 2) and a mature polypeptide of 155 amino acids (residue 21 to residue 175 of SEQ ID NO: 2) .
  • SEQ ID NO: 2 175 amino acids
  • the present invention also includes the polypeptides having amino acid sequences comprising amino acid residues 17-175 of SEQ ID NO : 2 , residues 18-175 of SEQ ID NO : 2 , residues 19-175 of SEQ ID NO:2, residues 20-175 of SEQ ID NO:2, residues 21-175 of SEQ ID NO:2, residues 22-175, residues 23-175 of SEQ ID NO: 2 and residues 24-175 of SEQ ID NO: 2 as well as the polynucleotides encoding them.
  • the C-terminal tail of the zsiglO polypeptide appears to be longer than that of XLU82110_1 (SEQ ID NO : 3 ) and about the same length as that of XLU76752_1 (SEQ ID NO : 4 ) . Also, the C-terminal tail region of aligned proteins zsiglO (SEQ ID NO: 2) and XLU76752_1 (SEQ ID NO : 4 ) , but not XLU82110_1 (SEQ ID NO:3), is highly positively charged, potentially indicating alternative regulation or specificity.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Amino acids 50-55 of SEQ ID NO: 2 (corresponding to nucleotides 210-227 of SEQ ID NO:l, nucleotides 148-165 of SEQ ID NO:14 and their complements) ;
  • Amino acids 43-48 of SEQ ID NO: 2 (corresponding to nucleotides 189-206 of SEQ ID NO:l, nucleotides 127-144 of SEQ ID NO:14 and their complements) .
  • the activity of polypeptides identified by such probes or of polypeptides encoded by polynucleotides identified by such probes can be determined by methods that are known in the art as generally described herein.
  • Oligonucleotide probes based on the polynucleotide sequence of SEQ ID NO : 1 can be used to localize the zsiglO gene to a particular chromosome.
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al . , Science 250 :245-50, 1990) . Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
  • Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) . These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs) , and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers .
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have .
  • Sequence tagged sites can also be used independently for chromosomal localization.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
  • An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example,
  • SEQ ID NO: 14 is a degenerate DNA sequence that encompasses all DNAs that encode the zsiglO polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 14 also provides all RNA sequences encoding SEQ ID NO : 2 by substituting U for T.
  • zsiglO polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 525 of SEQ ID NO: 14 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO : 14 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide (s) .
  • the code Y denotes either C or T
  • its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO: 14, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • any X NNN 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)
  • the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) .
  • some 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. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO : 14 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • zsiglO polypeptides may be involved in differentiation of neural tissue, such as in anterior/posterior axis formation.
  • zsiglO polypeptides of the present invention bear six putative protein kinase C phosphorylation sites. Otte et al., Nature 334 :618-20, 1988, have shown a correlation between neural induction and protein kinase C activation. Consequently, zsiglO polypeptides may be useful to evaluate the potential of mammalian neural tissue to grow, develop or differentiate.
  • zsiglO polypeptides may be involved in adhesion or other mucous-mediated functions. More specifically, zsiglO polypeptides may constitute a component of mucous or may be a factor influencing mucous production, mucous composition or mucous integrity. ZsiglO polypeptides may also serve a mucous-clearing function in conditions associated with pathological mucous deposition.
  • zsiglO polypeptides or antagonists or agonists thereof are expected be useful in circumstances where modulation of adhesion is desired.
  • adhesion-modulating function may be used in in vi tro experiments designed to study adhesion, such as inhibition of " adhesion of microorganisms to cells, tissue or mucous.
  • Enhancers and inhibitors of adhesion also have potential as therapeutics for conditions requiring such enhancement or inhibition. For example, enhanced tumor cell-tumor cell adhesion in a primary solid tumor does not favor metastasis thereof. Also, diminished tumor cell- endothelial cell adhesion also does not favor metastasis formation at a site distant from the primary tumor.
  • Adhesion may also be evaluated in assays assessing mucous samples for known indicia of adhesion, such as bacterial colonization, susceptibility to and persistence of infection and the like.
  • zsiglO polypeptides or agonists or antagonists thereof are expected to be useful in the modulation of mucous production, composition or integrity or in a mucous clearing role.
  • modulation may be useful in altering mucous composition or integrity for in vi tro study thereof, such as reducing integrity of mucous to evaluate the implication thereof on bacterial-mucous interaction.
  • modulation may be useful in the treatment of disease states characterized by inappropriate mucous production, composition or integrity. For example, cystic fibrosis is associated with dehydration of the mucous, which results in mucous thickening (reduction in viscosity).
  • chronic obstructive pulmonary disease asthma, and the like
  • chronic mucous hypersecretion See, for example, Prescott et al . , Uqeskr Laeqer 158 (45) : 6456-60, 1996; Gordon, Ear Nose Throat J. 75 (2) : 97-101, 1996; and Jeffery, Am. J. Respir. Crit . Care Med. 150 (5 Pt 2) : S6-13, 1994.
  • chronic obstructive pulmonary disease and sinonasal inflammatory disease are associated with changes in rhealogical properties or thickening of mucous. See, for example, Agliati, J. Int. Med. Res.
  • mucous structural integrity is adversely impacted in inflammatory bowel disease, possibly via increased proteolysis. See, for example, Playford et al . , Amer. J. Pathol. 146 (2) : 310-6, 1995. Certain forms of chronic obstructive pulmonary disease are associated with increased acidic mucous. See, for example, the Jeffery article cited above. Mucous clearing may be useful in a number of these conditions as well.
  • zsiglO polypeptides such as zsiglO polypeptides, agonists or antagonists are evaluated for mucosal integrity maintenance activity according to procedures known in the art. See, for example, Zahm et al . , Eur. Respir. J. 8 . : 381-6, 1995, which describes methods for measuring viscoelastic properties and surface properties of mucous as well as for evaluating mucous transport by cough and by ciliary activity. If desired, zsiglO polypeptide performance in this regard can be compared to mucins or the like.
  • Other assays for evaluating the properties of mucous are known to those of ordinary skill in the art. Such assays include those for determining mucin content, water content, carbohydrate content, intrinsic buffering capacity, acidity, barrier properties, ability to absorb water and the like.
  • zsiglO polypeptides in the serum, mucous or tissue biopsy of a patient undergoing evaluation for or disorders characterized by inappropriate mucous deposition, composition or properties, such as cystic fibrosis, asthma, bronchitis, inflammatory bowel disease, Crohn's disease, chronic obstructive pulmonary disease or the like, can be employed in a diagnostic application of the present invention.
  • Such zsiglO polypeptides can be detected using immunoassay techniques and antibodies capable of recognizing a zsiglO polypeptide epitope.
  • the present invention contemplates methods for detecting zsiglO polypeptide comprising: exposing a sample possibly containing zsiglO polypeptide to an antibody attached to a solid support, wherein said antibody binds to an epitope of a zsiglO polypeptide; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsiglO polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label. Elevated concentrations of zsiglO polypeptide (in comparison to normal concentrations thereof) in the test sample appears to be indicative of dysfunction.
  • compositions containing such mucosa-modulating agents may be employed in the treatment of disorders associated with alterations in mucosal production, composition or integrity, such as those described above.
  • Such patients will be given an effective amount of zsiglO polypeptide or agonist or antagonist thereof having mucosal-modulating activity to achieve a therapeutic benefit, generally manifested in a change in mucosal production, composition or integrity in the direction of the normal physiological state thereof.
  • the zsiglO polypeptides of the present invention are found in high abundance in digestive tissues, such as stomach, small intestine and colon. Thus, expression of zsiglO polypeptides may serve as a marker for digestive function or to promote digestive organ proliferation or differentiation. Also, zsiglO polypeptides or agonists or antagonists thereof may be useful in modulating the lubrication or barrier properties of digestive organ mucosa. ZsiglO polypeptides of the present invention or agonists or antagonists thereof may be used as antimicrobial agents to protect against pathological action of microorganisms. Such anti-bacterial agents are preferably active on mucosa-associated microorganisms, such as C. albicans , pneumonus , hemophilus , H. pylori , and the like.
  • anti-microbial protective agents may be directly acting or indirectly acting. Such agents operating via membrane association or pore forming mechanisms of action directly attach to the offending microbe. Anti-microbial agents can also act via an enzymatic mechanism, breaking down microbial protective substances or the cell wall/membrane thereof. Antimicrobial agents, capable of inhibiting microorganism proliferation or action or of disrupting microorganism integrity by either mechanism set forth above, are useful in methods for preventing contamination in cell culture by microbes susceptible to that anti-microbial activity. Such techniques involve culturing cells in the presence of an effective amount of said zsiglO polypeptide or an agonist or antagonist thereof .
  • zsiglO polypeptides or agonist or antagonists thereof as anti-microbial agents are known in the art .
  • detection of zsiglO polypeptides in the serum, mucous or tissue biopsy of a patient undergoing evaluation for microbial disorders, particularly those associated with mucosa can be employed in a diagnostic application of the present invention.
  • Such zsiglO polypeptides can be detected using immunoassay techniques and antibodies capable of recognizing a zsiglO polypeptide epitope.
  • the present invention contemplates methods for detecting zsiglO polypeptide comprising : exposing a sample possibly containing zsiglO polypeptide to an antibody attached to a solid support, wherein said antibody binds to an epitope of a zsiglO polypeptide; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsiglO polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Depressed concentrations of zsiglO polypeptide (in comparison to normal concentrations thereof) in the test sample appears to be indicative of dysfunction.
  • compositions containing such anti-microbial agents may be employed in the treatment of microbial disorders, particularly those associated with mucosa.
  • Such patients will be given an effective amount of zsiglO polypeptide or agonist or antagonist thereof having anti-microbial activity to achieve a therapeutic benefit, generally manifested in a decrease in proliferation or function of the pathogenic microbe.
  • Other conditions which may be addressed in accordance with the present invention are eye, nasal, oral and rectal conditions involving the mucosa and/or pathological microbial agents, chemotherapy side effects impacting the mucosa, AIDS complications relating to mucosa or the like.
  • the anti-microbial activity of zsiglO polypeptides, agonists or antagonists may be determined using known assays therefore.
  • zsiglO polypeptides of the present invention may also constitute a component of a known tissue glue, imparting additional adhesive and/or antimicrobial properties thereto.
  • purified zsiglO polypeptide would be used in combination with collagen or a form of gelatin, muscle adhesion protein, fibrinogen, thrombin, Factor XIII or the like.
  • tissue glues as well as the composition thereof are known in the art.
  • the present invention provides methods for identifying agonists or antagonists of the zsiglO polypeptides disclosed above, which agonists or antagonists may have valuable therapeutic properties as discussed further herein.
  • a method of identifying zsiglO polypeptide agonists comprising providing cells responsive to a zsiglO polypeptide as disclosed above, culturing the cells in the presence of a test compound and comparing the cellular response with the cell cultured in the presence of the zsiglO polypeptide, and selecting the test compounds for which the cellular response is of the same type. Agonists are therefore useful to mimic or augment the function of zsiglO polypeptides.
  • a method of identifying antagonists of zsiglO polypeptide comprising providing cells responsive to a zsiglO polypeptide, culturing a first portion of the cells in the presence of zsiglO polypeptide, culturing a second portion of the cells in the presence of the zsiglO polypeptide and a test compound, and detecting a decrease in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • Antagonists are therefore useful to inhibit or diminish zsiglO polypeptide function.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, SEQ ID NO: 6 (an oligonucleotide primer designated ZC11668) , SEQ ID NO: 7 (an oligonucleotide primer designated ZC12253) , SEQ ID NO: 8 (an oligonucleotide primer designated ZC12241) , other probes described herein, 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.
  • the 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 less than about 0.02 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from fetal liver or spleen, colon, ovary, prostate, stomach, small intestine, lung, fetal lung or trachea, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al . , Biochemistry JL8:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc .
  • cDNA is prepared from poly (A) + RNA using known methods.
  • Polynucleotides encoding zsiglO polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs) .
  • species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species .
  • zsiglO polypeptides from other mammalian species, including murine, rat, porcine, ovine, bovine, canine, feline, equine and other primate proteins.
  • Orthologs of the human proteins 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 the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue of cell line. A zsiglO- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences . A cDNA can also be cloned using the polymerase chain reaction, or PCR
  • 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 zsiglO polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequences disclosed in SEQ ID NO:l and SEQ ID NO: 2 represent a single allele of the human zsiglO gene and polypeptide, and that allelic variation and alternative splicing are expected to occur.
  • cDNAs generated from alternatively spliced mRNAs which retain the properties of the zsiglO polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Northern blot analysis revealed 1 kb and 2 kb mRNAs, wherein the 1 kb variant was more highly expressed (approximately 50 times higher in most tissues and approximately 25 times higher in trachea) .
  • Such mRNA species are likely to be splice variants.
  • allelic variants and splice variants can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures.
  • the present invention also provides isolated zsiglO polypeptides that are substantially homologous to the polypeptides of SEQ ID NO : 2 and their species homologs/orthologs .
  • the term "substantially homologous” is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO : 2 or their orthologs .
  • Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2 or its orthologs.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48 : 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci . USA 8_9:10915-9, 1992. 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 "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes) . The percent identity is then calculated as:
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), or the like. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Aromatic phenylalanine tryptophan tyrosine
  • the present invention further provides a variety of other polypeptide fusions [and related multimeric proteins comprising one or more polypeptide fusions] .
  • a zsiglO polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin- zsiglO polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric zsiglO analogs] .
  • Auxiliary domains can be fused to zsiglO polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen) .
  • a zsiglO polypeptide or protein could be targeted to a predetermined cell type by fusing a zsiglO polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell.
  • a zsiglO 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 . , Con . Tiss. Res. 3_4:l-9 1996.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, trans-4 -hydroxyproline, N-methyl - glycine, allo-threonine, methylthreonine, hydroxyethyl- cysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3- dimethylproline, tert-leucine, norvaline, 2-azaphenyl- alanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine .
  • an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tR ⁇ As .
  • Methods for synthesizing amino acids and aminoacylating tR ⁇ A are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J . Am . Chem . Soc .
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) .
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine
  • the non- naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 3_3: 7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification.
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zsiglO amino acid residues.
  • Essential amino acids in the zsiglO polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 : 1081-5, 1989) .
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244 : 1081-5, 1989.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., adhesion modulation, anti-microbial activity or the like) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al . , J. Biol. Chem. 271 :4699-708, 1996.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255 :306-312 , 1992; Smith et al . , J. Mol. Biol. 224 :899-904, 1992; Wlodaver et al . , FEBS Lett. 309 : 59-64 , 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related polypeptides.
  • Patent NO: 5,223,409; Huse , WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene 46 . : 14.5, 1986; Ner et al . , DNA 7:127, 1988).
  • variants of the disclosed zsiglO DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370 :389-91 , 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51. 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed above can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides e.g., capable of modulating adhesion, having anti-microbial activity or the like
  • 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.
  • polypeptides that are substantially homologous to residues 21-175 of SEQ ID NO : 2 or allelic variants thereof and retain the adhesion-modulating, anti-microbial or like properties of the wild-type protein.
  • polypeptides may include additional amino acids, such as affinity tags and the like.
  • polypeptides may also include additional polypeptide segments as generally disclosed herein.
  • polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A
  • a DNA sequence encoding a zsiglO polypeptide of the present invention is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers .
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the zsiglO polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo .
  • the secretory signal sequence is joined to the zsiglO DNA sequence in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al .
  • the secretory signal sequence portion of the zsiglO polypeptide (amino acids 1-20 of SEQ ID NO: 2) may be employed to direct the secretion of an alternative protein by analogous methods.
  • Cultured mammalian cells are also preferred hosts within the present invention.
  • Preferred cultured mammalian cells include the COS-1 (ATCC NO: CRL 1650), COS-7 (ATCC NO: CRL 1651), BHK 570 (ATCC NO: CRL 10314), 293 (ATCC NO: CRL 1573; Graham et al . , J . Gen . Virol. 36 . : 59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC NO: CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus.
  • Suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as " transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants . " A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin.
  • Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4 , CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells.
  • the use of Agroba cterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci . (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent NO: 5,162,222; Bang et al . , U.S. Patent NO: 4,775,624; and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) .
  • DNA encoding the zsiglO polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the zsiglO flanked by AcNPV sequences.
  • Suitable insect cells e.g.
  • SF9 cells are infected with wild-type AcNPV and transfected with a transfer vector comprising a zsiglO polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • a transfer vector comprising a zsiglO polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow et al . , J. Virol. 62:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsiglO polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zsiglO.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 21:971-6, 1990; Bonning et al . , J. Gen. Virol.
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native zsiglO secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT) , honey bee Melittin (Invitrogen, Carlsbad, CA) , or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zsiglO secretory signal sequence.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N- terminus of the expressed zsiglO polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. USA 8_2:7952-4, 1985).
  • a transfer vector containing zsiglO is transformed into E. coli , and 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, e.g.
  • Recombinant virus that expresses zsiglO 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, S . frugiperda . See, in general, Glick and
  • Another suitable cell line is the High FiveOTM celi line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOD of 0.1 to 10, more typically near 3.
  • the recombinant virus-infected cells typically produce the recombinant zsiglO polypeptide at 12-72 hours post- infection and secrete it with varying efficiency into the medium.
  • the culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant) .
  • the supernatant containing the zsiglO polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size.
  • Fungal cells including yeast cells, and particularly cells of the genus Saccharomyces , can also be used within the present invention, such as for producing zsiglO fragments or polypeptide fusions.
  • Methods for transforming yeast 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:
  • 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 yeast is the P0T1 vector system disclosed by Kawasaki et al . (U.S. Patent NO: 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent NO: 4,599,311; Kingsman et al . , U.S.
  • Patent NO: 4,615,974; and Bitter U.S. Patent NO: 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosa ccharomyces pombe , Kluyveromyces lactis , Kluyveromyces fragilis , Ustilago maydis , Pichia pastoris , P. methanolica , P. guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol .
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P . methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P . methanolica alcohol utilization gene
  • ⁇ AUG1 or AUG2 ⁇ AUG1 or AUG2
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS) , formate dehydrogenase (FMD) , and catalase (CAT) genes.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in Pichia methanolica is a P .
  • methanolica ADE2 gene which encodes phosphoribosyl- 5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes ⁇ PEP4 and PRB1) are preferred.
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P . methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant ( ⁇ ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) .
  • the polypeptide When expressing a zsiglO 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.
  • Expressed recombinant zsiglO polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryvilie, PA), Octyl -Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl-Sepharose FF Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryvilie, PA), Octyl -Sepharose (Pharmacia) and the like
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • the polypeptides of the present invention can be isolated by exploitation of their structural properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins or proteins containing a His tag. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 2:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adsorption
  • a fusion of the polypeptide of interest and an affinity tag e.g., FLAG, Glu-Glu, polyhistidine, maltose-binding protein, an immunoglobulin domain
  • an affinity tag e.g., FLAG, Glu-Glu, polyhistidine, maltose-binding protein, an immunoglobulin domain
  • a member of a complement/anti-complement pair may be constructed to facilitate purification.
  • ZsiglO fused to an N- or C- terminal FLAG tag or Glu-Glu tag can be purified by virtue of the affinity tags discussed in more detail in the examples below.
  • Such purification methods allow for purification of proteins where the structural properties are not known or are not amenable to exploitation for purification.
  • Protein refolding (and optionally reoxidation) procedures may be advantageously used. It is preferred to purify the protein to >80% purity, more preferably to >90% purity, even more preferably >95%, and particularly preferred is 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. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • ZsiglO polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZsiglO polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • a zsiglO-binding polypeptide can also be used for purification of the zsiglO polypeptide of the present invention.
  • the zsiglO-binding polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing zsiglO polypeptide are passed through the column one or more times to allow zsiglO polypeptide to bind to the receptor polypeptide.
  • the bound zsiglO polypeptide is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1) , or pH to disrupt ligand-receptor binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement/ anti-complement pair
  • a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ)
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and in Cunningham and Wells, J. Mol. Biol . 234 : 554-63 , 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • ZsiglO polypeptide and other ligand homologs can also be used within other assay systems known in the art .
  • Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al . , Science 253 : 545-48 , 1991; Cunningham et al . , Science 245 : 821-25, 1991).
  • zsiglO polypeptides may modulate the binding of factors or itself constitute a factor involved in the assembly of extracellular matrix or mucous-type secretions.
  • ZsiglO polypeptides can also be used to prepare antibodies that specifically bind to zsiglO epitopes, peptides or polypeptides.
  • Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al . (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R. , Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs and rats as well as transgenic animals such as transgenic sheep, cows, goats or pigs.
  • Antibodies may also be expressed in yeast and fungi in modified forms as well as in mammalian and insect cells.
  • the zsiglO polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal or elicit an immune response.
  • Suitable antigens would include the zsiglO polypeptide encoded by SEQ ID NO: 2 from amino acid residue 21-175 of SEQ ID NO: 2, or a contiguous 9-175 amino acid residue fragment thereof.
  • the immunogenicity of a zsiglO 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 zsiglO 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.
  • polypeptide portion is "hapten-like"
  • such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen- binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non- human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Human antibodies can also be made in mice having a humanized humoral immune system (Mendez et al., Nat. Genet. 1 ⁇ :146- 56, 1997).
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to zsiglO protein or peptide, and selection of antibody display libraries, in phage or similar vectors (for instance, through use of immobilized or labeled zsiglO protein or peptide) . Mutagenesis methods discussed herein, in particular domain shuffling, can be used to generate and mature antibodies.
  • the antibodies of the current invention can be used to direct molecules to a specific target.
  • T-bodies chimeric receptors combining antibody recognition with T cell effector function, (Eshhar et al . , Springer Semin Immunopathol . 1 ⁇ :199-209, 1996; Eshhar, Cancer Immunol. Imrrtunother . 4_5: 131-6, 1997).
  • Intrabodies engineered single-chain antibodies expressed inside the cell and having high affinity and specificity for intracellular targets.
  • Such molecules have use in gene therapy and treatment of infectious diseases (Marasco, Immunotechnology _1:1-19, 1995; Marasco et al., Gene Ther.
  • Diabodies bispecific non-covalent di ers of scFv antibodies useful for immunodiagnosis and therapeutically. In addition they can be constructed in bacteria (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993) .
  • Antibodies herein specifically bind if they bind to a zsiglO polypeptide, peptide or epitope with a binding affinity (K a ) of 10 M or greater, preferably 10 M or
  • the bmdmg affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-72, 1949).
  • binding proteins can be obtained by screening random or directed peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • constrained phage display libraries can also be produced.
  • These peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances .
  • Peptide display libraries can be screened using the zsiglO sequences disclosed herein to identify proteins which bind to zsiglO.
  • binding proteins which interact with zsiglO polypeptides can be used essentially like an antibody, for tagging cells; for isolating homolog polypeptides by affinity purification; directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity.
  • the binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. To increase the half- life of these binding proteins, they can be conjugated. Their biological properties may be modified by dimerizing or multimerizing for use as agonists or antagonists.
  • assays known to those skilled in the art can be utilized to detect antibodies and/or binding proteins which specifically bind to zsiglO proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zsiglO protein or polypeptide.
  • Antibodies and binding proteins to zsiglO may be used for tagging cells that express zsiglO; for isolating zsiglO by affinity purification; for diagnostic assays for determining circulating levels of zsiglO polypeptides; for detecting or quantitating soluble zsiglO as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zsiglO polypeptide adhesion modulating or anti-microbial or like activity in vitro and in vivo .
  • 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 to zsiglO or fragments thereof may be used in vi tro to detect denatured zsiglO or fragments thereof in assays, for example, Western Blots or other assays known in the art .
  • Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance) .
  • zsiglO polypeptides or anti-zsiglO antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) .
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues) .
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest .
  • the anti- complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti- complementary-detectable/ cytotoxic molecule conjugates.
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially, intraductally with DMSO, intramuscularly, subcutaneously, intraperitoneally, also by transdermal methods, by electro-transfer, orally or via inhalant.
  • Molecules of the present invention can be used to identify and isolate receptors involved in adherence.
  • proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques, Hermanson et al .
  • Proteins and peptides can also be radiolabeled (Methods in Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed. , Acad. Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al . , Ann. Rev. Biochem. £2:483-514, 1993 and Fedan et al . , Biochem. Pharmacol. 22:1167-80, 1984) and specific cell-surface proteins can be identified.
  • the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
  • pharmaceutical formulations will include a zsiglO protein 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.
  • Therapeutic doses will generally be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
  • the proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.
  • Polynucleotides encoding zsiglO polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zsiglO activity. If a mammal has a mutated or absent zsiglO gene, the zsiglO gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zsiglO polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillo avirus, 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 herpes simplex virus 1 (HSV1) vector (Kaplitt et al . , Molec. Cell. Neurosci . 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , J. Clin. Invest. £0 .
  • HSV1 herpes simplex virus 1
  • a zsiglO gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . Cell 22:153, 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.£:1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; WIPO Publication WO 95/07358; and Kuo et al . , Blood £2:845, 1993.
  • the vector can be introduced by lipofection in vivo using liposomes.
  • 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 .
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be 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 purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies
  • non-peptide molecules can be coupled to liposomes chemically.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al . , J. Biol. Chem. 267:963-7, 1992; Wu et al . , J. Biol. Chem. 263 :14621-4, 1988.
  • Antisense methodology can be used to inhibit zsiglO gene transcription, such as to inhibit cell proliferation in vivo .
  • Polynucleotides that are complementary to a segment of a zsiglO -encoding polynucleotide e.g., a polynucleotide as set froth in SEQ ID N0:1 are designed to bind to zsiglO -encoding mRNA and to inhibit translation of such mRNA.
  • Such antisense polynucleotides are used to inhibit expression of zsiglO polypeptide-encoding genes in cell culture or in a subject .
  • mice engineered to express the zsiglO gene, and mice that exhibit a complete absence of zsiglO gene function, referred to as "knockout mice"
  • mice may be employed to study the zsiglO gene and the protein encoded thereby in an in vivo system.
  • the present invention also provides reagents for use in diagnostic applications.
  • the zsiglO gene, a probe comprising zsiglO DNA or RNA, or a subsequence thereof can be used to determine if the zsiglO gene is present on chromosome 7 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the zsiglO gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
  • 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 (iii) 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 polymorphism (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.; Marian,
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • 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.
  • PCR assays a patient's 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
  • novel zsiglO polypeptide-encoding polynucleotides of the present invention were initially identified by querying an EST database for secretory signal sequences characterized by an upstream methionine start site, a hydrophobic region of approximately 13 amino acids and a cleavage site (SEQ ID NO: 5, wherein cleavage occurs between the alanine and arginine amino acid residues) in an effort to select for secreted proteins.
  • Polypeptides corresponding to ESTs meeting those search criteria were compared to known sequences to identify secreted proteins having homology to known ligands.
  • a single EST sequence was discovered and predicted to be related to secreted proteins found in Xenopus laevis .
  • Oligonucleotides ZC976 (SEQ ID NO: 9), ZC694 (SEQ ID NO: 10) and ZC6768 (SEQ ID NO: 11) to the LacZ, T7 and T3 promoters on the clone-containing vector were used as sequencing primers.
  • Oligonucleotides ZC11668 (SEQ ID NO: 6), ZC12253 (SEQ ID NO: 7) and ZC12241 (SEQ ID NO: 8) were used to complete the sequence from the clone.
  • SEQ ID NO: 1 was radioactively labeled with P using T4 polynucleotide kinase and forward reaction buffer (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's specifications.
  • the probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA) .
  • EXPRESSHYB (Clontech, Palo Alto, CA) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 42° C, and the blots were then washed in 2X SSC and 0.05% SDS at RT, followed by a wash in IX SSC and 0.1% SDS at 60°C.
  • the 1 kb message was detected in much higher abundance than the 2 kb message, with the 1 kb message expressed at least about 50 times higher in most tissues except trachea where the expression appeared to be approximately 25 times higher.
  • Signal intensity was highest for lung, prostate, small intestine, colon, trachea and stomach, with relatively less intense signals in uterus, pancreas and kidney.
  • the zsiglO gene was mapped to chromosome 7 using the commercially available version of the Whitehead Institute/MIT Center for Genome Research's GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) .
  • the GeneBridge 4 Radiation Hybrid Panel contained PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) .
  • a publicly available WWW server http://www- genome . wi . mit .
  • edu/cgi-bin/contig/rhmapper .pi allowed for mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR” radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.
  • ADVANTAGE KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x ⁇ l ddH20 for a total volume of 25 ⁇ l .
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at
  • Three expression vectors were prepared for the zsiglO polypeptide, zSIG10CF/pZP9 and zSIG10NF/pZP9 , wherein the constructs are designed to express a zsig25 polypeptide with a C- or N-terminal FLAG tag (SEQ ID NO:
  • a approximately 875 bp restriction digest fragment of ZSIG-10 DNA was derived from the clone described in Example 1 above. Five micrograms of the clone was digested with 1 ⁇ l each of the restriction enzymes Eco Rl and Xho I . The resultant ligation fragment was then run on a 0.8% LMP agarose gel (Seaplaque
  • Plasmid pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, multiple restriction sites for insertion of coding sequences, a stop codon and a human growth hormone terminator.
  • the plasmid also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • a 533 bp PCR generated ZSIG-10 DNA fragment was created using ZC13436 (SEQ ID NO: 36) and ZC13435 (SEQ ID NO: 37) as PCR primers and the template described in Example 1 above.
  • the PCR reaction was incubated at 94°C for 5 minutes, and then run for 10 cycles of 30 seconds at 94°C and 2 minutes at 75°C, followed by 15 cycles at 94°C o for 30 seconds and 62 C for 2 minutes.
  • the resultant PCR product was then run on a 0.9% GTG/TBE agarose gel with lx
  • the zSIG10/CFpZP9 expression vector uses the native zSIGlO signal peptide, and the FLAG epitope
  • Plasmid CF/pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, multiple restriction sites for insertion of coding sequences, a sequence encoding the FLAG tag (SEQ ID NO: 35), a stop codon and a human growth hormone terminator.
  • the plasmid also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • a 474 bp PCR generated zSIGlO/NF DNA fragment was created in accordance with the procedure set forth above using Z13441 (SEQ ID NO: 38) and ZC13442 (SEQ ID NO: 39) as PCR primers.
  • the purified PCR fragment was digested with the restriction enzymes Bam HI (Boehringer Mannheim) and Xho I (Gibco BRL) , followed by phenol/chloroform/isoamyl alcohol extraction and ETOH/glycogen precipitation.
  • the excised and restriction digested zSIGlO DNA was subcloned into plasmid NF/pZP9 which had been cut with Bam HI and Xho I.
  • the zSIG10/NFpZP9 expression vector incorporates the TPA leader and attaches the FLAG tag (SEQ ID NO: 35) to the N-terminal of the zsiglO polypeptide- encoding polynucleotide sequence.
  • Plasmid NF/pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, a TPA leader peptide followed by the sequence encoding the FLAG tag (SEQ ID NO: 35), multiple restriction sites for insertion of coding sequences, and a human growth hormone terminator.
  • the plasmid also contains an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.
  • the untagged zsiglO construct approximately 100 ng of the zsiglO insert and 100 ng of the Eco RI/Not I digested pZP9 vector were ligated as described for the tagged constructs.
  • the N- and C- tagged constructs about 10 ng of the restriction digested inserts and 10 ng of the corresponding vectors were ligated at room temperature for 4 hours.
  • One microliter of each ligation reaction was independently electroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, MD) according to manufacturer's direction and plated onto LB plates containing 50 mg/ml ampicillin, and incubated overnight . Colonies were screened by PCR as described above.
  • the primers were ZC6583 (SEQ ID NO:40) and ZC5020 (SEQ ID NO:41), for zSIG10CF/pZP9 screens the primers were, ZC13435 (SEQ ID NO: 37) and ZC13436 (SEQ ID NO:36) and for zSIG10NF/pZP9 screens the primers were ZC13442 (SEQ ID NO: 39) and
  • ZC13441 (SEQ ID NO: 38) .
  • the insert sequence of positive clones, 950 bp for zsiglO untagged, 474 bp fragment for zSIGlONF and a 533 bp fragment for zSIGlO/CF were verified by sequence analysis.
  • BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissue culture dishes and allowed to grow to approximately 50 to 70% confluency overnight at 37°C, 5% C0 2 , in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, MD) , 5% fetal bovine serum (Hyclone, Logan, UT) , 1 ⁇ M L-glutamine (JRH Biosciences, Lenexa, KS) , 1 ⁇ M sodium pyruvate (Gibco BRL) ) .
  • DMEM Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, MD)
  • 5% fetal bovine serum Hyclone, Logan, UT
  • JRH Biosciences, Lenexa, KS 1 ⁇ M sodium pyruvate
  • the cells were then transfected with the plasmid zsiglONF/pZP9 (N- terminal FLAG tag) , zsiglOCF/pZP9 (C-terminal FLAG tag) , or zsiglO/pZP9 (untagged) , using Lipofectamine (Gibco BRL) , in serum free (SF) media formulation (DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L- glutamine and 1% sodium pyruvate) .
  • DMEM serum free
  • SF medium 605 ⁇ l of SF medium.
  • the Lipofectamine TM mix was added to the DNA mix and allowed to incubate approximately 30 minutes at room temperature.
  • Five milliliters of SF media was added to the DNA: Lipofectamine TM mixture.
  • Three plates of cells were rinsed once with 5 ml of SF media, aspirated, and the DNA: Lipofectamine TM mixture was added. The cells were incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to each plate.
  • the plates were incubated at 37°C overnight and the DNA: ipofectami.neTM mixture was replaced with fresh 5% FBS/DMEM media the next day.
  • the cells were split into the selection media (DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50.
  • the cells were refed at day 5 post- transfection with fresh selection media.
  • two 150 mm culture dishes of methotrexate resistant colonies from each transfection were trypsinized and the cells were pooled and plated into a T-162 flask and transferred to large scale culture.
  • T-162 flask containing confluent cells expressing zsiglO/NF and one containing confluent cells expressing zsiglO-untagged, obtained from the expression procedure described above, were expanded into six T-162 flasks.
  • One of the six resulting flasks was used to freeze down four cryovials, and the other five flasks were used to generate a Nunc cell factory.
  • the cells from the five T-165 flasks were used to seed a Nunc cell factory (10 layers, commercially available from VWR) .
  • ESTEP1 media (668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvic acid, sodium salt 96% (Mallinckrodt) , 185.0 g/50L NaHC0 3 (Mallinkrodt) , 5.0 mg/ml and 25 ml/50L insulin (JRH Biosciences), 10.0 mg/ml and 25 ml/50L transferrin (JRH Biosciences), 2.5L/50L fetal bovine serum (characterized) (Hyclone) , 1 ⁇ M MTX, with pH adjusted to 7.05 +/- 0.05 ) prewarmed to 37°C.
  • ESTEP1 media 668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvic acid, sodium salt 96% (Mallinckrodt) , 185.0 g/50L NaHC0 3 (Mallinkrodt) , 5.0 mg/ml and 25 ml/50L
  • the cells from the T-162 flasks described above containing cells expressing untagged zsiglO were detached using trypsin, pooled, and added to 1.5 liters of SL6V2 media (13.3 g/1 DMEM, 0.11 g/1 Na- pyruvate, 3.7 g/1 NaHC0 3 , 5.96 g/1 HEPES (JRH Biosciences, Lenexa, KS) and 50 ml/1 FBS (Hyclone, Logan, UT) , pH 7.05) .
  • the media containing cells was then poured into Nunc cell factories via a funnel. The cell factories were placed in a 37°C/5.0% C02 incubator.
  • a visual contamination test (phenol red color change) was performed on the Nunc cell factories. Since no contamination was observed, supernatant from the confluent factories was poured into a small harvest container, sampled and discarded. The adherent cells were then washed once with 400 ml PBS. To detach the cells from the factories, 100 mis of trypsin was added to each and removed and the cells were then incubated for 5 to 10 minutes in the residual trypsin. The zsiglONF cells were collected following two, 200 ml washes of ESTEP1 media, the untagged zsiglO cells were collected in ESTEP Form, 5%HIA-FBS/DMEM media.
  • a visual contamination test (phenol red color change) was performed on the Nunc cell factories. Since no contamination was observed, supernatant from the confluent factories were poured into a small harvest container, sampled and discarded. Cells were then washed once with 400 ml PBS. To the factories containing zsiglO-NF cells, 1.5 liters of ESTEP2 media
  • An aseptically assembled filter train apparatus was used for aseptic filtration of the harvest supernatant (conditioned media) . Assembly was a follows: tubing was wire-tied to an Opti- Cap filter (Millipore Corp., Bedford, MA) and a Gelman Supercap 50 filter (Gelman Sciences, Ann Arbor, MI) . The Supercap 50 filter was also attached to a sterile capped container located in a hood; tubing located upstream of the Millipore Opti-cap filter was inserted into a peristaltic pump; and the free end of the tubing was placed in the large harvest container.
  • Opti- Cap filter Millipore Corp., Bedford, MA
  • Gelman Supercap 50 filter Gelman Sciences, Ann Arbor, MI
  • the peristaltic pump was run between 200 and 300 rpm, until all of the conditioned media passed through the 0.22 ⁇ m final filter into a sterile collection container.
  • the filtrate was placed in a 4 °C cold room pending purification.
  • Conditioned media containing zsiglO/NF and untagged zsiglO was collected for concentration at various time points (at the 5 T-162 flask stage; 1 factory, fetal bovine serum media; 10 factories, fetal bovine serum media; 10 factories, serum free media and a second 10 factory, serum free media time point) . Since the expected mass of the protein was in excess of 8 kDA, Millipore 5 kDa cut off concentrators were used.
  • the starting volume for each sample was 15 ml, which was concentrated to a final volume of 1.5 ml .
  • the concentrators were spun at 4°C in Beckman tabletop centrifuge at 2000 x g (3000 rpm) for 40 minutes.
  • the concentrate was transferred to a 1.5 ml non-stick microfuge tube, and the volume was adjusted to 1 ml using flow through media to achieve a lOx concentration.
  • the lOx concentrate was split into two Costar Spin-X tubes, and the tubes were spun at 8000 x g for two minutes in a Eppendorf 5415 microfuge (VWR, Seattle, WA) .
  • Expression of zsiglO in Pichia methanolica utilizes the expression system described in co-assigned WIPO publication WO 97/17450.
  • An expression plasmid containing all or part of a polynucleotide encoding zsiglO is constructed via homologous recombination.
  • An expression vector was built from pCZR204 to express C- terminal Glu-Glu-tagged (CEE) zsiglO polypeptides.
  • the pCZR204 vector contains the AUG1 promoter, followed by the ⁇ Fpp leader sequence, followed by a blunt-ended Sma I restriction site, a carboxy-terminal peptide tag (Glu- Glu) , a translational STOP codon, followed by the AUG1 terminator, the ADE2 selectable marker, and finally the AUG1 3' untranslated region. Also included in this vector are the URA3 and CEN-ARS sequences required for selection and replication in S . cerevisiae, and the AmpR and colEl ori sequences required for selection and replication in E. coli .
  • a second expression vector was built from zCZR191 to express a N-terminal Glu-Glu-tagged (NEE) zsiglO polypeptides.
  • the zCZR191 expression vector is as described above, having an amino terminal Glu-Glu tag.
  • the zsiglO sequence inserted into these vectors begins at residue 21 (Arg) of the zsiglO amino acid sequence (SEQ ID NO : 2 ) .
  • the untagged N- terminal linker spans 70 base pairs of the alpha factor prepro (aFpp) coding sequence on one end and joins it to the 70 base pairs of the amino-terminus coding sequence from the mature zsiglO sequence on the other.
  • the NEE-tagged linker joins Glu-Glu tag (SEQ ID NO: 42) between the aFpp coding sequence and the zsiglO sequence.
  • the untagged C-terminal linker spans about 70 base pairs of carboxy terminus coding sequence of the zsiglO on one end with 70 base pairs of AUG1 terminator sequence.
  • the CEE-tagged linker inserts the Glu-Glu tag (SEQ ID NO: 42) between the C-terminal end of zsiglO and the AUG1 terminator region.
  • NEE-tagged- zsiglO plasmid was made by homologously recombining 100 ng of the Smal digested pCZR190 acceptor vector, 1 ⁇ g of Eco Rl-Xho I zsiglO cDNA donor fragment, 1 ⁇ g NEE-tagged-zsiglO linker (SEQ ID NO: 19) and 1 ⁇ g of C-terminal untagged linker (SEQ ID NO: 24) in S . cerevisiae .
  • the NEE- zsiglO linker was synthesized by a PCR reaction. To a final reaction volume of 100 ⁇ l was added 1 pmol each of linkers, ZC13,731 (SEQ ID NO: 16) and
  • the C-terminal untagged zsiglO linker was made via a PCR reaction as described using oligonucleotides ZC13,734 (SEQ ID NO:23), ZC13,732 (SEQ ID NO:20), ZC13,728
  • a CEE-zsiglO plasmid was made by homologously recombining 100 ng of Sma I digested pCZR204 acceptor vector, the l ⁇ g of Eco Rl-Xho I zsiglO cDNA donor fragment, 1 ⁇ g of N-terminal untagged zsiglO linker (SEQ ID NO:29) and 1 ⁇ g of CEE-tagged linker (SEQ ID NO:34) in a S. cerevisiae .
  • the N-terminal untagged zsiglO linker was made via a PCR reaction as described above using oligonucleotides ZC14,822 (SEQ ID NO:25), ZC14,821 (SEQ ID NO:26), ZC14,832 (SEQ ID NO:27) and ZC14,833 (SEQ ID NO:28).
  • the resulting 147 bp double stranded, N-terminal untagged linker is disclosed in SEQ ID NO: 29.
  • the CEE-tagged linker was made via a PCR reaction as described above using ZC14,834 (SEQ ID NO: 30), ZC15,957 (SEQ ID NO:31), ZC15,632 (SEQ ID NO:32) and ZC
  • yeast/DNA mixtures were electropulsed at 0.75 kV (5 kV/cm) , ⁇ ohms, 25 ⁇ F .
  • To each cuvette was added 600 ⁇ l of 1.2 M sorbitol and the yeast was plated in two 300 ⁇ l aliquots onto two URA D plates and incubated at 30°C.
  • Ura + yeast transformants from a single plate were resuspended in 2.5 ml H 2 O and spun briefly to pellet the yeast cells.
  • the cell pellet was 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 was 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, followed by a 5 minute spin in a Eppendorf centrifuge as maximum speed.
  • Three hundred microliters of the aqueous phase was transferred to a fresh tube and the DNA precipitated with 600 ⁇
  • DH10B Gibco BRL
  • DH10B Gibco BRL
  • SOC 2% BactoTM Tryptone (Difco, Detroit, Ml), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgS0 4 , 20 mM glucose
  • LB AMP plates LB broth (Lennox), 1.8% BactoTM Agar (Difco) , 100 mg/L Ampicillin
  • plasmid DNA was isolated using the Qiagen Maxi kit (Qiagen) according to manufacturer's instruction and the DNA was digested with Not I to liberate the Pichia-ZsiglO expression cassette from the vector backbone.
  • the Not I- restriction digested DNA fragment was then transformed into the Pichia methanolica expression host, PMAD16. This was done by mixing 100 ⁇ l of prepared competent PMAD16 cells with 10 ⁇ g of Not I restriction digested zsiglO and transferred to a 0.2 cm electroporation cuvette.
  • the yeast/DNA mixture was electropulsed at 0.75 kV, 25 ⁇ F, infinite ohms.
  • ADE DS 0.056% -Ade -Trp -Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, 0.5% 200X tryptophan, threonine solution, and 18.22% D-sorbitol
  • the resulting NEE-tagged-zsiglO plasmid containing yeast cells were designated PMAD16 : :pSDH112-5 and the CEE-tagged-zsiglO plasmid containing yeast cells were designated PMAD16 : :pTAP13.
  • the transformed yeast cells were plated on ADE DS plates for selection. Clones were picked and screened via Western blot for high-level ZsiglO expression and subjected to fermentation.
  • GAA ACA ACT GAC AAA CAC CTT TCT CCT GAT GGC CAG TAT GTC CCC AGG 443 Glu Thr Thr Asp Lys His Leu Ser Pro Asp Gly Gin Tyr Val Pro Arg 115 120 125
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Other (vi i) IMMEDIATE SOURCE- (B) CLONE. ZC694

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Abstract

La présente invention concerne un polynucléotide zsig10 et les nouveaux polypeptides zsig10 sécrétés codés par ce polynucléotide. Ces nouveaux polypeptides zsig10 sont supposés présenter une activité anti-microbienne, ainsi que des propriétés de modulation des sécrétions muqueuses et/ou de modulation de l'adhérence d'agents pathogènes, et peuvent donc être utilisées dans une culture cellulaire afin d'évaluer ces propriétés. La présente invention concerne également des anticorps desdits polypeptides zsig10.
PCT/US1998/005251 1997-03-19 1998-03-18 Polypeptides secretes presentant une homologie avec les proteines de la glande cementaire du xenopus WO1998041627A1 (fr)

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
US6171816B1 (en) 1996-08-23 2001-01-09 Human Genome Sciences, Inc. Human XAG-1 polynucleotides and polypeptides
WO2004031239A3 (fr) * 2002-10-02 2004-05-27 Univ Liverpool Composes inducteurs de metastases
WO2004056858A3 (fr) * 2002-12-23 2004-08-12 Ingenium Pharmaceuticals Ag Methodes et agents pour le diagnostic et la prevention, l'amelioration ou le traitement de troubles lies aux cellules caliciformes
EP0929575A4 (fr) * 1996-08-23 2004-09-08 Human Genome Sciences Inc Nouveaux facteurs de croissance humains
WO2006061418A3 (fr) * 2004-12-09 2006-08-03 Ingenium Pharmaceuticals Ag Procedes et agents de traitement d'etats se caracterisant par une hyperproduction/hypersecretion de mucus
JP2007289196A (ja) * 2000-10-02 2007-11-08 Bayer Corp 癌組織でディファレンシャルに発現される核酸配列

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WO1998011217A2 (fr) * 1996-09-13 1998-03-19 Sagami Chemical Research Center Proteines humaines comportant des sequences de signaux secretoires et adn codant ces proteines

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TASHIRO K ET AL: "SIGNAL SEQUENCE TRAP: A CLONING STRATEGY FOR SECRETED PROTEINS AND TYPE I MEMBRANE PROTEINS", SCIENCE, vol. 261, 30 July 1993 (1993-07-30), pages 600 - 603, XP000673204 *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171816B1 (en) 1996-08-23 2001-01-09 Human Genome Sciences, Inc. Human XAG-1 polynucleotides and polypeptides
EP0929575A4 (fr) * 1996-08-23 2004-09-08 Human Genome Sciences Inc Nouveaux facteurs de croissance humains
US6818412B2 (en) 1996-08-23 2004-11-16 Human Genome Sciences, Inc. Human growth factors
US7060801B2 (en) 1996-08-23 2006-06-13 Human Genome Sciences, Inc. Antibodies to human growth factor huXAG-3 and methods of use
US7611846B2 (en) 1996-08-23 2009-11-03 Human Genome Sciences, Inc. Diagnostic methods involving human growth factor huXAG-1
JP2007289196A (ja) * 2000-10-02 2007-11-08 Bayer Corp 癌組織でディファレンシャルに発現される核酸配列
WO2004031239A3 (fr) * 2002-10-02 2004-05-27 Univ Liverpool Composes inducteurs de metastases
WO2004056858A3 (fr) * 2002-12-23 2004-08-12 Ingenium Pharmaceuticals Ag Methodes et agents pour le diagnostic et la prevention, l'amelioration ou le traitement de troubles lies aux cellules caliciformes
WO2006061418A3 (fr) * 2004-12-09 2006-08-03 Ingenium Pharmaceuticals Ag Procedes et agents de traitement d'etats se caracterisant par une hyperproduction/hypersecretion de mucus

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