WO2000029576A1 - Lipocalin homologs - Google Patents

Abstract

The present invention is directed to polynucleotides and polypeptides for a novel member of the lipocalin family. The expression of this novel polynucleotide is primarily restricted to thyroid and thyroid tumor tissue. The polypeptide has been designated zlipo3. The present invention includes methods of producing the polypeptides and antibodies to the ziplo3 polypeptides. The invention also includes use of the polypeptide as anti-microbial and delivery agent.

Description

Description LIPOCALIN HOMOLOGS

BACKGROUND OF THE INVENTION

Lipocalins are small secreted proteins that are believed to be involved in the transport of small, hydrophobic molecules. The lipocalin family is characterized by the structural motif of a barrel formed by eight, anti-parallel, beta-sheets, which are arranged as two orthogonal sheets. The lipocalin family is diverse at the sequence level . The most related members of the family share three characteristic conserved sequence motifs. Members of this group include: retinol -binding protein; purpurin; retinoic acid-binding protein; α_2u-globin; major urinary protein; bilin-binding protein; α-crustacyanin; pregnancy protein 14; β-lactoglobin; neutrophil gelatinase lipocalin, prostaglandin D₂ synthase, and choroid plexus protein. Outlier lipocalins are classified as such because they have 2 or less sequence motifs conserved and these proteins include: odorant-binding protein, von Ebner's gland protein, probasin and aphrodisin.

The lipocalins are members of the superfamily known as calycins, all of which are ligand-binding proteins for hydrophobic molecules . Other members of the calycin family are fatty acid-binding proteins (FABPs) and avidins . The members of this super-family share some conformational homology, with little sequence homology (Flower, FEBS Letters 354:7-11, 1994; and Flower, J. Molec. Recognition 8:185-195, 1995) .

Prostaglandin D₂ synthase is lipocalin family member involved in the synthesis of prostaglandin D₂ in the

^rain by catalyzing prostaglandin H₂ into prostaglandin D₂, the presence of thiol compounds. PD₂ synthase is unusual in that enzymatic activity has not been associated with other lipocalins (Flower, Biochem. J. 318 : 1-14 , 1996) . PD₂ synthase has been identified in choroid plexus, meninges and oligodendrocytes and as a major component of cerbrospinal fluid. It has also been found in genital organs (Tanaka et al . , J. Biol. Chem. 272 : 15789-15795 , 1997) .

Similar to other lipocalins, PD₂ synthase is a carrier for hydrophobic compounds . PD₂ synthase binds retinol in vitro, and has been proposed as a secretory retinoid transporter, that circulate retinoids in a variety of body fluids and transport them to their intracellular transporters. Once inside the cells, the retinoids bind to a dimerized receptor and ultimately play a biological role in the regulation of diverse processes, such as morphogenesis, differentiation, and mitogenesis (Tanaka et al . , 1997, ibid. ) .

Other activities associated with members of the lipocalin family include antimicobial, pheromone transport, olfaction and regulation of immune response. One lipocalin associated with immune modulation is neutrophil gelatinase associated lipocalin (NGAL) . NGAL has been localized to specific granules in neutrophils as both monomers and dimers (Bartsch et al . , FEBS Letters 357:255-259, 1995) . NGAL is typical of lipocalins in that it binds small hydrophobic molecules to transport through hydrophilic fluids. While the physiological ligand for NGAL has not been identified, it has been shown to bind the bacterial chemotaxic factor FMLP, suggesting that the molecule binds lipophilic inflammatory mediators (Bungaard et al., Biochem. Biophys . Res. Comm. 202:1468-1475, 1994).

Discovery of a novel protein, with specific expression patterns, provides a new mediator of the diverse biological processes associated with the lipocalin family. These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an illustration of the secondary protein structure of zlipo3. Within the Figure, "H" designates a loop with helical potential; "B" are !^'- strands; "A" designates an -helix; "!" designates a ligand binding residue based on alignment with the crystal structure; "* *" designates disulfide bond; and "#" free cysteine with potential for dimerization involvement. Figure 2 is an illustration of a multiple alignment of chicken quiesence specific protein (QSP; SEQ ID NO: 8 ) ; human neutrophil gelatinase associated protein (NGAL; SEQ ID NO : 9) ; human prostaglandin D₂ synthase (PD₂ synthase; SEQ ID NO:10), human zlipo3 (SEQ ID NO: 2).. Figure 3 is an illustration of the ''-strands forming loops in most members of the lipocalin family (3A) and zlipo3 (3B) . In zlipo3, strands B-C form loop2, strands C-D form loop3, strands D-E form loop4 , strands E- F form loop5, strands F-I form loop6. " " represents putative disulfide bonds that may form monomeric or dimeric molecules.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:

The term "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. In principal, 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 (Nilsson et al . , EMBO J . 4:1075, 1985; Nilsson et al . , Methods Enzymol . 198 : 3 , 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag™ peptide (Hopp et al . , Biotechnology 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al . , Protein Expression and Purification 2 : 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ) . The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene . The terms "amino-terminal" and "carboxyl- terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl- terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide. The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) 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. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10⁹ M^-1. The term "complements of a polynucleotide molecule" is a polynucleotide molecule 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 ' .

The term "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 ' . The term "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) .

The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators . The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985) .

An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

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. 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.

A "polynucleotide" is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

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".

The term "promoter" is used herein for its art- recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. A "protein" is a macromolecule comprising one or more polypeptide chains . A protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. The term "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- peptide 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 . In general, 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) .

The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene .

Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%. All references cited herein are incorporated by reference in their entirety.

Analysis of a cDNA encoding a new member of the lipocalin family, designated zlipo3, revealed a polynucleotide sequence with an open reading frame from nucleotide 11 to nucleotide 472 of SEQ ID NO: 1, encoding a polypeptide of 153 amino acids (SEQ ID NO: 2) . The polypeptide comprises a putative signal peptide of 19 amino acid residues (residue 1 to residue 19 of SEQ ID NO: 2) , and a mature polypeptide of 134 amino acids (residue 20 to residue 153 of SEQ ID NO: 2), and is predicted to have a molecular weight of approximately 14.776 kD. Analysis of the tissue distribution of the mRNA corresponding to this novel DNA showed that expression was highest in thyroid, followed by decreased expression in testis, with some expression in liver and kidney. The nucleotide sequence of an 5 ' expressed sequence tag is described in SEQ ID NO. 1, polynucleotide number 7 to 244.

Multiple alignment of zlipo3 with neutrophil gelatase associated protein (NGAL) revealed regions of high identity corresponding to amino acid residues 39-43,

45-49, 84-89, and 137-142 of SEQ ID NO: 2, and as is shown in Figure 2.

The proteins in the lipocalin family have the structure of a single eight-stranded continuously hydrogen-bonded antiparallel barrel (Flower, J. Molec. Recognition 8_.:185-195, 1995). The β-strands (designated A- I in Figure 3) form a calyx- or cup-shaped antiparallel β- barrel (Flower et al . , Protein Science 2_.:753-761, 1993). The 3-10 helix, just C-terminal to the A strand, closes off one end of the barrel and with the A and B strands forming LI, is involved in formation of the of a cap for the internal ligand-binding site. Additional loops are formed by B-C (L2) , C-D (L3), D-E (L4), E-F (L5) as shown in Figure 3. Unlike other members of the lipocalin family which contain Loops L6 connecting strands F and G and L7 connecting strands G and H and L8 connecting strands H and Helix 1, zlipo3 is missing strands G and H forming only L6 connecting strand F to the helix (see Figure 3) . At the C-terminus, after strand F is an α-helix, which folds back against the barrel. Based on X-ray crystal monomer structures of bovine β-lactoglobulin (Brownlow et al . , Structure 5:481-495, 1997) and mouse pheromone-binding protein (Bocskei et al . , J Mol Biol. 218 (4) :699-701, 1991) Cysl53 in zlipo3 may form a disulfide bond with Cys88. There is evidence, based on the crystal structure of odorant binding protein (Monaco et al . , Biopolymers 3_2:457-465, 1992) , that dimerization may cause the C- terminal helix and strand I to associate with a dimer partner. Additionally, in dimerized forms of zlipo3, alternative disulfide patterns including inter-monomer, as well as intra-monomer, covalent connections are possible. The structural conformations, including L7 and L8 , define the lipocalin family (Flower et al, ibid. 1993), thereby suggesting zlipo3 is a related, but structurally novel member of the lipocalin family. Lipocalins are characterized by a multi-domain structure comprising a ligand binding domain that is typically involved in binding small, hydrophobic molecules; a conserved cell -surface receptor-binding domain that is typically involved in binding some putative cell-surface receptor that may be common to more than one lipocalin; and open end of the fold structure that forms a macromolecular complex, perhaps involving the cell-surface receptor. For example, retinol binding protein, a member of the lipocalin family, is characterized by the presence of a ligand binding site that binds retinol, a small hydrophobic molecule. While many of the lipocalins bind retinol, there is a diverse range of other ligands that bind lipocalins. Some of the other ligands include odorants, bilirubin, prostaglandins and pheromones . However, the lipophilic region for binding appears to be well conserved. Therefore, based on homology with other lipocalins, beta strand formation is predicted for regions designated as A-F and I in Figure 1 and corresponds to amino acid residues 41-49, 61-68, 75-82, 89-96, 103-108, 112-116 and 137-140, as shown in SEQ ID NO: 2.

Based on the a structure and sequence analysis of the lipocalin family, a putative ligand-binding cavity is formed that includes amino acid residues 45, 48, 61, 63, 65, 78, 80, 93, 110 and 111-115, as shown in SEQ ID NO: 2, and in Figure 1 as represented by "!".

In addition to the β-strands and loop structures, the zlipo3 proteins are characterized by the presence of conserved motifs at positions corresponding to: (1) residues 39-49 of SEQ ID NO: 2

(2) residues 84-89 of SEQ ID NO: 2 (3) residues 135-141 of SEQ ID NO: 2 The motifs are shown in Table 1 using the standard single-letter codes for amino acid residues.

Table 1

Additional residues that would likely be conserved include: residue 41 (Gly) , residue 43 (Trp) , residue 88 (Cys) , residue 102 (Gly) , residue 140 (Phe) , and residue 153 (Cys). Thus, molecules of the present invention include polypeptides that are at least 90% identical to motifs 1-3 (disclosed above) , which correspond to residues: motif 1) 39-49 of SEQ ID NO: 2 motif 2) 84-89 of SEQ ID NO: 2 motif 3) 135-141 of SEQ ID NO: 2 and β-strands at positions corresponding to residue 41-49, 61-68, 75-82, 89-96, 103-108, 112-116, and 137-140 of SEQ ID NO: 2, such that these β-strands are separated by loops. Variations within the identified motifs are can be defined as those that occur in the lipocalin family, and are shown in SEQ ID NOS: 4, 5 and 6. It is often the case that structure and function are retained when structurally conserved residues that family members have in common are exchanged. Therefore, the present invention includes molecules such as those shown in SEQ ID NOS: 7, 30, 31 and 32, where substitutions correspond to one or more regions and/or residues conserved within the lipocalin family. By varying the lengths of the various regions of the molecule, in particular sequences not defined by the β-strands or motifs, the length of the zlipo3 protein can be varied. Small extensions can also be made in the β- strands . The present invention thus provides proteins and polypeptides as defined above as comprising at least 114 amino acids residues, or preferably 123 amino acid residues, and more preferably 134 amino acid residues. Zlipo3 proteins can be further extended by joining them to linkers, affinity tags or other polypeptides to produce fusion proteins as disclosed in more detail below.

Those skilled in the art will recognize that predicted domain boundaries are somewhat imprecise and may vary by up to ± 5 amino acid residues.

Polypeptides of the present invention comprise at least 6, preferably at least 9, more preferably at least 15 contiguous amino acid residues of SEQ ID NO: 2. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO: 2, up to the entire predicted mature polypeptide (residues 20 to 153 of SEQ ID NO: 2) or the primary translation product (residues 1 to 153 of SEQ ID NO: 2) . As disclosed in more detail below, these polypeptides can further comprise additional, non- zlipo3, polypeptide sequence (s) .

Within the polypeptides of the present invention are polypeptides that comprise an epitope-bearing portion of a protein as shown in SEQ ID NO: 2. An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al . , Proc . Natl . Acad. Sci . USA

11:3998-4002, 1984. Epitopes can be linear or conformational , the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al . , Science 219 : 660-666 , 1983. Antibodies that recognize short, linear epitopes are particularly useful in analytic and diagnostic applications that employ denatured protein, such as Western blotting (Tobin, Proc . Natl . Acad . Sci .

USA 76:4350-4356, 1979) , or in the analysis of fixed cells or tissue samples. Antibodies to linear epitopes are also useful for detecting fragments of zlipo3, such as might occur in body fluids or cell culture media.

Antigenic, epitope-bearing polypeptides of the present invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a zlipo3 protein. Antigenic, epitope-bearing polypeptides contain a sequence of at least, six, nine, from 15 to about 30 contiguous amino acid residues of a zlipo3 protein (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zlipo3 protein, i.e. from 30 to 50 residues up to the entire sequence, are included. Generally, the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is, the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided. Such regions include the interdomain loops of zlipo3 and fragments thereof. Specific zlipo3 polypeptides would include those encoded by SEQ ID NO: 2 from amino acid residues 55-59, residues 72-77, residues 53-59, residues 69-74 and residues 52-57.

Polypeptides of the present invention can be prepared with one or more amino acid substitutions, deletions or additions as compared to SEQ ID NO: 2. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other changes that do not significantly affect the folding or activity of the protein or polypeptide, and include amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, an amino or carboxyl-terminal cysteine residue to facilitate subsequent linking to maleimide- activated keyhole limpet hemocyanin, a small linker peptide of up to about 20-25 residues, or an extension that facilitates purification (an affinity tag) as disclosed above. Two or more affinity tags may be used in combination. Polypeptides comprising affinity tags can further comprise a polypeptide linker and/or a proteolytic cleavage site between the zlipo3 polypeptide and the affinity tag. Preferred cleavage sites include thro bin cleavage sites and factor Xa cleavage sites.

The present invention further provides a variety of other polypeptide fusions. For example, a zlipo3 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-zlipo3 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zlipo3 analogs. In addition, a zlipo3 polypeptide can be joined to another bioactive molecule, such as a cytokine, to provide a multi-functional molecule. Auxiliary domains can be fused to zlipo3 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a zlipo3 polypeptide or protein can be targeted to a predetermined cell type by fusing a zlipo3 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes . A zlipo3 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al . , Connective Tissue Research 34:1-9, 1996.

Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, preferably not more than about 1,200 residues, more preferably not more than about 1,000 residues, and will in many cases be considerably smaller. For example, a zlipo3 polypeptide of 134 residues (residues 20-153 of SEQ ID NO:2) can be fused to E. coli ?-galactosidase (1,021 residues; see Casadaban et al . , J^".

Bacteriol . 143.: 971-980, 1980), a 10-residue spacer, and a

4 -residue factor Xa cleavage site to yield a polypeptide of 1,166 residues. In a second example, residues 20-153 of SEQ ID NO: 2 can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.

As disclosed above, the polypeptides of the present invention comprise at least 6 contiguous residues of SEQ ID NO: 2. These polypeptides may further comprise additional residues as shown in SEQ ID NO: 2, a variant of SEQ ID NO : 2 , or another protein as disclosed herein. When variants of SEQ ID NO: 2 are employed, the resulting polypeptide will be at least 90%, or at least 95% identical to the corresponding region of SEQ ID NO:2. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull . Math .

Bio . 4_.8:603-616, 1986, and Henikoff and Henikoff, Proc . Natl . Acad. Sci . USA 89:10915-10919, 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 "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid. ) as shown in Table 2 (amino acids are indicated by the standard one-letter codes) . The percent identity is then calculated as :

Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

> <# H ι H

1

≥ H CN ro H 1

En LT) CN CN 0 1 1 O <tf H ro CN CN

1 1 1

fo S CN CN H ro rH

1 1 1 1

2 _π O CN H H H H H 1 1 1 1 1 i LΠ H ro rH 0 H ro CN CN

1 1 1 1 1 1 1 ] J ^•^ N CN σ ro CN H CN H rH

(ϋ 1 1 1 1 1 1 rH <# CN ro H 0 ro CN H ro H ro rd 1 1 1 1 1 1

EH ffi 00 ro ro H CN H CN H CN CN CN ro 1 1 1 1 1 1 1 1 1 1

O ω CN CN ro ro CN O CN CN ro ro 1 1 1 1 1 1 1 1 1 1 1

H LΠ CN O ro ro rH CN ro H O H ro CN CN

1 1 1 1 1 1 1 1 1 1 σ in CN CN O ro CN H 0 ro H O H CN H CN

1 1 1 1 1 1 1 1 1 u as ro ro ro H ro H CN ro rH H CN CN 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Q 1X1 en 0 CN H ro rH ro ro H O H ro ro 1 1 1 1 1 1 1 1 1 1 1 1 1

£ SD H ro 0 σ 0 H ro ro O CN ro CN H O CN ro

1 1 1 1 1 1 1 1 1 tf LO O CN ro H σ CN O ro CN CN H ro CN H H ro CN ro

1 1 1 1 1 1 1 1 1 1 1 1 1

< H CN CN 0 H H O CN H H H H CN H H 0 ro CN 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

<: £ Q U α W O X J * _€ El! C O EH Eε H >

LO O LO O

H CN The level of identity between amino acid sequences can be determined using the "FASTA" similarity search algorithm disclosed by Pearson and Lipman { Proc .

Natl . Acad. Sci . USA 85:2444, 1988) and by Pearson (Meth . Enzymol . 183:63, 1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2) , without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff" value (calculated by a predetermined formula based upon the length of the sequence and the ktup value) , then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol . Biol . 48_.: 444, 1970;

Sellers, SIAM J. Appl . Math . .26:787, 1974), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, 1990 ( ibid. ) .

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default. The present invention includes polypeptides having one or more conservative amino acid changes as compared with the amino acid sequence of SEQ ID NO: 2. The BLOSUM62 matrix (Table 1) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, ibid. ) . Thus, the

BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the term "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3 ) .

The proteins of the present invention can also comprise non-naturally occuring amino acid residues. Non- naturally occuring amino acids include, without limitation, trarzs-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N- methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine , hydroxyethylhomocysteine , nitroglutamine, homoglutamine, pipecolic acid, tert- leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4 -azaphenylalanine, and 4- fluorophenylalanine . Several methods are known in the art for incorporating non-naturally occuring amino acid residues into proteins. For example, an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs . Methods for synthesizing amino acids and aminoacylating tRNA 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 . 113 :2722 ,

1991; Ellman et al . , Methods Enzymol . 202:301, 1991; Chung et al . , Science 259 :806-809, 1993; and Chung et al . , Proc. Natl . Acad. Sci . USA 90:10145-10149, 1993) . In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRΝAs (Turcatti et al . , J. Biol .

Chem . 271:19991-19998, 1996) . Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occuring amino acid(s) (e.g., 2-azaphenylalanine, 3 -azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) . The non- naturally occuring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occuring amino acid residues can be converted to non-naturally occuring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci . 2:395-403, 1993) .

Amino acid sequence changes are made in zlipo3 polypeptides so as to minimize disruption of higher order structure essential to biological activity. Amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can identify specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity, secondary structure propensities, binary patterns, complementary packing, and buried polar interactions (Barton, Current Opin . Struct . Biol . 5:372-376 , 1995 and

Cordes et al . , Current Opin . Struct . Biol . 6:3-10, 1996).

In general, determination of structure will be accompanied by evaluation of activity of modified molecules. For example, changes in amino acid residues will be made so as not to disrupt the domain structure geometry of the protein family. The effects of amino acid sequence changes can be predicted by, for example, computer modeling using available software (e.g., the Insight II^® viewer and homology modeling tools; MSI, San Diego, CA) or determined by analysis of crystal structure (see, e.g., Lapthorn et al , Nature 369:455-461, 1994; Lapthorn et al . ,

Nat . Struct . Biol . 2:266-268, 1995). Protein folding can be measured by circular dichroism (CD) . Measuring and comparing the CD spectra generated by a modified molecule and standard molecule are routine in the art (Johnson, Proteins 7:205-214, 1990) . Crystallography is another well known and accepted method for analyzing folding and structure. Nuclear magnetic resonance (NMR) , digestive peptide mapping and epitope mapping are other known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al . , Science 257:961-964, 1992). Mass spectrometry and chemical modification using reduction and alkylation can be used to identify cysteine residues that are associated with disulfide bonds or are free of such associations (Bean et al., Anal . Biochem. 201:216-226, 1992; Gray, Protein Sci .

2:1732-1748, 1993; and Patterson et al . , Anal . Chem. 66:3727-3732, 1994). Alterations in disulfide bonding will be expected to affect protein folding. These techniques can be employed individually or in combination to analyze and compare the structural features that affect folding of a variant protein or polypeptide to a standard molecule to determine whether such modifications would be significant .

Those skilled in the art will recognize that this hydrophilicity will be taken into account when designing alterations in the amino acid sequence of a zlipo3 polypeptide, so as not to disrupt the overall profile. Residues within the core of the four-helix bundle can be replaced with some combination of hydrophobic residues selected from the group consisting of Leu, lie, Val, Met, Phe, Trp, Gly, and Ala. Cysteine residues at positions 88, 126, and 153 of SEQ ID NO: 2; beta strand formation predicted for regions corresponding to amino acid residues 41-49, 61-68, 75-82, 89-96, 103- 108, 112-116 and 137-140, as shown in SEQ ID NO: 2; a putative ligand-binding cavity that includes amino acid residues 45, 48, 61, 63, 65, 78, 80, 93, 110 and 111-115, as shown in SEQ ID NO: 2; and conserved motifs at positions corresponding to residues 39-43 of SEQ ID NO: 2, residues 44-49 of SEQ ID NO : 2, residues 84-89 of SEQ ID NO: 2, and residues 135-141 of SEQ ID NO : 2 will be relatively intolerant of substitution. The length and amino acid composition of the interdomain loops are also expected to be important for receptor binding (and therefore biological activity) ; conservative substitutions and relatively small insertions and deletions are thus preferred within the loops, and the insertion of bulky amino acid residues (e.g., Phe) will in general be avoided.

Essential amino acids in the polypeptides of the present invention can be identified experimentally according to procedures known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 , 1081-1085, 1989; Bass et al., Proc. Natl . Acad. Sci . USA 88_.:4498-4502 , 1991).

In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule . Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc.

Natl . Acad. Sci . USA 86:2152-2156, 1989) . Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al . , U.S.

Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene

46:145, 1986; Ner et al . , DNA 7:127, 1988). Variants of the disclosed zlipo3 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Na ture 3_70: 389-391,

1994 and Stemmer, Proc. Natl . Acad. Sci . USA 91:10747-

10751, 1994. Briefly, variant genes are generated by in vi tro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

In many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a zlipo3 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide. For example, expressing the complete zlipo3 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved. Differential processing of individual chains may result in heterogeneity of expressed polypeptides.

The human zlipo3 polypeptide sequence (SEQ ID NO: 2) contains three cysteine residues, at positions 88, 126 and 153. Structural predictions indicate that Cys residues 88 and 153 may form an intrachain disulfide bond, and that and residue 126 may be free to form interchain disulfide bonds, resulting in dimerization. Actual conformation will depend in part upon the cell in which in the polypeptide is expressed. The polypeptides of the present invention thus include those comprising these cysteine residues, such as polypeptides comprising residues 88-153 of SEQ ID NO : 2. Mutagenesis methods as disclosed above can be combined with high volume or high-throughput screening methods to detect biological activity of zlipo3 variant polypeptides. Assays that can be scaled up for high throughput include mitogenesis assays, which can be run in a 96-well format. Mutagenized DNA molecules that encode active zlipo3 polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptide fragments or variants of SEQ ID NO: 2 that retain the activity of wild-type zlipo3. The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the zlipo3 polypeptides disclosed above. A representative DNA sequence encoding the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO:l. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO : 3 is a degenerate DNA sequence that encompasses all DNAs that encode the zlipo3 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 3 also provides all RNA sequences encoding SEQ ID NO: 2 by substituting U for T. Thus, zlipo3 polypeptide-encoding polynucleotides comprising nucleotides 1-459 or nucleotides 58-459 of SEQ ID NO: 3, and their RNA equivalents are contemplated by the present invention, as are segments of SEQ ID NO: 3 encoding other zlipo3 polypeptides disclosed herein. Table 3 sets forth the one-letter codes used within SEQ ID NO: 3 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide (s) . For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

TABLE 3

Nucleotide Resolutions Complement Resolutions

A A T T

C C G G

G G C C

T T A A

R A|G Y C|T

Y c|τ R A|G

M A|C K G|T

K G|T M A|C

S C|G S C|G

W A|T W A|T

H A|C|T D A|G|T

B C|G|T V A|C|G

V A|C|G B C|G|T

D A|G|T H A|C|T

N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO: 3, encompassing all possible codons for a given amino acid, are set forth in Table 4, below.

TABLE 4

Amino One- Degenerate

Acid Letter Codons Codon

Code

Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT CAN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gin Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

He I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F TTC TTT TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter . TAA TAG TGA TRR

Asn | Asp B RAY

Glu | Gin Z SAR

Any X NNN

Gap -

One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR) , and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) . A similar relationship exists between codons encoding phenylalanine and leucine. Thus, 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. One of ordinary skill in the art will also appreciate that different species can exhibit preferential codon usage. See, in general, Grantham et al . , Nuc . Acids

Res . 8:1893-912, 1980; Haas et al . Curr. Biol . 6:315-24,

1996; Wain-Hobson et al . , Gene 13_.: 355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc . Acids Res .

14:3075-87, 1986; and Ikemura, J. Mol . Biol . 158:573-97,

1982. Introduction of preferred 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: 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA from human and other species . Methods for preparing DNA and RNA are well known in the art. RNA is isolated from a tissue or cell that produces large amounts of zlipo3 mRNA, as described herein. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980) , and include thyroid, thyroid tumor tissue, as well as, skeletal muscle, colon and intestine. Polynucleotides encoding zlipo3 polypeptides are then identified and isolated by, for example, hybridization or PCR using a variety of methods. One method is probing mRNA with full or partial cDNA sequences encoding zlipo3, or one or more sets of degenerate probes based on the disclosed sequences Hybridization will generally be done under low stringency conditions, wherein washing is carried in 1 x SSC with an initial wash at 40 °C , with subsequent washes at 5°C higher intervals until background is suitably reduced. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primer designed from the representative sequence human zlipo3 sequence disclosed herein. Within additional method, 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 the zlipo3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Preferred probes and primers are shown below in Table 6. These probes and primers are derived from least degenerate regions of an alignment of zlipo3 (SEQ ID NO: 1), NGAL (SEQ ID NO: 8) and PD₂ synthase (SEQ ID NO: 9). The "consensus" and "complement" primers are useful for cloning both orthologs and paralogs, while the "zlipo3" primers will, in general, be more selective for orthologs. Within Table 5, primers are grouped with the amino acid sequence of the corresponding region of zlipo3 (SEQ ID NO: 2) . Amino acid residues are designated by one-letter code.

Table 5

Sequence Primer SEQ ID NO:

E V A G K W 2 (residues 38 to 43) AR GTN GCN GGN AAR TGG QSP chick 11 AR KTN BHN GGN VRN TGG CONSENSUS 12 TY MAN VDN CCN BYN ACC COMPLEMENT 13

D E M V A V 2 (residues 135 to 140)

GAY GAR ATG GTN GCN GT QSP chick 14 GAN RAN MHN RTN GYN KT CONSENSUS 15

CTN YTN KDN YAN CRN MA COMPLEMENT 16

Y I V A L A 2 (residues 44 to 49) TAY ATN GTN GCN YTN GC QSP chick 17 TWY DBN BYN GSN YTN GC CONSENSUS 18 AWR HVN VRN CSN RAN CG COMPLEMENT 19

K G C R K W 2 (residues 87 to 91) AAR GGN TGY MGN AAR TG QSP chick 20 MAN VRN TGY VRN WMN HG CONSENSUS 21 KTN BYN ACR BYN WKN DC COMPLEMENT 22

A full-length clone encoding zlipo3 is obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zlipo3, receptor fragments, or other specific binding partners. Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO: 1, or a sequence complementary thereto, under stringent hybridization and wash conditions. In general, stringent hybridization 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. Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 5X SSC, about 5X Denhardt's solution, up to about 10% dextran sulfate, and about 10-20 μg/ml denatured commercially-available carrier DNA; hybridization is then followed by washing filters in up to about 2X SSC. For example, a suitable wash stringency is equivalent to 0. IX SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C. Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.

Zlipo3 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5 ' non- coding regions of a zlipo3 gene. Promoter elements from a zlipo3 gene can thus be used to direct the expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zlipo3 proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zlipo3 gene in a cell is altered by introducing into the zlipo3 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zlipo3 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zlipo3 locus, whereby the sequences within the construct become operably linked with the endogenous zlipo3 coding sequence. In this way, an endogenous zlipo3 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS : 1 and 2 represent a single allele of human zlipo3. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures .

The present invention further provides counterpart polypeptides and polynucleotides from other species ("orthologs"). Of particular interest are zlipo3 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zlipo3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zlipo3 as disclosed above. A library is then prepared from mRNA of a positive tissue or cell line. A zlipo3 -encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zlipo3 sequence disclosed herein. Within an additional method, 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 zlipo3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones .

For any zlipo3 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 3 and 4, above. Moreover, those of skill in the art can use standard software to devise zlipo3 variants based upon the nucleotide and amino acid sequences described herein. The present invention thus provides a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l and SEQ ID NO: 2, and portions thereof. Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT) , a disk cache, and a ZIP™ disk. Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW) , and CD-recordable) , and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW) .

The polynucleotides of the present invention can also be synthesized using methods that are well known to those ordinarily skilled the art. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994) ; Itakura et al . , Annu. Rev. Biochem. 53 : 323-56, 1984 and Climie et al . , Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

The zlipo3 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, 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 Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al . eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a zlipo3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art . Many such elements are described in the literature and are available through commercial suppliers. To direct a zlipo3 polypeptide into the secretory pathway of a host cell, 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 zlipo3 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo . The secretory signal sequence is operably linked to the zlipo3 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell . Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al . , U.S. Patent No. 5,143,830).

Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-19 of SEQ ID NO: 2 is be operably linked to another polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 14:725, 1978; Corsaro et al . , Somatic Cell Genetics 7:603, 1981: Graham et al . , Virology 52:456, 1973), electroporation (Neumann et al . , EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid.), and liposome-mediated transfection (Hawley-Nelson et al . , Focus 15:73, 1993; Ciccarone et al . , Focus 15:80, 1993), and viral vectors (Miller et al . , BioTechniques 7:980-90, 1989; Wang et al . , Nature Med. 2:714-716, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al . , U.S. Patent No. 4,784,950; Palmiter et al . , U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650) , COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. 36_.: 59-72, 1977) and Chinese hamster ovary (e.g. CH0-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. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter .

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants" . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate . Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology. Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci. ("Bangalore J 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; and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) . See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method of making recombinant zlipo3 baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al . , J Virol 67:4566-79, 1993). This system, which utilizes transfer vectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zlipo3 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See, Hill-Perkins, M.S. and Possee, R.D., J Gen Virol 71:971-6, 1990; Bonning, B.C. et al . , J Gen Virol 7_5:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B., J Biol Chem 270:1543-9, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zlipo3 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al . , Proc. Natl. Acad. Sci. 82 :7952- 4, 1985) . Using a technique known in the art, a transfer vector containing zlipo3 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. Sf9 cells. Recombinant virus that expresses zlipo3 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art .

The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S.

Patent 5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™

(Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid. ; O'Reilly, D.R. et al . , ibid. ; Richardson, C. D., ibid. ) . Subsequent purification of the zlipo3 polypeptide from the supernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica .

Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) . A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092), and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccharomyces pombe,

Kluyveromyces lactis, Kluyveromyces fragilis , Ustilago maydis, Pichia pastoris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol . 132:3459-3465, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S.

Patent No. 4,486,533. The use of 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. For polypeptide production in P. methanolica, it is preferred that 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) . Other useful promoters include those of the dihydroxyacetone synthase (DHAS) , formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences . 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 . 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 (τ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds. Prokaryotic host cells, including strains of the bacteria Escherichia coli , Bacill us and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid.). When expressing a zlipo3 polypeptide in bacteria such as E . coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, 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. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors . A preferred culture medium for P. methanolica is YEPD (2% D-glucose,

2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine) .

It is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, 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 polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. Expressed recombinant zlipo3 polypeptides (or chimeric zlipo3 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl , butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or 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. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography : Principles & Methods, Pharmacia LKB

Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploitation of for example, their ligand- binding or complex forming properties. For example, affinity chromatography using retinoids can used to bind zlipo3 to the retinoids (Ferrari et al . , FEBS Lett . 401:73-77, 1997) .

Alternatively, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3_.:l-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. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39) . Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e .g. , maltose- binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

Moreover, using methods described in the art, polypeptide fusions, or hybrid zlipo3 proteins, are constructed using regions or domains of the zlipo3 in combination with those of other human lipocalin family proteins, or heterologous proteins (Sambrook et al . , ibid. , Altschul et al . , ibid. , Picard, Cur . Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain (s) conferring a biological function may be swapped between zlipo3 of the present invention with the functionally equivalent domain (s) from another family member, such as NGAL or PD₂ synthase. Such domains include, but are not limited to, the secretory signal sequence, conserved motifs (e.g., beta strands, helices, and alpha helices) and corresponding structures in the other members of the lipocalin family. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known lipocalin family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.

Zlipo3 polypeptides or fragments thereof may also be prepared through chemical synthesis, for example as described by Merrifield, J . Am . Chem . Soc . 85: 2149, 1963; Stewart et al . , "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford, IL, 1984) and Bayer & Rapp Chem. Pept . Prot . 3:3 (1986); and Atherton et al . , Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989. zlipo3 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. Proteins of the present invention are useful for their antimicrobial properties. Antimicrobial activity can be measured in vi tro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model . Assays for testing antimicrobial activity are specific to the microbe and are generally known by those ordinarily skilled in the art. For example, in vivo testing for antimicrobial activity is done by inoculating mice intraperitoneally with pathogenic microorganisms in an appropriate broth. Shortly after inoculation, a composition containing zlipo3 polypeptide is administered and death during the subsequent 7 days is recorded. Generally adminstration is intravenous, subcutaneous, intraperitoneal or by mouth. See, for example, Musiek et al . , Antimicrobial Agents Chemother. 3_.:40, 1973, for discussion of in vivo and in vi tro testing of antimicrobials.

To test zlipo3 molecules of the present invention for in vivo activity host cells expressing zlipo3 polypeptides can be implanted into appropriate animal models. For example, one in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno- associated virus (AAV) . Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al . , Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44- 53, 1997) . The adenovirus system offers several advantages: adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts may be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell human 293 cell line) . When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (i.e., liver) will express and process (and, if signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

The adenovirus system can also be used for protein production in vi tro . By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown in adherent or suspension cultures at relatively high cell density to produce significant amounts of protein (see Garnier et al . , Cytotechnol . 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins may also be effectively obtained. The activity of molecules of the present invention can be measured using a variety of assays that measure the ability to bind small hydrophobic molecules. Such assays include, but are not limited to assays measuring changes in fluorescence intensity (Cogan et al . , Eur. J. Biochem. 65 : 71-78, 1976) and equilibrium dialysis of water soluble compounds (Hase et al . , J. Biochem. 79:373-380, 1976) .

In view of the tissue distribution observed for zlipo3, agonists and antagonists have enormous potential in both in vi tro and in vivo applications. Compounds identified as zlipo3 agonists, including zlipo3, are useful for transportation of small hydrophobic molecules either in vi tro or in vivo . For example, agonist compounds are useful as components of defined cell culture media, to delivery small, hydrophobic molecules to cells and protect them from degradation by enzymes present in serum. Agonists are thus useful in specifically promoting the growth and/or development of thyroid-specific cell lineages in culture.

Zlipo3 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zlipo3. In addition to those assays disclosed herein, samples can be tested for inhibition of zlipo3 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zlipo3 -dependent cellular responses. For example, zlipo3 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a zlipo3 -stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zlipo3-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE) , hormone response elements (HRE) insulin response element (IRE) (Nasrin et al . , Proc. Natl. Acad. Sci . USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al . Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al . , J. Biol. Chem. 263 (19) : 9063-6; 1988 and Habener, Molec. Endocrinol . 4 (8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zlipo3 on the target cells as evidenced by a decrease in zlipo3 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zlipo3 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zlipo3 binding to receptor using zlipo3 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, and the like) . Within assays of this type, the ability of a test sample to inhibit the binding of labeled zlipo3 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.

A zlipo3 polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F_c fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify ligand, in vi tro assay tool, and as antagonists. For use in assays, the chimeras are bound to a support via the F_c region and used in an ELISA format . A zlipo3 ligand-binding polypeptide can also be used for purification of ligand. The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl) , or pH to disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145.: 229-40, 1991 and 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.

Ligand-binding receptor polypeptides can also be used within other assay systems known in the art . Such systems include Scatchard analysis for determination of binding affinity (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) .

Zlipo3 polypeptides can also be used to prepare antibodies that specifically bind to zlipo3 epitopes, peptides or polypeptides. The zlipo3 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. Hydrophilicity can be used to determine regions that have the most antigenic potential . Suitable antigens would be the zlipo3 polypeptide encoded by SEQ ID NO: 2 from amino acid residues 55-59, residues 72-77, residues 53-59, residues 69-74 and residues 52-57, or about a contiguous 9 to 30 amino acid fragment thereof. 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.

As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zlipo3 polypeptide or a fragment thereof. The immunogenicity of a zlipo3 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 zlipo3 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.

As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 ^anc 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) . In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.

Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules. First, antibodies herein specifically bind if they bind to a zlipo3 polypeptide, peptide or epitope with a binding affinity

/~ -| "7 __- 1 (K_a) of 10 M or greater, preferably 10 M or greater, more preferably 10 8 M—1 or greater, and most preferably 10 9 M-1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949). A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zlipo3 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, radioim uno-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 zlipo3 protein or polypeptide .

Antibodies to zlipo3 may be used for tagging cells that express zlipo3; for isolating zlipo3 by affinity purification; for diagnostic assays for determining circulating levels of zlipo3 polypeptides; for detecting or quantitating soluble zlipo3 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 zlipo3 in vi tro 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 herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to zlipo3 or fragments thereof may be used in vi tro to detect denatured zlipo3 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. For instance, 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) . More specifically, zlipo3 polypeptides or anti-zlipo3 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. Molecules of the present invention can be used to identify and isolate receptors involved in forming a ligand-receptor complex with zlipo3. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Technigues, Hermanson et al . , eds., Academic Press, San Diego, CA, 1992, pp.195-202). 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. 62_.: 483 -514 , 1993 and Fedan et al . , Biochem. Pharmacol. 33.: 1167-1180 , 1984) and specific cell- surface proteins can be identified.

Another utility for molecules of the present invention is as a delivery system to transport and/or stabilize small lipophilic molecules. For example, molecules of the present invention can be used to microencapsulate a small lipophilic molecule that in an active pharmacological agent, and thus protect the agent from extreme pH in the gut, exposure to powerful digestive enzymes and impermeability of gastrointestinal membranes to the active ingredient. Other advantages as encapsulation of the pharmacologic agent can include; preventing premature activation of the agent or protection from gastric irritants. Molecules of the present invention can be used for binding small fatty acids in blood or tissues to modulate their biological function. Molecules of the present invention can be used to transport retinoids or steroids to receptors, in particular as part of the therapy for breast cancer, emphysema and diseases of the skin and play and important role in reproduction. Other uses include modulation of anti -inflammatory responses

(Flower, ibid. 1996), activity as a microbial, either as a enhancer of enzymes (Glasgow, Arch. Clin. Exp. Opthalmol . 233:513-522, 1995) or as an enzyme-like molecule itself.

Based on the tissue distribution being present in thyroid tumor zlipo3 would have utility as a diagnostic for tyroid carcinomas and as a tool for predicting tumor aggressiveness . Polynucleotides encoding zlipo3 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit zlipo3 activity. If a mammal has a mutated or absent zlipo3 gene, the zlipo3 gene can be introduced into the cells of the mammal . In one embodiment, a gene encoding a zlipo3 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or , almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective 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 . , JJ_ Clin. Invest. 90:626-30, 1992; and a defective adeno- associated virus vector (Samulski et al . , J. Virol. 61:3096-101, 1987; Samulski et al . , J. Virol. 63:3822-8,

1989) .

In another embodiment, a zlipo3 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 33:153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol. 62_.: 1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al . ; and Kuo et al . , Blood 82_.: 845, 1993. Alternatively, 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. USA 84:7413-7, 1987; Mackey et al . , Proc. Natl. Acad. Sci . USA 85:8027-31, 1988) . The use of 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, or non-peptide molecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked 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 zlipo3 gene transcription, such as to inhibit cell proliferation in vivo . Polynucleotides that are complementary to a segment of a zlipo3 -encoding polynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO.-l) are designed to bind to zlipo3 -encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zlipo3 polypeptide-encoding genes in cell culture or in a subject .

Transgenic mice, engineered to express the zlipo3 gene, and mice that exhibit a complete absence of zlipo3 gene function, referred to as "knockout mice" (Snouwaert et al . , Science 257 :1083 , 1992), may also be generated (Lowell et al . , Nature 366 : 740-42 , 1993). These mice may be employed to study the zlipo3 gene and the protein encoded thereby in an in vivo system.

For pharmaceutical use, 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. In general, pharmaceutical formulations will include a zlipo3 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. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed. , Mack Publishing Co.,Easton, PA, 19th ed. , 1995. Therapeutic doses will generally be in the range of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20 μg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. 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.

Some embodiments of the present invention can therefore be summarized to include, an isolated polynucleotide molecule encoding a polypeptide comprising at least 114 amino acids as shown in SEQ ID NO : 2 from amino acid residue 39 to amino acid residue 153.

The present invention also embodies an isolated polynucleotide molecule encoding a polypeptide comprising at least 90% identity to 134 amino acids as shown in SEQ ID NO: 2 selected from the group consisting of: (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO : 1 from nucleotide 68 to nucleotide 472; (b) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 3 from nucleotide 58 to nucleotide 459; and (c) polynucleotide molecules encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 20 (Gin) to residue 153 (Cys) Additional polynucleotides of the present invention include an isolated polynucleotide molecule comprising nucleotides encoding for a polypeptide as shown in SEQ ID NO: 7, and wherein the encoded polypeptide comprises a sequence as shown in SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32.

Another isolated polynucleotide molecule embodied in the present invention comprises a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 11 to 472 or from nucleotide 68 to nucleotide 472. Also included are expression vectors operably linked elements including a transcription promoter; a DNA segment encoding a polypeptide as described herein and a transcription terminator, and cultured cells expressing the expression vectors . Included in the present are also methods of producing a polypeptide comprising culturing a cell into which has been introduced an expression vector described herein, and whereby said cell expresses said polypeptide encoded by said DNA segment ; and then recovering said expressed polypeptide.

Further aspects of the present invention include an isolated polypeptide comprising a sequence of amino acid residues of at least 114 amino acids as shown in SEQ ID NO: 2 from amino acid residue 39 (Phe) to amino acid residue 153 (Cys) , including wherein the amino acid residues comprise a sequence as shown in SEQ ID NO: 2 are from amino acid residue 20 (Gin) to residue 153. Also included are isolated polypeptides wherein the amino acid residues comprise a sequence as shown in SEQ ID NO : 2 from amino acid residue 1 (Met) to residue 153 (Cys) .

An isolated polypeptide comprising a sequence of amino acid residues of at least 90% identity to 134 amino acids as shown in SEQ ID NO: 2 from amino acid residue 20

(Gin) to residue 153 (Cys) is included in the present invention

Furthermore, the present invention provides an isolated polypeptide comprising at least 15 or more contiguous amino acids residues of SEQ ID NO : 2, and also provides an isolated polypeptide comprising at least 114 amino acids as shown in SEQ ID NO : 2 from amino acid residue 39 to amino acid residue 153. The present invention provides the polypeptides described herein as comprising a composition in an pharmaceutically acceptable vehicle.

The present invention provides antibodies that specifically bind to an epitope of the polypeptide described herein, and includes antibodies selected from the group consisting of: (a) a polyclonal antibody; (b) a murine monoclonal antibody; (c) a humanized antibody derived from (b) ; and (d) a human monoclonal antibody.

The present invention also provides a fusion protein comprising a secretory signal sequence comprising the amino acid sequence as shown in SEQ ID NO: 2 from residue 1 to 19, wherein said secretory signal sequence is operably linked to an additional polypeptide, and a fusion protein comprising a first portion and a second portion, wherein said first or second peptide comprises a sequence of amino acids as shown in SEQ ID NO : 2 from residues 2 to 153 and said first portion is operably linked to said second portion.

The invention is further illustrated by the following non-limiting examples. Example 1

Scanning of a translated DNA for secreted proteins identified an EST, which upon sequencing of the cDNA associated with the EST revealed a novel member of the lipocalin family.

Northerns were performed using Human Multiple Tissue blots I, II, and III (Clontech, Palo Alto, CA) and a Human RNA Master Dot Blot (Clontech) . A 250 bp probe was generated by PCR using primers ZC13138 (SEQ ID NO: 23) and ZC13137 (SEQ ID NO: 24) with human thyroid cDNA as a template. Cycling conditions consisted of one cycle at 94°C for 1 minute, 35 cycles of 94°C for 20 sec, 60°C for 30 sec and 72 °C for 30 sec followed by a final cycle of 72 °C for 10 minutes. The DNA probe was purified from a 2% GTG-agarose (FMC) gel using a Qiaquick gel extraction kit

(Qiagen) and radioactively labeled with ³²P using a random primer system (Multiprime, Amersham) according to the manufacturer's specifications. Free counts were removed from the probe using a NUCTRAP push column (Stratagene, La Jolla, CA) . EXPRESSHYB (Clontech, Palo Alto CA) solution was used for prehybridization and as a hybridizing solution for the Northern blots. The blots were prehybridized for three hours at 65 °C. Hybridization took place overnight at 65°C, and the blots were then washed four times in 2X SSC and 0.5% SDS at RT, followed by two washes in 0. IX SSC and 0.1% SDS at 50 °C and a final wash at 55 °C. in 0. IX SSC and 0.1% SDS. In the multiple tissue blots the highest expression was found in thyroid with weaker expression in testis and very weak expression in kidney and liver. The RNA master dot blot showed a similar expression pattern with high expression in thyroid, and weaker expression in kidney, liver and testis. The predominant transcript size was ~800-900 bp . Example 2

Zlipo3 was mapped to chromosome 9 using the commercially available "GeneBridge 4 Radiation Hybrid Panel" (Research Genetics, Inc., Huntsville, AL) . The GeneBridge 4 Radiation Hybrid Panel contains 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) allows 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 .

For the mapping of zlipo3 with the "GeneBridge 4 RH Panel", 25 μl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene) . Each of the 95 PCR reactions consisted of 2.5 μl 10X PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA) , 2 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA) , 1.25 μl sense primer, ZC13139 (SEQ ID NO: 25), 1.25 μl antisense primer, ZC13137 (SEQ ID NO: 24), 2.5 μl "RediLoad" (Research Genetics, Inc., Huntsville, AL) , 0.5 μl "Advantage KlenTaq Polymerase Mix" (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH20 for a total volume of 25 μl . The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 4 minute denaturation at 94 °C, 35 cycles of a 1 minute denaturation at 94 °C, 1.5 minute annealing at 60 °C and 1.5 minute extension at 72 °C, followed by a final 1 cycle extension of 7 minutes at 72 °C. The reactions were separated by electrophoresis on a 3% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME) .

The results showed that zlipo3 maps 3.05 cR_3000 from the framework marker D9S158 on the chromosome 9 WICGR radiation hybrid map. Proximal and distal framework markers were D9S158 and WI-14048, respectively. The use of surrounding markers positions Zlipo3 in the 9q34.3 region on the integrated LDB chromosome 9 map (The Genetic Location Database, University of Southhampton, WWW server: http: //cedar .genetics . soton.ac.uk/public_html/) .

Example 3

A. Mammalian Expression Constructs An expression plasmid containing all or part of a polynucleotide encoding zlipo3 is constructed via homologous recombination. A fragment of zlipo3 cDNA is isolated using PCR that includes the polynucleotide sequence from nucleotide 1 to nucleotide 472 of SEQ ID NO: 1 with flanking regions at the 5 ' and 3 ' ends corresponding to the vectors sequences flanking the zlipo3 insertion point . The primers for PCR each include from 5 ' to 3' end: 40 bp of flanking sequence from the vector and 17 bp corresponding to the amino and carboxyl termini from the open reading frame of zlipo3.

Ten μl of the 100 μl PCR reaction is run on a 0.8% LMP agarose gel (Seaplaque GTG) with 1 x TBE buffer for analysis. The remaining 90 μl of PCR reaction is precipitated with the addition of 5 μl 1 M NaCl and 250 μl of absolute ethanol. The plasmid pCZR199 which has been cut with Smal. Plasmid pCZR199 was constructed from pZP9 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and is designated No. 98668) with the yeast genetic elements taken from pRS316 (deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, and is designated No. 77145) pCZR199 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, the SV40 terminator, as well as the URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae .

One hundred microliters of competent yeast cells (S. cerevisiae) are independently combined with 10 μl of the various DNA mixtures from above and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtures are electropulsed at 0.75 kV (5 kV/cm) , > ohms, 25 μF. To each cuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two 300 μl aliquots onto two URA-D plates and incubated at 30°C. After about 48 hours, the Ura+ yeast transformants from a single plate are resuspended in 1 ml H2O and spun briefly to pellet the yeast cells. The cell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture is added to an Eppendorf tube containing 300 μl acid washed glass beads and 200 μl phenol-chloroform, vortexed for 1 minute intervals two or three times, followed by a 5 minute spin in a Eppendorf centrifuge at maximum speed. Three hundred microliters of the aqueous phase is transferred to a fresh tube, and the DNA precipitated with 600 μl ethanol (EtOH) , followed by centrifugation for 10 minutes at 4°C. The DNA pellet is resuspended in 10 μl H2O.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B cells. The cells are electropulsed at 1.7 kV, 25 μF and 400 ohms. Following electroporation, 1 ml SOC (2% Bacto" Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl , 10 mM MgCl2, 10 mM MgS04, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates (LB broth (Lennox), 1.8% Bacto Agar (Difco) , 100 mg/L Ampicillin) .

Individual clones harboring the correct expression construct for zlipo3 are identified by restriction digest to verify the presence of the zlipo3 insert and to confirm that the various DNA sequences have been joined correctly to one another. The insert of positive clones are subjected to sequence analysis. Larger scale plasmid DNA is isolated using the Qiagen Maxi kit (Qiagen) according to manufacturer's instruction.

Example 4

A. Mammalian Expression of zlipo3

CHO DG44 (Chasin et al . , Som. Cell. Molec. Genet . 12:555-666, 1986) are plated in 10 cm tissue culture dishes and allowed to grow to approximately 50 to

70% confluency overnight at 37 °C , 5% C0₂ , in Ham's

F12/FBS media (Ham's F12 medium, (Gibco BRL, Gaithersburg,

MD) , 5% fetal bovine serum (Hyclone, Logan, UT) , 1% L- glutamine (JRH Biosciences, Lenexa, KS) , 1% sodium pyruvate (Gibco BRL) ) . The cells are then transfected with the plasmid zlipo3/pCZR199 , using Lipofectamine™ (Gibco BRL), in serum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate) . zlipo3/pCZR199 is diluted into 15 ml tubes to a total final volume of 640 μl with SF media. 35 μl of

Lipofectamine™ (Gibco BRL) is mixed with 605 μl of SF medium. The Lipofectamine™ mix is added to the DNA mix and allowed to incubate approximately 30 minutes at room temperature. Five milliliters of SF media is added to the

DNA: Lipofectamine™ mixture. The cells are rinsed once with 5 ml of SF media, aspirated, and the

DNA: Lipofectamine™ mixture is added. The cells are incubated at 37 °C for five hours, then 6.4 ml of Ham's

F12/10% FBS, 1% PSN media is added to each plate. The plates are incubated at 37 °C overnight and the

DNA: Lipofectamine™ mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 2 post-transfection, the cells are split into the selection media (nucleoside- free Alpha MEM/dialyzed FBS media with the addition of 50 nM methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50. The cells are refed at day 5 post -transfection with fresh selection media. Approximately 10 days post-transfection, two 150 mm culture dishes of methotrexate resistant colonies from each transfection are trypsinized and the cells are pooled and plated into a T-162 flask and transferred to large scale culture for scale-up and dilution cloning.

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells per well in 96 well dishes in selection medium and allowed to grow out for approximately two weeks. The wells are checked for evaporation of medium and brought back to 200 μl per well as necessary during this process . When a large percentage of the colonies in the plate are near confluency, 100 μl of medium is collected from each well for analysis by dot blot, and the are fed with fresh selection medium. The supernatant is applied to nitrocellulose filter in a dot blot apparatus and the filter is treated at 100°C in a vacuum oven to denature the protein. The filter was incubated in 625 mM tris glycine, pH 9.1, 5mM βmercaptoethanol, at 65°C, 10 minutes, then in 2.5% non-fat dry milk Western A Buffer

(0.25% gelatin, 50 mM TrisHCl pH 7.4, 150 mM NaCl, 5 mM

EDTA, 0.05% Igepal CA-630) overnight at 4°C on a rotating shaker. The filter was incubated with the antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer for 1 hour at room temperature on a rotating shaker. The filter was washed three times at room temperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filter was developed with ECL reagent according to manufacturer's directions (Amersham, Arlington Heights, IL) and exposed to film (Hyperfilm ECL, (Amersham) approximately 5 minutes. Positive clones are trypsinized from the 96 well dish and transferred to 6 well dishes in selection medium for scaleup and analysis by Western blot .

B. Yeast Expression

Expression of zlipo3 in Pichia methanolica utilizes the expression system described in commonly- assigned WIPO publication WO 97/17450. An expression plasmid containing all or part of a polynucleotide encoding zlipo3 is constructed via homologous recombination .

An expression vector is built from pCZR190 to express N-terminal tagged zlipo3 polypeptides. The pCZR190 vector contains the AUGl promoter, followed by the aFpp leader sequence and an amino-terminal peptide tag (FLAG) , followed by a blunt-ended Sma I restriction site, a translational STOP codon, followed by the AUGl terminator, the ADE2 selectable marker, and finally the AUGl 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 . For each construct two linkers are prepared, and along with zlipo3, are homologously recombined into the yeast expression vectors described herein.

One hundred microliters of competent yeast cells (S. cerevisiae) are independently combined with 10 μl of the various DNA mixtures from above and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtures are electropulsed at 0.75 kV (5 kV/cm) , ∞ ohms, 25 μF. To each cuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two 300 μl aliquots onto two URA-D plates and incubated at 30°C. After about 48 hours, the Ura+ yeast transformants from a single plate are resuspended in 1 ml H₂0 and spun briefly to pellet the yeast cells. The cell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0 , 1 mM EDTA) . Five hundred microliters of the lysis mixture is added to an Eppendorf tube containing 300 μl acid washed glass beads and 200 μl phenol -chloroform, vortexed for 1 minute intervals two or three times, followed by a 5 minute spin in a Eppendorf centrifuge at maximum speed. Three hundred microliters of the aqueous phase is transferred to a fresh tube, and the DNA precipitated with 600 μl ethanol (EtOH) , followed by centrifugation for 10 minutes at 4°C. The DNA pellet is resuspended in 100 μl H₂0.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) is done with 0.5-2 μl yeast DNA prep and 40 ul of DH10B cells. The cells is electropulsed at 2.0 kV, 25 mF and 400 ohms. Following electroporation, 1 ml SOC (2% Bacto" Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KC1 , 10 mM MgCl2, 10 mM MgS04, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates (LB broth (Lennox), 1.8% Bacto" Agar (Difco) , 100 mg/L Ampicillin) .

Individual clones harboring the correct expression construct for zlipo3 are identified by PCR analysis or restriction digest to verify the presence of the zlipo3 insert and to confirm that the various DNA sequences have been joined correctly to one another. The insert of positive clones are subjected to sequence analysis. Larger scale plasmid DNA is isolated using the Qiagen Maxi kit (Qiagen) according to manufacturer's instruction, and the DNA is digested with Not I to liberate the Pichia-Zlipo3 expression cassette from the vector backbone. The Not I -restriction digested DNA fragment is then transformed into the Pichia methanolica expression host, PMAD16. This is done by mixing 100 ml of prepared competent PMAD16 cells with 10 μg of Not I restriction digested zlipo3 and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixture is electropulsed at 0.75 kV, 25 mF, infinite ohms. To the cuvette is added 1 ml of IX Yeast Nitrogen Base and 500 ml aliquots are plated onto two 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) plates for selection and incubated at 30 °C . Clones are picked and screened via Western blot for high-level Zlipo3 expression and fermented.

Example 5

Protein Purification of zlipo3

Unless otherwise noted, all operations will be carried out at 4°C. A total of 25 liters of conditioned medium from Chinese hamster ovary (CHO) cells or baby hamster kidney cells (BHK) is be sequentially sterile filtered through a 4 inch, 0.2 mM Millipore (Bedford, MA) OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, MI) Supercap 50. The material is then be concentrated to about 1.3 liters using a Millipore ProFlux A30 tangential flow concentrator fitted with a 3000 kDa cutoff Amicon (Bedford, MA) S10Y3 membrane. The concentrated material is sterile-filtered with the Gelman filter again as described above. A mixture of protease inhibitors is added to the concentrated conditioned medium to final concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim) .

Generic screening for protein capture is carried out using a BioCad 700E, Sprint, or Vision workstation (PE Biosystems, Framingham, MA) using the column screening module according to the manufacturer's instructions. A 50 ml sample of the concentrated CHO conditioned medium is brought to the appropriate pH by in-line dilution with screening buffer (25 mM Tris, 25 mM MOPS, 25 mM MES, and 25 mM acetate adjusted to the appropriate pH as described below) and pumped sequentially at a flow rate of 2-5 ml/min onto a 1.7 ml Poros HS (PE Biosystems) column equilibrated at pH 4.0, 5.0, and 6.0, onto a 1.7 ml Poros HQ (PE Biosystems) column equilibrated at pH 7.0, 8.0, and 9.0, onto a 1.7 ml Poros HE (PE Biosystems) column at pH 7.4, and onto a 1.7 ml Poros HP2 (PE Biosystems) column equilibrated at pH 7.4 and 1.0-4.0 M NaCl. After sample application, each column is washed with the appropriate equilibration buffer and when the absorbance at 280 nm of the effluent is below 0.05, the Poros HS, HQ, and HE columns are eluted stepwise with 1.0-2.0 M NaCl. The Poros HP2 column is eluted stepwise with water. 1.0 ml fractions is collected and the target protein in each of the column eluates is identified by the automated proteolysis-mass spec procedure described below. Positive identities are confirmed by SDS-PAGE analysis of each eluate fraction according to standard procedures. Once the binding conditions are established for a particular protein, these conditions are used for its large batch purification. Purity at each step of the purification is assessed by SDS-PAGE and Western blotting with anti-peptide antibodies directed against a linear peptide sequence of the target protein.

Proteins eluted as described above are detected independent of western blotting or other antibody related strategies. The presence of the desired protein is determined as either a single component or in a complex mixture by analysis of the eluate of a column, collected over several fractions and resulting in a relative quantitation of the amount of zlipo3 protein present in each fraction.

The system uses a stepwise combination of proteolytic digestion of protein samples (module 1) , chromatographic separation (module 2) and mass spectral analysis (module 3) of the digestion mixture. The three modules of this process are used individually for analysis of protein samples in a manual fashion, resulting in maximal data output, or in the stepwise process of 3 modules in a fully automated set-up, resulting in maximal high throughput .

In the automated set-up, module 1 and 2 are combined in the INTEGRAL Workstation (PE Biosystems, Farmington, MA) which is on-line connected to module 3. Module 3 is an LCQ ion-trap mass spectrometer (Finnigan, San Jose, CA) equipped with an electrospray source.

To maximize data output, module 1 and 2 are separated and the proteolytic digestion of samples is removed from the automated procedure. A MAGIC HPLC system (Michrom BioResources, Inc., Auburn, CA) serves as module 2. Samples are injected either manually or via autoinjector . Module 3 is an LCQ ion-trap mass spectrometer equipped with an electrospray source. Module 3 is on-line connected to module 2.

Module 1: Proteolytic Digestion

Typically, samples are proteolytically digested with trypsin, however, other proteases with defined specificity can be utilized. If necessary, samples are filtered or centrifuged to remove aggregates or other potential particulate matter. In some cases, samples are applied to a size exclusion step by filtration prior to analysis to simplify the resulting digestion mixture and make the identification of peptides related to the desired protein easier. All necessary buffer adjustments are made before proteolytic digestion.

In the automated set-up, the samples are digested on-line on an immobilized trypsin column (PE Biosystems) . The injection onto the column is done using the INTEGRAL autoinjector and the resulting peptides are chromatographically separated on module 2. In the manual approach, samples are digested overnight in solution and injected by hand or via autoinjector onto module 2.

Module 2 : Chromatographic Separation

The chromatographic separation of peptides is carried out on a 1 mm ID reverse phase (POROS, PE Biosystems) column (LC-Packings, San Francisco, CA) . Typically, the column is eluted with a trifluoroacetic acid (TFA) /water, TFA/acetonitrile gradient and the elution of peptides is monitored by UV. In the automated, as well as the manual approach, peptides are analyzed online on module 3 as they elute off the column.

Module 3 : Mass Spectral Analysis

The mass spectral analysis of peptides is carried out using the "triple play" approach. First, full mass range scans are taken as the column eluate is sprayed into the source of the mass spectrometer. If a signal above a predetermined intensity threshold is detected, the instrument switches to a setting which provides a high resolution mass measurement, followed by an MS/MS scan.

The MS/MS scan provides the fragmentation pattern which is used to derive the primary sequence of the peptide. Peptide sequences are then used for the identification of the protein. Typically, primary sequence and the nature of the protein is determined using the search algorithm SEQUEST (Finnigan) . Mass spectral sample and data analysis are carried out automatically. If necessary, data interpretation to derive peptide sequences is done manually and the protein is identified using a variety of standard database search algorithms.

Ion intensities and number of peptides detected for one protein are used to determine the relative abundance of this protein in different fractions.

If the mass spectral analysis is carried out on all ions observed leading to the analysis of all components in the digestion mixture. In order to simplify the analysis, the mass spectrometer is typically set to analyze only those ions which can be expected following the proteolysis of the desired protein. Through this filter, the analysis becomes amenable to very complex mixtures which potentially contains the desired protein as only a minor component

Example 6 Transgenic Expression

To make transgenic animals expressing zlipo3 genes requires adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4- 5 weeks, (Taconic Farms) ) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).

The donors are acclimated for 1 week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma, St. Louis, MO) I. P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin

(hCG (Sigma)) I. P. to induce superovulation. Donors are mated with studs subsequent to hormone injections.

Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating.

Fertilized eggs are collected under a surgical scope (Leica MZ12 Stereo Microscope, Leica, Wetzlar, DE) . The oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma) . Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (Table 6) that has been incubated with 5% CO2 ,

5% 0₂, and 90% N₂ at 37°C. The eggs are then stored in a

37°C/5% C0₂ incubator until microinjection. 10-20 micrograms of plasmid DNA containing a cDNA of the zlipo3 gene is linearized, gel -purified, and resuspended in 10 mM Tris pH 7.4, 0.25 mM EDTA pH 8.0, at a final concentration of 5-10 nanograms per microliter for microinj ection .

Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO2 - equilibrated mineral oil. The DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary) , and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei .

Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue- culture dish with pregassed W640 medium for storage overnight in a 37°C/5% C0₂ incubator.

The following day 2-cell embryos are transferred into pseudopregnant recipients. The recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds. Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope. A small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen. The reproductive organs are exteriorized onto a small surgical drape. The fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.

With a fine transfer pipette containing mineral oil followed by alternating W640 and air bubbles, 12-17 healthy 2-cell embryos from the previous day's injection are transferred into the recipient. The swollen ampulla is located and holding the oviduct between the ampulla and the bursa, a nick in the oviduct is made with a 28 g needle close to the bursa, making sure not to tear the ampulla or the bursa. The pipette is transferred into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette. The fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in. The peritoneal wall is closed with one suture and the skin closed with a wound clip. The mice recuperate on a 37°C slide warmer for a minimum of 4 hours .

The recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning. The weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.

Genomic DNA is prepared from the tail snips using a Qiagen Dneasy kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to the human growth hormone (hGH) 3 ' UTR portion of the transgenic vector. A region unique to the human sequence was identified from an alignment of the human and mouse growth hormone 3' UTR DNA sequences, ensuring that the PCR reaction does not amplify the mouse sequence. Primers zcl7251 (SEQ ID NO: 26) and zcl7252 (SEQ ID NO: 27) amplify a 368 base pair fragment of hGH. In addition, primers zcl7156 (SEQ ID NO: 28) and zcl7157 (SEQ ID NO: 29) , which hybridize to vector sequences and amplify the cDNA insert, are often used along with the hGH primers. In these experiments, DNA from animals positive for the transgene will generate two bands, a 368 base pair band corresponding to the hGH 3 ' UTR fragment and a band of variable size corresponding to the cDNA insert.

Once animals are confirmed to be transgenic (TG) , they are back-crossed into an inbred strain by placing a TG female with a wild-type male, or a TG male with one or two wild-type female (s) . As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping. To check for expression of a transgene in a live animal, a partial hepatectomy is performed. A surgical prep is made of the upper abdomen directly below the xiphoid process. Using sterile technique, a small 1.5- 2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized. Using 4-0 silk, a tie is made around the lower lobe securing it outside the body cavity. An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid, Wayne, N.J.) is placed proximal to the first tie. A distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish. The excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice. The surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hr post operatively. The animal is checked daily post operatively and the wound clips removed 7-10 days after surgery . Analysis of the mRNA expression level of each transgene is done using an RNA solution hybridization assay or real-time PCR on an ABI Prism 7700 (PE Applied Biosystems, Inc., Foster City, CA) following manufacturer's instructions. Table 6

WHITTEN'S 640 MEDIA

KC1 72 180

KH₂P0₄ 32 80

MgS0₄»7H₂0 60 150

Glucose 200 500

Ca²⁺ Lactate 106 265

Benzylpenicill .in 15 37.5

Streptomycin S0₄ 10 25

NaHC0₃ 380 950

Na Pyruvate 5 12.5

H₂0 200 ml 500 ml

500 mM EDTA 100 μl 250 μl

5% Phenol Red 200 μl 500 μl

BSA 600 1500

All reagents are available from Sigma. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMS What is claimed is:

1. An isolated polynucleotide molecule encoding a polypeptide comprising at least 114 amino acids as shown in SEQ ID NO: 2 from amino acid residue 39 (Phe) to amino acid residue 153 (Cys) .

2. An isolated polynucleotide molecule encoding a polypeptide comprising at least 90% identity to 134 amino acids as shown in SEQ ID NO: 2 selected from the group consisting of:

(a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 68 to nucleotide 472;

(b) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 3 from nucleotide 58 to nucleotide 459; and

(c) polynucleotide molecules encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 20 (Gin) to residue 153 (Cys) .

3. An isolated polynucleotide molecule comprising nucleotides encoding for a polypeptide as shown in SEQ ID NO: 7.

4. The polynucleotide molecule of claim 3, wherein the encoded polypeptide comprises a sequence as shown in SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32.

5. An isolated polynucleotide molecule comprising a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 11 to 472 or from nucleotide 68 to nucleotide 472.

6. An isolated polynucleotide molecule encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 20 (Gin) to residue 153 (Cys) .

7. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator.

8. A cultured cell into which has been introduced an expression vector according to claim 7, wherein said cell expresses said polypeptide encoded by said DNA segment.

9. A method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector according to claim 7, whereby said cell expresses said polypeptide encoded by said DNA segment; and recovering said expressed polypeptide.

10. An isolated polypeptide comprising a sequence of amino acid residues of at least 114 amino acids as shown in SEQ ID NO: 2 from amino acid residue 39 (Phe) to amino acid residue 153 (Cys) .

11. An isolated polypeptide comprising a sequence of amino acid residues of at least 90% identity to 134 amino acids as shown in SEQ ID NO: 2 from amino acid residue 20 (Gin) to residue 153 (Cys) .

12. The polypeptide of claim 10, wherein the amino acid residues comprise a sequence as shown in SEQ ID NO: 2 are from amino acid residue 20 (Gin) to residue 153.

13. The polypeptide of claim 10, wherein the amino acid residues comprise a sequence as shown in SEQ ID NO: 2 from amino acid residue 1 (Met) to residue 153 (Cys) .

14. An isolated polypeptide comprising at least 15 or more contiguous amino acids residues of SEQ ID NO: 2.

15. An isolated polypeptide comprising a sequence of amino acids as shown in SEQ ID NO: 7 from amino acid residue 1 to amino acid residue 134.

16. A composition comprising a polypeptide according to claim 10, in an pharmaceutically acceptable vehicle .

17. An antibody that specifically binds to an epitope of the polypeptide of claim 13.

18. An antibody according to claim 17, wherein said antibody is selected from the group consisting of:

(a) a polyclonal antibody;

(b) a murine monoclonal antibody;

(c) a humanized antibody derived from (b) ; and

(d) a human monoclonal antibody.

19. A fusion protein comprising a secretory signal sequence comprising the amino acid sequence as shown in SEQ ID NO: 2 from residue 1 to 19, wherein said secretory signal sequence is operably linked to an additional polypeptide.

20. A fusion protein comprising a first portion and a second portion, wherein said first or second peptide comprises a sequence of amino acids as shown in SEQ ID NO: 2 from residues 2 to 153 and said first portion is operably linked to said second portion.