JP2002527065A - Follistatin-related protein ZFSTA2 - Google Patents

Abstract

  (57) [Summary] The present invention relates to polynucleotides and polypeptides of zfsta2, a novel member of the follistatin family, the polypeptides and polynucleotides encoding them, for binding to members of the TGF-β family, and for the central nervous system, Useful for mediating reproductive, hematopoietic and bone-related activities. The invention also encompasses antibodies to the zfsta2 polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION: Follistatin is a glycosylated monomeric protein originally identified in porcine follicular fluid as a potential inhibitor of pituitary follicle stimulating hormone (FSH) synthesis and secretion. Statins later, FSH
-Has been shown to exert some of its biological effects by specifically binding the inducer activin.

Other follistatin family members include follistatin-related proteins or FRP (Zwijsen et al., Eur. J. Biochem. 225: 937-46, 1994), SPARC (also osteonectin) or EM-40. Or SPARC, known as the human ortho-form of the mask TSC-36 (Lane and Sage, supra), Agrin (Patthy an
d Nikolics, TINS 16: 76-81, 1993), Hevin (Girard and Springer, Immunit)
y 2: 113-23, 1995), chicken Flik protein (Amthor et al., Dev. Biology
176: 343-62, 1996), and rat brain protein SC1 (Mendis et al., Brain Re.
s. 730: 95-106, 1996).

[0003] However, follistatin is an "activin conjugate" because follistatin-deficient mice prepared by gene targeting have a more complex and different phenotype than activin gene knockout animals. (Mazuk et al., Nature 374: 360-3, 1995, and Mazuk et al., Nature 374: 356-
9, 1995).

Activins and inhibitors are respective potential activators and inhibitors of pituitary FSH secretion and are members of the TGF-β family of peptide growth factors (Mather et al., Proc. Soc. . Exp. Biol. Med. 215: 2
09-22, 1997). Activin and inhibin family hormones are pituitary FSH
Although originally described as a gonad-produced regulator of secretion, it is now known to have a wide range of effects within and outside the reproductive system (
Mather et al., Supra).

Inhibin consists of a normal α subunit covalently linked to one of two different β subunits (inhibin A: α / B _A ; inhibin B: α / B _B ); a covalently bound dimer species of B- subunit, and therefore, three different forms (activin _a: B a / B a; activin _B: B B / B B; activin AB
: Activin and inhibin present in B _A / B _B ) bind to faristatin with high affinity and the structure of the activin binding site is not completely defined,
Preliminary data (Inouye et al., Biochem. Biophys. Res. Commun. 179: 352-8, 1991
) Suggest that residues in the first amino-terminal cysteine-rich follistatin domain are involved in hormone binding.

[0006] Thus, the binding of activin to follistatin appears to limit its biological effect by the release of peptide hormones. Thus, the wide range of biological effects of activin and inhibin, and possible other members of the TGF-β family, can be modulated by binding to the follistatin family of proteins. The different binding proteins are individual TGF-β family members, as follistatin binds activin with high affinity (nM), binds inhibin with low affinity, and does not appear to bind TGF-β at all. (Mather et al., Supra). Follistatin family members can regulate the activity of other growth factors, for example, SPARC or BM-40 have been shown to bind platelet-derived growth factors (PDGF-AB, PDGF-BB) ( Lane and Sage,
FASEB J. 8: 163-73, 1994).

The present application provides a novel member of the forstatin family, zfsta2, which probably plays a major role in regulating the biological activity of TGF-β growth factor. Like other members of the follistatin family, afsta2 is involved in the pathogenesis of atherosclerosis, regulation of the gonad-pituitary-hypothalamic axis, formation of teeth and bone, regulation of gonad hormone production, spermatogenesis, hypothalamic oxytomin secretion. , Can play a wide role in erythroid progenitor proliferation and differentiation, hematopoiesis, host cell and neuron survival. The present invention provides such polypeptides for their and other uses which will be apparent to those skilled in the art.

SUMMARY OF THE INVENTION: In one aspect, the invention relates to amino acid residues 6 of the amino acid sequence of SEQ ID NO: 2.
Provided is an isolated polypeptide comprising a follistatin homology domain comprising 5-133. In one aspect, the polypeptide further comprises an α-helical linker region located at the carboxyl-terminal position to the follistatin homology domain, wherein the α-helical linker region has the amino acid sequence of SEQ ID NO: 2. Comprising amino acid residues 134-174.

In a related aspect, the polypeptide further comprises a calmodulin homology domain located at a carboxyl-terminal position relative to the α-helix linker region, wherein the calmodulin homology domain is the amino acid sequence of SEQ ID NO: 2. Comprises amino acid residues 175-250 of In another embodiment, the polypeptide further comprises two I-set Ig domains located at the carboxyl-terminal position relative to the calmodulin homology domain, wherein the I-set Ig domain is SEQ ID NO: 2 comprising amino acid residues 251-431.

[0010] In another embodiment, the polypeptide further comprises a carboxy-terminal domain present at a carboxyl-terminal position relative to the I-group Ig domain, wherein the carboxy-terminal domain is SEQ ID NO: 2 comprises amino acid residues 433-847. In yet another embodiment, the polypeptide further comprises a hydrophobic linker region located at an amino-terminal position to the follistatin homology domain, wherein the hydrophobic linker region is an amino acid of SEQ ID NO: 2. It comprises amino acid residues 21-64 of the sequence.

[0011] In another embodiment, the polypeptide further comprises a secretory signal sequence located at an amino-terminal position relative to the hydrophobic linker region, wherein the secretory signal sequence is an amino acid of SEQ ID NO: 2. It comprises amino acid residues 1-20 of the sequence. In another embodiment, the present invention is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, which specifically binds to an antibody that specifically binds a polypeptide having the amino acid sequence of SEQ ID NO: 2. An isolated polypeptide having an amino acid sequence is provided.

[0012] In one embodiment, the isolated polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. In another embodiment, the isolated polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. Further, in another aspect, any difference between the amino acid sequence and the corresponding amino acid sequence of SEQ ID NO: 2 is due to one or more conservative amino acid substitutions.

In another embodiment, the percent identity of the amino acids is ktup = 1, gap opening penalty = 10, gap extension penalty = 1, and substitution matrix = bl
Determined using the FASTA program with osum62, as well as other parameters set as errors. The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

The present invention further provides a polypeptide comprising a) a sequence of amino acid residues 21-64 of SEQ ID NO: 2; b) a polypeptide consisting of a sequence of amino acid residues 65-133 of SEQ ID NO: 2 Peptide; c) a polypeptide consisting of the sequence of residues 134 to 174 of SEQ ID NO: 2; d) a polypeptide consisting of the sequence of residues 175 to 250 of SEQ ID NO: 2; e) SEQ ID NO: A polypeptide consisting of the sequence of the amino acid residues of residues 251 to 334 of SEQ ID NO: 2; f) a polypeptide consisting of the sequence of the amino acid residues of residues 335 to 432 of SEQ ID NO: 2; A polypeptide consisting of the sequence of 487 amino acid residues; h) a polypeptide consisting of the sequence of amino acid residues 251 to 432 of SEQ ID NO: 2;
i) a polypeptide consisting of the sequence of amino acid residues 65-174 of SEQ ID NO: 2; j
A) a polypeptide consisting of the sequence of amino acid residues 65 to 250 of SEQ ID NO: 2; k)
A polypeptide consisting of the sequence of residues 65-334 of SEQ ID NO: 2; l) a polypeptide consisting of the sequence of amino acids residues 65-847 of SEQ ID NO: 2; m) the residue of SEQ ID NO: 2 A polypeptide consisting of the sequence of amino acid residues 134 to 250; n) a polypeptide consisting of the sequence of amino acids 134 to 334 of SEQ ID NO: 2; o) an amino acid residue of residues 134 to 432 of SEQ ID NO: 2 P) SEQ ID NO: 2
A) a polypeptide comprising the sequence of amino acid residues from residues 134 to 847 of SEQ ID NO: 2; q) a polypeptide comprising a sequence of amino acid residues from residues 175 to 334 of SEQ ID NO: 2; And s) a polypeptide consisting of the sequence of amino acid residues 175-847 of SEQ ID NO: 2. .

[0015] The present invention also provides an isolated polypeptide as described above further comprising an affinity label or binding domain. In another embodiment, the invention provides a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-20 of SEQ ID NO: 2 operably linked to an additional polypeptide. . The present invention also provides a fusion protein consisting essentially of a first portion and a second portion linked by a peptide bond, wherein said first portion comprises a polypeptide as described above, and The two parts comprise another polypeptide.

[0016] In another aspect, the invention also provides an isolated polynucleotide encoding a polypeptide as described above. In one embodiment, the polypeptide further comprises an affinity label or a binding domain. The invention also provides an isolated polynucleotide molecule that is a degenerate nucleotide sequence that encodes a polypeptide as described above.

The invention further provides an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, which specifically binds to an antibody to which the polypeptide having the amino acid sequence of SEQ ID NO: 2 specifically binds. An isolated polynucleotide encoding a polypeptide having the same is provided. In one embodiment, the isolated polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. In another embodiment, the isolated polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2.

In yet another embodiment, any difference between the amino acid sequence and the corresponding amino acid sequence of SEQ ID NO: 2 is due to one or more conservative amino acid substitutions. In yet another embodiment, the percent identity of the amino acids is ktup =
1, FA with gap opening penalty = 10, gap extension penalty = 1 and substitution matrix = blosum62, and other parameters set as errors
Determined using the STA program. The present invention provides an isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotides 58 to 3006. The present invention also provides an isolated polynucleotide molecule of SEQ ID NO: 1.

The present invention further provides: a) a polynucleotide consisting of nucleotides 58-117 of SEQ ID NO: 1; b) a polynucleotide consisting of nucleotides 118-249 of SEQ ID NO: 1; c
A) a polynucleotide consisting of nucleotides 457-579 of SEQ ID NO: 1; e) a polynucleotide consisting of nucleotides 580-810 of SEQ ID NO: 1; f) a polynucleotide consisting of nucleotides 580-810 of SEQ ID NO: 1. Nucleotide 811
G) a polynucleotide consisting of nucleotides 1060 to 1353 of SEQ ID NO: 1; h) a polynucleotide consisting of nucleotides 1354 to 3006 of SEQ ID NO: 1; i) a polynucleotide consisting of nucleotides 250 to 579 of SEQ ID NO: 1. Nucleotides; j) a polynucleotide consisting of nucleotides 250-810 of SEQ ID NO: 1; k)
A polynucleotide consisting of nucleotides 250-1059 of SEQ ID NO: 1; m) a polynucleotide consisting of nucleotides 250-3006 of SEQ ID NO: 1; n) a polynucleotide consisting of nucleotides 250-3006 of SEQ ID NO: 1. Four
O) a polynucleotide consisting of nucleotides 457-1059 of SEQ ID NO: 1; p) a polynucleotide consisting of nucleotides 457-1353 of SEQ ID NO: 1; q) a polynucleotide consisting of nucleotides 457-3006 of SEQ ID NO: 1. A polynucleotide consisting of nucleotides 580-1059 of SEQ ID NO: 1;
s) a polynucleotide consisting of nucleotides 580-1353 of SEQ ID NO: 1; t) a polynucleotide consisting of nucleotides 580-3006 of SEQ ID NO: 1; and u) a polynucleotide consisting of nucleotides 811-1353 of SEQ ID NO: 1. Provided are isolated and isolated polynucleotides.

The present invention also provides a polynucleotide encoding a fusion protein comprising a secretory signal sequence having an amino acid sequence of amino acid residues 1 to 20 of SEQ ID NO: 2 operably linked to an additional polypeptide. provide. The invention further provides a polynucleotide molecule encoding a fusion protein consisting essentially of a first portion and a second portion linked by a peptide bond, wherein said polynucleotide molecule comprises
One part comprises a polypeptide as described above, and the second part comprises another polypeptide.
Comprising two polypeptides.

[0021] In another aspect, the present invention provides an expression vector comprising: an operably linked element: a transcription promoter; a polynucleotide molecule encoding the polypeptide of claim 1; and a transcription terminator. I will provide a. In another embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment. In another embodiment, the polynucleotide encodes a polypeptide covalently attached at the amino terminus or at the carboxy terminus to an affinity label.

[0022] In another aspect, the invention provides a operably linked element: a transcription promoter; a polynucleotide molecule encoding a polypeptide as described above;
And a cultured cell expressing the polypeptide encoded by the polynucleotide segment, into which an expression vector comprising a transcription terminator has been introduced.

In yet another aspect, the present invention provides a method for producing a polypeptide, wherein said method comprises the following operably linked elements: a transcription promoter;
Culturing a cell into which an expression vector comprising a polynucleotide molecule encoding a polypeptide as described above; and a transcription terminator has been introduced, whereby the cell comprises the polypeptide encoded by the polynucleotide segment. Expressing; and recovering said expressed potypeptide.

[0024] In another aspect, the invention provides an antibody or antibody fragment that specifically binds to a polypeptide as described above. In one aspect,
Said antibodies are a) polyclonal antibodies; b) murine monoclonal antibodies; c) b
And d) a human monoclonal antibody. In another aspect, the antibody fragment is F (ab '
), F (ab), Fab ', Fab, Fv, scFv and a minimal recognition unit.
In another embodiment, an anti-idiotype antibody that specifically binds to an antibody as described above is provided.

[0025] In another aspect, there is provided a polypeptide as described above in combination with a pharmaceutically acceptable vehicle. These and other aspects of the invention will be apparent based on the following specific description of the invention.

Specific Description of the Invention: Before describing the present invention in detail, the following terms may be used to assist in understanding the invention: “Affinity tag” refers to the purification of a second polypeptide. Or used herein to indicate a polypeptide segment that can be attached to a second polypeptide to provide detection or provide a site for binding of the second polypeptide to a substrate. Primarily, antibodies or any peptide or protein for which other specific binding agents are available can be used as an affinity label.

Affinity labels are poly-histidine based, ie, protein A (Nilsson et al.,
(EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol. 198: 3, 1991).
, Glutathione S transferase (Smits and Johnson, Gene 67; 31, 19
88), Glu-Glu affinity label (Grussenmeyer, etc., Proc. Natl. Acad. Sci. USA
82: 7952-4, 1985), substance P, ie Flag ™ peptide (Hopp et al., B
iotechnology 6: 1204-1210, 1988), streptavidin binding peptides, or other antigenic epitopes or binding domains. Generally, Ford etc., Prot
See ein Expression and Purification 2: 95-107, 1991. DNA encoding the affinity tag can be obtained from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ).
Eastman Kodak, New Heven, CT; New England Biolabs, Beverly, MA).

[0028] The term "allelic variant" is used herein to indicate any of a plurality of alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation, and can result in phenotypic polymorphisms within a population. 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 indicate the protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein to indicate position within a polypeptide. Where the context allows, those terms are used in reference to a particular sequence or portion of a polypeptide to indicate accessibility or relative position. For example, certain sequences located at the carboxyl terminus of a subject sequence in a polypeptide are located adjacent to the carboxyl terminus of the subject sequence, but are not required at the carboxyl terminus of the complete polypeptide.

The term “complement / anti-complement pair” refers to non-identical components that, under appropriate conditions, form a stable pair that are non-covalently associated. For example, biotin and avidin (or streptavidin) are the prototype 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. If complement pair followed dissociation is desired, the complement / anti - - complement / anti-complement pair preferably has a binding affinity of <10 ⁹ M ^-1.

The term “complement of a polynucleotide molecule” is a polypeptide molecule that has a complementary base sequence and an opposite orientation as compared to a reference sequence. For example, the sequence 5 ′ ATGCACGG
G 3 ′ is complementary to 5 ′ CCCGTGCAT 3 ′. The term "contig" refers to a polynucleotide having a contiguous sequence of identical or complementary sequences to another polynucleotide. A contiguous sequence is said to "overlap" a length of a polynucleotide sequence, in whole or along a portion of the polynucleotide. For example, a polynucleotide sequence
Representative contigs for 5'-ATGGCTTAGCTT-3 'are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '.

The term “degenerate nucleotide sequence” refers to a sequence of nucleotides (as compared to a control polynucleotide that encodes a polypeptide) that includes one or more degenerate codons. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (ie, GAU and GAC triplets are each Asp
Code).

The term “expression vector” is used to indicate a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest operably linked to an additional segment that provides for its transcription. You. Such additional segments include promoter and terminator sequences and one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors generally can be derived from plasmid or viral DNA, or can include elements of both.

The term “isolated” when applied to a polynucleotide, removes the polynucleotide from its natural genetic environment and, therefore, has no other extraneous or unwanted coding sequences. And exist in a form suitable for use in a genetically engineered protein production system. Such isolated molecules are those that have been separated from their natural environment and include cDNA and genomic clones. An isolated DNA molecule of the present invention does not contain other genes which are not normally involved, but can contain naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. Identification of relevant regions will be apparent to those of skill in the art (see, for example, Dynan and Tijan, Nature 316: 774-78, 1985).

An “isolated” polypeptide or protein may have conditions other than its native environment,
For example, polypeptides or proteins found under conditions that are separate from blood and animal tissues. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, especially other polypeptides of animal origin. Highly refined form,
That is, it is preferable to supply the polypeptide with a purity of 95% or more, more preferably 99% or more. As used in this context, the term "isolated" excludes the presence of the same polypeptide in other physical forms, such as a dimeric form or other glycosylated or derivatized form do not do.

“Operably linked” when applied to a DNA segment, said segments function cooperatively for their intended purpose, eg, transcription is initiated at a promoter, and Indicates that it is arranged to proceed to the terminator through the code segment. The term "orthology" refers to a polypeptide or protein from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences between ortho-forms are the result of specification.

“Polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 ′ end to the 3 ′ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Polynucleotide sizes are expressed as base pairs (abbreviation "bp"), nucleotides ("nt"), or kilobases ("kb").

Here, the latter two terms describe a polynucleotide that is single-stranded or double-stranded. When the term is applied to double-stranded molecules, it will be used to indicate the overall length and will be understood to be equivalent to the term "base pairs". It is understood by those skilled in the art that the two strands of a double-stranded polynucleotide differ slightly in length, and that their ends differ as a result of enzymatic degradation;
Not all nucleotides within a double-stranded polynucleotide molecule can be paired. Such unpaired ends do not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically. Polypeptides of up to about 10 amino acid residues are commonly referred to as "polypeptides." The term "promoter" is used herein to indicate a portion of a gene that contains a DNA sequence that provides for the binding of RNA polymerase and initiation of transcription. A promoter sequence is usually, but not necessarily, found in the 5 'non-coding region of a gene.

A “protein” is a macromolecule that comprises one or more polypeptide chains.
Proteins can also contain non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents will be added by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined herein by their amino acid backbone; substituents, such as carbohydrate groups, are generally not specified, but can nevertheless be present.

The term “receptor” refers to a cell-associated protein that binds to a bioactive molecule (ie, “ligand”) and mediates the effect of the ligand on a cell. Membrane-bound receptors
It is characterized by a multi-peptide structure comprising an extracellular ligand binding domain and, typically, an intracellular effector domain involved in signal transduction. Binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell.

This interaction induces a change in the metabolism of the cell. Metabolic phenomena linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased AMP production,
Includes cellular calcium metabolism, membrane lipid metabolism, cell attachment, inositol lipid hydrolysis, and phospholipid hydrolysis. Generally, receptors are membrane-bound, cytosolic or nuclear; monomers (eg, thyroid stimulating hormone receptor, β-
Adrenergic receptors) or multimers (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor) Can be

The term “secretory signal sequence” refers to a DNA encoding a polypeptide (“secretory peptide”) that directs a larger polypeptide as a component of a larger polypeptide through the secretory pathway of the cell in which it is synthesized. Shows the sequence. The larger polypeptide is usually degraded during transit through the secretory tract to remove the secreted peptide.

The term “splice variant” is used herein to indicate an alternative form of RNA transcribed from a gene. Splice mutations occur in transcribed RNA
Through the use of alternative splicing sites between intramolecular or normally low, but separately transcribed RNA molecules, some mRNA may occur naturally and be transcribed from the same gene. Splice variants can encode polypeptides having an altered amino acid sequence. The term splice variant is also used herein to indicate the protein encoded by the splice variant of mRNA transcribed from the gene.

It will be appreciated that the molecular weight and length of the polymer as determined by inaccurate analytical methods (eg, gel electrophoresis) are approximate. Where such a value is expressed as “about” X or “approximately” X, it will be understood that the stated value of X is exactly ± 10%. All documents cited herein are incorporated by reference in their entirety.

A new member of the follistatin family of proteins, namely zfata2,
Identified from the human hypothalamus library. zfsta2 protein is about 86,000 Da
Exhibiting a specific amino-terminal cysteine-rich follistatin domain with an estimated molecular weight of (Hohenester et al., EMBO J. 16: 3778-86, 199
7).

Other follistatin family members include follistatin-related proteins or FRP (Zwijsen et al., Eur. J. Biochem. 225: 937-46, 1994), SPARC (also osteonectin or EM-40, or mask). SPARC, known as the human orthoform of TSC-36 (Lane and Sage, supra), Agrin (Patthy and Nikolics, TINS)
16: 76-81, 1993), Hevin (Girard and Springer, Immunity 2: 113-23, 199)
5), chicken Flik protein (Amthor et al., Dev. Biology 176: 343-62, 19
96), and rat brain protein SC1 (Mendis et al., Brain Res. 730: 95-106,
1996).

The present invention is based, in part, on the expression of a novel DNA sequence that encodes a polypeptide having homology to the family of follistatin. The zfsta2 polynucleotide sequence is disclosed in SEQ ID NO: 1 and encodes a 847 amino acid (SEQ ID NO: 2) multidomain secretory protein. As shown by SEQ ID NO: 2, sequence analysis of the deduced amino acid sequence of zfsta2 revealed a signal sequence of 20 amino acid residues (amino acid residues 1-20 of SEQ ID NO: 2, nucleotides 58-117 of SEQ ID NO: 1). ), Followed by a known dominantly hydrophilic short linker domain without homology (amino acid residues 21-26 of SEQ ID NO: 2, nucleotides 118-246 of SEQ ID NO: 1), a follistatin homology domain ( Amino acid residues 65-133 of SEQ ID NO: 2, nucleotides 250-456 of SEQ ID NO: 1), α-helical linker region (amino acid residue 134 of SEQ ID NO: 2)
-174, nucleotides 457-579 of SEQ ID NO: 1), calmodulin domain (amino acid residues 175-250 of SEQ ID NO: 2, nucleotides 580-810 of SEQ ID NO: 1), I-Set I
G domain # 1 (amino acid residues 251-334 of SEQ ID NO: 2, nucleotides 811-1059 of SEQ ID NO: 1), I-group IG domain # 2 (amino acid residues 335-432 of SEQ ID NO: 2, nucleotides of SEQ ID NO: 1 1060-1353), and C- with no known homology
Terminal domain (amino acid residues 443-847 of SEQ ID NO: 2, nucleotide of SEQ ID NO: 1)
1354-3006). One skilled in the art will recognize that the predicted domain boundaries are approximations based on the major sequence content and can vary slightly, however, such estimates are generally ± 5 Accurate within amino acid residues.

The follistatin homology domain is a SPARC (also known as BM-40 or osteonectin, Swiss-Prot SPRC_HUMAN, PDB 1BMO, Hohenester et al., 1997).
Is predicted to fold into a structure similar to that determined for the follistatin homology domain. This is the small hydrophobic core of the β hairpin structure followed by the α / β structure. Unlike SPARC, which is glycosylated with Asn99, zfsta2
Does not have a predicted glycosylation site.

Based on the disulfide bond pattern in SPARC, the disulfide pair ring in zfsta2 is: Cys65-Cys76, Cys70-Cys87, Cys89- of SEQ ID NO: 2
Cys119, Cys93-cysll2 and Cys101-Cys133. The zfsta2 follistatin homology domain has 47% identity to the follistatin domain in the human follistatin-related protein (Swiss-Prot FRP_HUMAN); the mouse orthoform of this protein is known as TSC-36 ( Swiss-Prot FRP_MOUSE).

The follistatin homology domain is a serine proteinase inhibitor, Kazal
It has substantial sequence similarity to the family (Bode and Huber., Eur. J. Biochem. 204, 433-51, 1992). Serine proteinase inhibitors regulate the proteolytic activity of target proteinases by dominating the active site,
And thereby hinders normal domination by the substrate. Although serine proteinase inhibitors fall into several unrelated structural similarities, they are all exposed loops that are stabilized by molecular interactions between binding loops and residues flanking the protein core ("" Inhibitor loops, "reaction cores", "reaction sites", "binding loops") (Bode and Huber, supra).

The interaction between the inhibitor and the enzyme dissociates very slowly, leading to a stable complex that produces either intact or modified inhibitors that are degraded at the scissile bond of the binding loop. Generate. Proteinase inhibitor PEC
Based on similarities to the crystal structures for -60 (PDB 1PCE) and ovomucoid (PDB 1OVO), the putative proteinase binding site in the follistatin homology domain of zfsta2 is amino acid residue Cys93 (P3) of SEQ ID NO: 2. , Lys94 (P2), Arg95 (P
1), including His96 (P1 ') and Tyr97 (P2'). Thus, a scissile bond of the binding loop will be present between the P1 and P1 ′ residues of SEQ ID NO: 2, Arg95 and His96.

[0053] The calmodulin homology domain can be found in SPARC (Hohnester et al., RMBO J. 16: 3778-
86, 1997) is predicted to fold into a structure similar to that determined for EC (EF hand calcium binding; calmodulin-like). Calmodulin (Swiss-Prot CALM_HUMAN, PDB 1CLI) binds calcium ions through a helix-loop-helix substructure loop known as the EF hand.
-A helical protein. Calmodulin has two structurally similar regions, each containing two EF hands linked by a connecting helix segment. As used herein, "calmodulin homology domain" is meant to describe one of those two regions.

[0054] The calmodulin homology domain of zfsta2 is predicted to contain two EF hand forms and thus two predicted calcium ion binding sites. Based on that type of analysis, the loops of the two EF hands are between Asp amino acid residue 188 and Leu amino acid residue 200 of SEQ ID NO: 2, and Asp amino acid residue 226 and phe
It is predicted to be between amino acid residue 238.

The last residues of the EF handle loop are always hydrophobic, ie, in zfsta2, they are leu amino acid residue 200 and Phe amino acid residue 238 of SEQ ID NO: 2. With respect to sequence homology, the calmodulin homology domain of zfsta2 has 24% identity at the amino acid level to the double EF hand segment of the human protein phosphatase PPEE-2 (GenBank accession AFO23456). The Zfsta2 calmodulin homology domain has no detectable sequence homology to the carbodulin domain of SPARC.

The second EF hand of the calmodulin domain of SPARC is stabilized by a disulfide bond extending the EF hand loop. When two Cys residues in this EF hand were mutagenized to Leu residues, a 100-fold decrease in calcium ion affinity was shown (Hohenester et al., Nat. Struct. Biol., 3:67). -73, 1996). The present application also discloses that the second EF hand is stabilized by replacing Asp amino acid residue 225 and Ala amino acid residue 241 of SEQ ID NO: 2 with a cysteine residue.
Also provided are mutated forms of This suddenly triggered form has a higher calcium affinity.

The homologous domain between forstatin and calmodulin is a short segment called an α-helical linker that can form a short linker peptide between the two segments. This linker is Glu amino acid residue 1 of SEQ ID NO: 2.
It is predicted to have an α-helical structure of 44-Glu amino acid residues 166. At the C-terminus of the linker, there are three basic residues that may be at proteolytic sites. Processing at this site of the secreted protein releases domains B and C containing the follistatin homology domain from the rest of the protein. Amino acid residue 140 of the α-helix linker peptide (Cys in SEQ ID NO: 2)
) Can form a disulfide bond with amino acid residue 216 (Cys in SEQ ID NO: 2), which precedes the second EF hand in the calmodulin homology domain of zfsta2.

The I-set IG domains # 1 and # 2 of zfsta2 are predicted to fold into structures similar to those determined for the telokin peptides (Swiss-Prot KMLS_HUMAN, PDB
1TLK). Telokin peptides are classified as immunoglobulins, which are all beta proteins that fold into a beta-sandwich-like structure (Bork et al., J. Mol. B
iol. 242: 309-20, 1994). They have two beta sheets comprising a 3 + 4 beta chain.

[0059] In addition, telokin peptides have been subdivided as "I" class immunoglobulin (IG) domains. Other proteins with group I immunoglobulin domains include titin, vascular and nerve cell adhesion molecules, and twitchin
Is included. In Zfsta2 domain I-set IG # 1 and # 2, there are two types of intradomain disulfide bonds, one is SEQ ID NO: 2 in I-set IG domain # 1
And between cysteine residues 270 and 321 of the I-group IG domain #
2 between cysteine residues 362 and 413 of SEQ ID NO: 2.

The C-terminal domain of zfsta2 shows no recognizable sequence or structural similarity to any known proteins. This segment, in the extracellular matrix,
Alternatively, they can act to immobilize proteins on cell surface membranes. Northern blot analysis of various human tissues reveals approximately 5 kb found in brain, placenta and spinal cord.
Resulted in a transcript. RNA dot blot analysis showed expression in the cerebellum, occipital lobe and pituitary.

The results of chromosome localization showed that zfsta2 was located at 2.84 cR_3000 from framework marker WI-5113 on chromosome 4 WICGR radiation hybrid map.
The proximal and distal framework markers were WI-5113 and CHLC.GATA4CO5, respectively.
It was 17. The use of the surrounding marker is based on the integrated 4q 2 on LDB chromosome 4 map.
8.3 Locate zfsta2 in the region.

[0062] The present invention further provides polynucleotide molecules, such as DNA and RNA molecules, that encode the zfsta2 protein. The polynucleotides of the present invention include DNA as a duplex having a sense strand; an antisense strand; and both sense and antisense strands that are annealed together by their respective hydrogen bonds. A representative DNA sequence encoding the Zfsta2 protein is shown in SEQ ID NO: 1. DNA sequences encoding other zfsta2 proteins can be readily generated by those skilled in the art based on the genetic code. The RNA sequence of the counterpart can be generated by substituting U for T instead of T.

One skilled in the art will readily recognize that, in light of the degeneracy of the genetic code, considerable sequence variation is possible between those polynucleotide molecules. SEQ ID NO: 3
2 is a degenerate DNA sequence encompassing all DNAs encoding the zfsta2 polypeptide of SEQ ID NO: 2. One of skill in the art will also appreciate that the degenerate sequence of SEQ ID NO: 3 also provides all RNA sequences encoding SEQ ID NO: 2 by replacing U and T. Accordingly, zfsta2 polypeptide-encoding polynucleotides comprising nucleotides 1-2949 of SEQ ID NO: 3 and their RNA equivalents are encompassed by the present invention. Table 1 shows the ones used in SEQ ID NO: 3 to indicate degenerate nucleotide positions.
Indicates the character code. "Determination" is the nucleotide denoted by a code letter. "Complement" indicates the code for the complementary nucleotide. For example, code Y
Indicates either C or T, and its complement R indicates A or G, where A is complementary to T and G is complementary to C.

[0064]

[Table 1] The degenerate codons used in SEQ ID NO: 3, including all possible codons for a given amino acid, are shown in Table 2.

[0065]

[Table 2]

One skilled in the art will appreciate that some ambiguity is introduced in the determination of degenerate codons, which are representative of all possible codons encoding an individual amino acid. For example, a degenerate codon for serine (WSN) can, under certain circumstances, encode arginine (AGR), and a degenerate codon for arginine (MGN) under certain circumstances, serine ( AGY) can be coded. A similar relationship exists between the codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by a degenerate sequence may encode a variant amino acid sequence, but those skilled in the art will recognize such variant sequences by reference to the amino acid sequence of SEQ ID NO: 2. It can be easily identified. Variant sequences can be readily tested for functionality as described herein.

The skilled artisan will also appreciate that different species exhibit “alternative codon usage”. Generally, Grantham, et al., Nuc. Acids Res. 8: 1893-912, 1980;
Haas, et al., Curr. Biol. 6: 315-24, 1996; Wain-Hobson, et al., Gene 1.
3: 355-64, 1981; Grosjean and Fiera, Gene 18: 199-209, 1982; Holm, N.
uc. Acids Res. 14: 3075-87, 1986; Mol. Biol. 158: 573-97
1982. As used herein, the term "alternative codon usage" or "alternative codon" refers to the possible codons that are used most frequently in certain types of cells and thus encode individual amino acids. Technical term referring to protein translation codons that favor one or a few representatives (see Table 2).

For example, the amino acid threonine (Thr) is encoded by ACA, ACC, ACG, or ACT, but in mammalian cells, ACC is the most commonly used codon; in other species, For example, in insect cells, yeast, viruses or bacteria, different Thr codons are preferred. Alternative codons for a particular species can be introduced into a polynucleotide of the invention by various methods known in the art.

For example, the introduction of alternative codon sequences into recombinant DNA enhances the production of the protein by making it more efficient for translation in certain cell types or species. Thus, the degenerate codon sequence disclosed in SEQ ID NO: 3 is commonly used in the art and acts as a template to optimize expression of the polypeptide in the various cell types and species disclosed herein. I do. Sequences containing selection codons can be tested for expression in various species and tested for functionality disclosed herein.

In a preferred embodiment of the invention, the isolated polynucleotide comprises a similarly sized region of SEQ ID NO: 1, other polynucleotide probes, primers, fragments and sequences cited herein, or Will hybridize to the complementary sequence. Polynucleotide hybridization is well known in the art and is widely used for many applications;

For example, Sambrook et al., Molecular Cloning; A Laboratory Manual, Second E
dition, Cold Spring Harbor, NY, 1989; Ausubel et al., eds., Current Proto
cols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987; Berger a
nd Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzym
ology, Volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 2
27-59, 1990. See also. Polynucleotide hybridization exploits the ability of single-stranded complementary sequences to form double-stranded hybrids. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.

Hybridization will occur between sequences that contain some degree of complementarity. Hybrids can tolerate mismatched base pairs in a double helix, but the stability of the hybrid is affected by the degree of mismatch. The mismatched hybrid T _m is reduced 1 ℃ for base pair mismatches any 1 to 1.5%. Varying the stringency of the hybridization conditions allows adjustment for the degree of mismatch that may be present in the hybrid. As the hybridization temperature decreases and the ionic strength of the hybridization buffer decreases, the degree of stringency increases.

The hybridization buffer is generally a blocking agent such as Denhardt's solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, milk powder (B
LOTTO), heparin or SDS, and a source of Na ⁺ , such as SSC (1 × SSC: 0.15: N NaCl,
15 mM sodium citrate) or SSPE (1 × SSPE: 1.8 M NaCl, 10 mM NaH ₂ PO ₄₎
, 1 mM EDTA, pH 7.7). By lowering the ionic concentration of the buffer, the stability of the hybrid is reduced. Typically, the hybridization buffer contains 10 mM to ¹ M Na ⁺ . The premixed hybridization solution also
Commercial sources such as Clonech Laboratories (Palo Alto, CA) and Promega Corporat
ion (Madison, WI) for use according to the manufacturer's instructions.

The addition of a destabilizing or denaturing agent, such as formamide, a tetraalkylammonium salt, a guanidium cation or a thiocyanate cation, to the hybridization solution will alter the _Tm of the hybrid. Typically, formamide is used at a concentration of up to 50% to allow more convenient and lower temperature incubations to be performed. Formamide also acts to reduce non-specific background when using RNA probes.

Stringent hybridization conditions include a temperature of about 5 to 25 ° C. below the thermal melting point (T _m ) of the hybrid, and a hybridization buffer with up to 1 M Na ⁺ . Higher temperature stringency at lower temperatures can be achieved by the addition of formamide, which lowers the hybrid Tm by about 1 ° C. for each 1% formamide in buffer solution. Generally, such stringent conditions include a temperature of 20-70 ° C. and a hybrid buffer containing 6 × SSC and 0-50% formamide.
A high degree of stringency can be achieved at a temperature of 40-70 ° C. and with a hybridization buffer having 4 × SSC and 0-50% formamide.

High stringency conditions typically include a temperature of 42-70 ° C. and a hybridization buffer with 1 × SSC and 0-50% formamide. Different degrees of stringent fluid can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, washing following hybridization is performed with increasing degrees of stringency to remove unhybridized polynucleotide probes from the hybridized complexes.

The above conditions are meant to act as a guide, and are well within the ability of those skilled in the art to adapt those conditions for use with a particular polypeptide hybrid. The T _m for a specific target sequence is the will 50% of the target sequence hybridizes to a perfectly matched probe sequence temperature (under the conditions defined). Those conditions that affect the _Tm include the size and base pair content of the polynucleotide probe, the ionic temperature of the hybridization solution, and the presence of destabilizing agents in the hybridization solution.

Many equations for calculating Tm are known in the art (eg, Sa
Ausubel et al., supra; Berger and Kimmel, supra and Wetmu.
r, supra), and specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of various lengths. Sequence analysis software, such as Oligo 4.0 and Primer Premier, and sites on the Internet, are available tools for analyzing a given sequence and calculating the _Tm based on user-defined criteria. .

[0079] Such programs also analyze a given sequence under defined conditions and provide an appropriate probe sequence. Typically, hybridization of polynucleotide sequences longer than 50 bp is performed at a temperature about 20-25 ° C. below the calculated T _m . For probes shorter than 50 bp or less, hybridization is typically performed at the T _m or at a temperature 5-10 ° C. lower. This allows for the highest rate of hybridization for DNA-DNA and DNA-RNA hybrids.

As indicated above, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. Generally, RNA is isolated from tissues or cells that produce large amounts of zfsta2 RNA. Such tissues and cells are obtained from Northern blots (Thomas, Proc.
USA 77: 5201, 1980) and includes the brain and spinal cord. Total RNA can be prepared by guanidinium isothiocyanate extraction followed by isolation by centrifugation on a CsCl gradient (Chirgwin et al., Biochemi
stry 18: 52-94, 1979).

Poly (A) ⁺ RNA was obtained from Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-
1412, 1972). Complementary DNA (cDNA) is prepared from poly (A) ⁺ RNA using known methods. On the other hand, genomic DNA can be isolated. Next, a polynucleotide encoding a zfsta2 polypeptide is identified and isolated, for example, by hybridization or polymerase chain reaction.

[0082] Full-length clones encoding zfsta2 polypeptides can be obtained by conventional cloning methods. Complementary DNA (cDNA) clones are preferred, however, for some uses (eg, expression in transgenic animals), using genomic clones or modifying cDNA clones to include at least one genomic intron. Is preferred. Methods for preparing cDNA and genomic clones are well-known and within the level of ordinary skill in the art, and the sequences disclosed herein for probing or sensitizing libraries. Or a partial use thereof. The expression library can be probed with antibodies to zfsta2, receptor fragments, or other specific binding partners.

[0083] The polynucleotides of the present invention can also be synthesized using automated equipment. Current,
The method of selection is the phosphoramidite method. When chemically synthesized double-stranded DNA is required for the synthesis of a gene or gene fragment, the individual complementary strands are produced separately. The generation of short genes (60-80 bp) is technically straightforward and can be achieved by synthesis of complementary strands and subsequent annealing thereof.

However, for the production of longer genes (over 300 bp),
During the synthesis, the coupling efficiency of the individual cycles is rarely 100%,
A particular method is desired. To overcome this problem, synthetic genes (double-stranded)
Are assembled in a controlled fashion from single-stranded fragments that are 20 to 100 nucleotides in length. Gene synthesis methods are well known in the art. For example, Glick and Pasternak, Molecular Biotechnology, Principles & Applicati
ons of Recombinant DNA, (ASM Press, Washington, DC 1994); Itakura et al.
., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad.
Sa. USA 87: 633-7, 1990.

The invention further provides counterpart ligands and polynucleotides from other species (ortho-forms). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species.
Of particular interest are zfsta2 polypeptides from other mammalian species, for example, mice, pigs, sheep, cows, dogs, cats, horses, and other primate ligands. Orthoforms of the human zfsta2 polypeptide can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques.

[0086] For example, cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zfsta2. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. Next, a library is prepared from mRNA of positive tissues or cell lines. Then, z
The cDNA encoding the fsta2 polypeptide can be isolated by various methods, for example, by probing with full or partial human cDNA, or with one or more degenerate probes based on the disclosed sequences.

The cDNA can also be prepared using the polymerase chain reaction (PCR) (Mullis, 1988) using primers designed from the representative human zfsta2 polynucleotide sequences disclosed herein.
U.S. Pat. No. 4,683,202) can also be cloned. In a further method, a cDNA library is used to transform or transfect host cells, and expression of the cDNA of interest can be detected by an antibody against the zfsta2 polypeptide. Similar techniques can also be applied to isolate genomic clones.

One skilled in the art will recognize that the sequence disclosed in SEQ ID NO: 1 represents a single allele of human zfsta2 and that allelic variation and alternating splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard methods. Allelic variants of the DNA sequence set forth in SEQ ID NO: 1, for example, those that include silent mutations and those in which the mutation results in an amino acid sequence change, are proteins that are allelic variants of SEQ ID NO: 2. As is within the scope of the present invention.

A cDNA generated from another spliced mRNA that retains the properties of a zfsta2 polypeptide, as well as the polypeptides encoded by such cDNA and mRNA, are included within the scope of the invention. Is done. Splice variants are known in the follistatin family, and follistatin exists in at least three forms (32,000, 35,000 and 39,000 Da) for other splicing. Allelic and splice variants of those sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard methods known in the art.

The present invention also provides SEQ ID NO: 2 polypeptides, and isolated zfsta2 polypeptides that are substantially identical to their orthoforms. The term "substantially identical" refers to 50% relative to the sequence set forth in SEQ ID NO: 2 or their ortho forms.
, Preferably 60%, and more preferably at least 80% are used herein to indicate polypeptides having sequence identity. Such a polypeptide is more preferably at least 90% relative to SEQ ID NO: 2 or its ortho form.
And most preferably 95% or more.

[0091] Percent sequence identity is determined by conventional methods. For example, Altschul., Bul
l. Math. Bio. 48: 603-616, 1986 and henikoff and Henikoff, Pruc. Natl. A
cad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences have a gap initiation penalty of 10, a gap extension penalty of 1, and a Henikoff and Henikoff (as described in Table 3 (amino acids are indicated by the standard one-letter code)). ) Using the "blosum 62" scoring matrix to optimize its matching scoring. Next, the percent identity is calculated as follows:

[0092]

(Equation 1)

[0093]

[Table 3] The sequence identity of the polynucleotide molecules is determined using the proportions disclosed above,
Determined in a similar manner.

[0094] Those skilled in the art understand that there are many established algorithms for aligning two amino acid sequences. The Pearson and Lipman “FASTA” similarity search algorithm uses the amino acid sequence disclosed herein and the putative variant zfsta2
Is a suitable protein alignment method to test the level of identity shared by the amino acid sequences of The FASTA algorithm is based on Pearson and Lipman, Proc.
at'l Acad. Sci. USA 85: 2444 (1988), and Pearson, Meth. Enzymol. 183: 6
3 (1990).

[0095] Briefly, FASTA first determines the highest density of identity (when the ktup variable is 1) without considering the sequence in question (eg, SEQ ID NO: 2) and conservative amino acid substitutions, insertions or deletions. Alternatively, characterize the sequence by identifying regions shared by test sequences having either pairwise identity (if ktup = 2). Next, the ten regions with the highest density of identity are reevaluated by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions It is "trimmed" to include only those residues that contribute to the score.

If there are some regions with a score higher than the “cutoff” value (calculated by a formula that is based on the length of the sequence and the ktup value), the trimmed initial region is , Is tested to determine if the region can be joined to form an approximate alignment with the gap. Finally, Needleman-Wunsch, where the highest scoring regions of the two amino acid sequences allow for amino acid insertions and deletions.
Algorithm (Needleman and winsch, J. Mol. Biol. 48: 444, 1970; Sellers
, SIAM J. Appl. Math. 26: 787, 1974). Exemplary parameters for FASTA analysis are: ktup = 1, gap opening penalty = 10, gap extension penalty = 1 and substitution matrix = BLOSUM
62. Those parameters can be introduced into the FASTA program by adjusting the rating matrix, as described in Appendix 2 of Pearson, 1990 (supra).

[0097] FASTA can also be used to determine the sequence identity of nucleic acid molecules, using ratios as disclosed above. For nucleotide sequence comparisons, ktup values range from 1-6, preferably 4-6, with other parameters set as errors.
Can be

The BLOSUM62 table is an amino acid substitution matrix from approximately 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of over 500 groups of related proteins [Henikoff and Henikoff, Proc. Nat'l Acad.
Sci. USA 89: 10915 (1992)]. Thus, the BLOSUM62 substitution frequency can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the invention.

While it is possible to design amino acid substitutions only based on chemical properties (as described above), the term “conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value greater than −1. To mention. For example, for an amino acid substitution,
Or conservative if characterized by a BLOSUM62 value of 3. According to this system, preferred conservative amino acid substitutions are at least 1 (eg, 1, 2 or 3
), But more preferably the conservative substitutions are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3).

[0100] Conservative amino acid changes in zfsta2 can be introduced by substituting nucleotides for the nucleotides listed in SEQ ID NO: 1. Such "conservative amino acid" variants include, for example, oligonucleotide-directed mutagenesis,
Obtained by linker scanning mutagenesis, mutagenesis using the polymerase chain reaction and similar methods (Ausubel (1995) pages 8-10-8-22; and McPhrson (ed.
.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)). To select for mutants with wild-type protein properties,
It can be performed using standard methods, for example, the assays described herein. On the other hand, mutant zfsta2 polypeptides can be identified by their ability to specifically bind anti-zfsta2 antibodies.

[0101] Variant zfsta2 polypeptides and substantially homologous zfsta2 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably conservative amino acid substitutions (see Table 4) and other substitutions that do not substantially affect the folding or activity of the protein and polypeptide; small deletions, typically 1 ~ 30 amino acid deletions; and small amino-
Or a carboxyl-terminal extension, such as an amino-terminal methionine residue, about 20-
Extension of a small linker peptide of up to 25 residues, or extension of an affinity label. Polypeptides comprising an affinity tag further comprise a proteolytic site between the zfsta2 polypeptide and the affinity tag. Preferred such sites include thrombin degradation sites and factor Xa degradation sites.

[0102]

[Table 4]

The present invention further provides various other polypeptide fusions. For example, zfsta2
Polypeptides can be prepared as fusions to dimeric proteins as disclosed in US Pat. Nos. 5,155,027 and 5,567,584. Preferred dimer proteins therefor include an immunoglobulin constant region domain. Immunoglobulin-zfsta2 polypeptide fusions can be expressed in genetically engineered cells to produce various multimeric zfsta2 analogs. Auxiliary domains can be fused to zfsta2 polypeptides to target them to particular cells, tissues or macromolecules.

For example, by fusing a zfsta2 polypeptide or protein to a ligand that specifically binds to a receptor on the surface of a target cell,
It can be targeted to a predetermined cell type. In this case, the polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A zfsta2 polypeptide can be fused to multiple components, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also include one or more cleavage sites, particularly between domains. See Tuan et al., Connective Tissue Research 34: 1-9, 1996.

The proteins of the present invention also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include trans-3-methylproline, 2,4-metaproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N
-Methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-
Methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2
-Azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine.

Several methods are known in the art for introducing non-naturally occurring amino acid residues into proteins. For example, an in vitro system in which nonsense mutations are suppressed using a chemically aminoacylated suppressor tRNA can be used. Methods for synthesizing amino acids and aminoacylating tRNA are known to those skilled in the art. Transcription and translation of the plasmid containing the nonsense mutation is performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography.

For example, Rovertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al.
, Meth. Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-09, 1993;
And Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-49, 1993. In a second method, translation is performed in Xenopus oocytes by microinjection of mutagenized mRNA and chemically aminoamylated suppressor-tRNA (Turcatti et al., J. Biol. Chem. Two
71: 1991-98, 1996).

In a third method, E. coli cells are used in the absence of the natural amino acid to be replaced (eg, phenylalanine) and the desired non-naturally occurring amino acid (eg, 2-azaphenylalanine, (3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine).
Non-naturally occurring amino acids are introduced into proteins in place of their natural counterparts. See Koide et al., Biochem. 33: 7470-46, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modifications can be combined with site-directed mutagenesis to further extend the scope of the substitution (Wynn and Richards, Protein Sci. 2: 395-403, 19
93).

A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can be replaced by a zfsta2 amino acid. Essential amino acids in the polypeptides of the invention can be identified by methods known in the art, for example, site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085, 1989). Bass et al., Proc. Natl. Scad. Sci. USA 88: 4498-502, 1991). In the latter technique, a single alanine mutation is introduced at every residue in the molecule, and the resulting mutant molecule is used to identify amino acid residues that are critical for the activity of the molecule. , Tested for biological activity as disclosed below.

See also Hilton et al., J. Biol. Chem. 271: 4699-5708, 1996.
The site of ligand-receptor interaction can also be determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling for mutations of putative contact site amino acids. For example, de Vos, etc., Science 255: 306-312, 1992; Sm
ith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett.
309: 59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homologs by related proteins, eg, human CMRF35.

Multiple amino acid substitutions can be made by known methods of mutagenesis and screening, eg, Re
idhaar-Olson and Sauer (science 241: 53-57, 1988) or Bowie and Sauer
(Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989). Briefly, the authors found that co-randomizing multiple positions in a polypeptide, screening for functional polypeptides, and then suddenly determining the range of possible substitutions at individual positions. Disclosed are methods for sequencing a mutagenized polypeptide. Other methods that can be used include phage display (eg, Lowman et al., Biochem. 30: 10832-10837).
Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/062.
No. 04), and region-directed mutagenesis (Derbyshire et al., Gene 46:14).
5, 1986; Ner et al., DNA 7: 127, 1988).

Variants of the disclosed zfsta2 DNA and polypeptide sequences are described in Stemmer, Nature 3
70: 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-51, 1
994 and WIPO publication WI97 / 20078 as disclosed by DNA shuffling. Briefly, mutant DNA is produced by random fragmentation of the parent DNA, resulting in randomly introduced point mutations, followed by in vitro homologous recombination by assembly using PCR. This technique can be improved with families of parent DNA, eg, allelic variants or DNA from different species, to introduce additional variability during the process. The subsequent interaction of the selection or screening, mutagenesis and assay of the desired activity is performed by selecting for the desired mutation while simultaneously selecting for deleterious changes.
Provides rapid "evolution" of sequences.

The mutagenesis methods as disclosed herein combine high throughput automated screening methods to detect the activity of the cloned mutagenized polypeptide in a host cell. Can be done. Mutagenized DNA molecules encoding the active polypeptide can be recovered from the host cells and immediately sequenced using modern equipment. These methods allow for 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 herein, one of skill in the art has identified various polypeptide fragments of SEQ ID NO: 2 that retain the properties of the wild-type zfsta2 protein,
And / or can be prepared. Such a polypeptide fragment is
-Terminal region, ferristatin and / or calmodulin homology domain, set I-IG
Domain # 1 and / or # 2, α-helical linker and C-terminal region can be included. Amino acid truncations or additions can also be present. For any zfsta2 polypeptide, including variants and fusion proteins, one of skill in the art can use the information provided in Tables 1 and 2 above to readily prepare a sufficient degenerate polynucleotide sequence encoding the variant. Can be generated.

The zfsta2 polypeptides of the present invention, eg, full-length polypeptides, biologically active fragments and fusion polypeptides, can be produced in genetically constructed 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, especially cultured cells of multicellular organisms, are preferred. Cloned
Techniques for manipulating DNA molecules and introducing exogenous DNA into various host cells are disclosed in Sambrool et al., Molecular Cloning: A Laboratory M
^{anual, 2 nd ed., Cold} Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, 1989, and Ausubel et al., Eds., Current Protocol in Molecular Biolog.
y, John Wiley and Sons, Ins., NY, 1987.

In general, a DNA sequence encoding a zfsta2 polypeptide of the present invention acts on other genetic elements required for its expression, such as, in general, transcription promoters and terminators in expression vectors. Linked as possible. The vector will also usually include one or more selectable markers and one or more origins of replication,
However, one of skill in the art will recognize that, within certain systems, the selectable marker can be provided on a separate vector, and replication of the exogenous DNA can be provided by integration into the host cell genome. Promoters, terminators, selectable markers,
The choice of vector and element is routine 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 zfsta2 polypeptide into the secretory tract of a host cell, a secretory signal sequence (or also known as a signal sequence, leader sequence, prepro sequence or pre sequence) is provided in the expression vector. Is done. The secretory signal sequence is zfst
a2, or may be derived from another secreted protein (eg, t-PA) or newly synthesized.

The secretory signal sequence is operably linked to the zfsta2 DNA sequence, ie, the two sequences are joined in the correct reading frame and direct the newly synthesized polynucleotide into the secretory pathway of the host cell. Placed in Secretory signal sequences are usually located 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences can be located elsewhere in the DNA sequence of interest (eg, Welch U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

On the other hand, a secretory signal sequence contained in a polypeptide of the present invention is used to direct another polypeptide into the secretory tract. The present invention provides such fusion polypeptides. A signal fusion polypeptide can be produced, wherein the secretory signal sequence derived from amino acid residues 1-20 of SEQ ID NO: 2 can be prepared using methods known in the art and those disclosed herein. It is operably linked to one polypeptide.

The secretory signal sequence contained in a fusion polypeptide of the invention is preferably fused amino-terminally to an additional peptide in the secretory tract to direct the additional peptide. Such structures have many uses known in the art. For example, such novel secretion signal sequence fusion constructs can direct the secretion of active components of proteins that are not normally secreted. Such fusions can be used in vivo or in vitro to direct the peptide through the secretory tract.

A cultured mammalian cell or a suitable host within the present invention. Exogenous DNA,
Methods for introduction into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Som.
atic 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., Supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15: 73, 1).
993; Ciccarone et al., Focus 15:80, 1993).

The production of recombinant polypeptides in cultured mammalian cells has been described, for example, by levinson
U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950
No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells are COS-
1 (ATCC No. CRL 165), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 16
32), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham
J. Gen. Viro. 36: 59-72, 1977), and the Chinese hamster ovary (eg, CHO-K1; ATCC No. CCL61) cell line.

Additional suitable cell lines are known in the art and are available from public depositories, for example, the American Type Culture Collection, Manassas, VA. Generally, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, for example, U.S. Pat. No. 4,956,288. Other suitable promoters include those from the metallothionein gene (U.S. Pat. Nos. 4,579,821 and 4,601,978), the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which exogenous DNA has been inserted. Such cells are usually "transfectants"
Is referred to as Cells that are cultured in the presence of the selection agent and are capable of transmitting the gene of interest to their progeny are referred to as "suitable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. The selection is performed in the presence of a neomycin-type drug, such as G-418 or the like.

The selection system, a method referred to as “amplification,” is also used to increase the level of expression of the gene of interest. Amplification involves culturing the transfectant in the presence of low levels of the selection agent, and then increasing the amount of the selection agent to select for cells that produce high levels of the introduced gene product. Will be implemented.
A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.

Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Other 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, can be trans-transformed by means such as FACS classification or magnetic bead separation techniques. It can be used to classify untransfected and transfected cells.

[0127] Other higher eukaryotic cells, such as insect cells, plant cells and bird cells, can also be used as hosts. The use of Agrobacterium rhizogenes as a vector to express genes in plant cells is described by Sinka
r, et al., J. Biosci. (Bangalore) 11: 47-58, 1987.
Transformation of insect cells, and the production of foreign polypeptides therein, has been described by Guarino
No. 5,162,222; and WIPO Publication WO 94/06463.

Insect cells can be infected with a recombinant baculovirus normally derived from Autographa californica nuclear polyhedrosis virus (AcNPV). DNA encoding the zfsta2 polypeptide can be obtained by one of two methods.
It is inserted into the baculovirus genome in place of the AcNPV polyhedrin gene coding sequence. The first method is a conventional method of homologous DNA recombination between a wild-type AcNPV and a transfer vector containing zfsta2 having an AcNPV sequence at its end. Suitable insect cells, eg, SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a zfsta2 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator and flanking sequences.

King, LA and Possee, RD, The Baculovirus Exprossion System: A Lab
oratory Guide, London, Chapman &Hall;O'Reilly, DR., Baculovirus E
xpression Vector: A Laboratory Manual, New York, Oxford University Press
., 1994; and Richardson, CD, Ed., Baculovirus Expression Protocols. M
See ethods in Molecular Biology, Totowa, NJ, Humana Press, 1995. Natural recombination in insect cells is achieved by zfst driven by the polyhedrin promoter.
would result in a recombinant baculovirus containing a2. Recombinant virus stocks are produced by methods commonly used in the art.

A second method for producing recombinant baculovirus is described in Luckow (Luckow, V
A, et al., J. Virol 67: 4566-79, 1993). This system is based on the Bac-to-Bac ™ kit (Lif
e Technologies, Rockville, MD). This system is
To transfer the DNA encoding the zfsta2 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called the “bacmid”, a transfer vector containing the Tn7 transposon, pFastBacI (TM) (Life
Technologies). The PFastBacl ™ transfer vector uses the AcNPV polyhydrin promoter to drive expression of the gene of interest, in this case, zfsta2.

However, pFastBacl ™ can be modified to a considerable degree. The polyhydrin promoter is removed and expressed early in baculovirus infection, and the baculovirus basic protein promoter known to be advantageous for expressing secreted proteins (also Pcor, p6
.9 or MP promoter). Hill-Perk
ins, MS and Possee, RD, J. Gen. Virol. 71: 971-6, 1990; Bonning, B.
C. et al., J. Gen. Virol. 75: 1551-6, 1994; and Chazenbalk, GD, and
See Rapoport, B., J. Biol Chem. 270: 1543-9,1995.

In such transfer vector constructs, short or long versions of the basic protein promoter may be used. Further, a transfer vector in which a natural zfsta2 secretory signal sequence is replaced by a secretory signal sequence derived from an insect protein can be constructed. For example, ecdysteroid glucosyltransferase (EGT), honeybee Melittin (Invitrogen, Carlsbad, C
A) Or baculovirus gp67 (PharMingem, San Diego, CA) can be used in the construct to replace the native secretory signal sequence.

Further, the transfer vector may be a C- or N-type of the expressed zfsta2 polypeptide.
-Can include an in-frame fusion with an epitope tag at the terminus, such as a DNA encoding a Glu-Glu epitope tag (Grussenmeyer, T. et al., Peoc.
Natl. Acad. Sci. 82: 7952-6, 1985). Using techniques known in the art, the transfer vector containing zfsta2 is used to transform E. coli and screened for a bamida containing the intermittent lacZ gene, a representation of a recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is
Isolated using standard techniques, and Spodopter frugiperda (Spodopter
a frugiperda) cells, such as Sf9 cells. A recombinant virus expressing zfsta2 results. Recombinant virus
Stocks are manufactured by methods commonly used in the art.

The recombinant virus is used to infect host cells, typically cell lines derived from the armyworm larva, Spodoptera frugiperda. In general, Glick and Pasternak, Molecular Biotechnology: Principles and Applicati
See on of Recombinant DNA, ASM Prss, Washington, DC, 1994. Another suitable cell line is H from Trichoplusia ni.
igh FiveO ™ cell line (Invitrogen) (US Pat. No. 5,300,435).

Commercially available serum-free media is used to grow and maintain the cells.
Suitable media are SF900II ™ (Life Technologies), or EST 921 ™ (Expression Systems) for Sf9 cells; and Ex-C for T. ni cells.
ellO405 ™ (JRH Biosciences, Lenza, KS) or Express FiveO ™ (L
ife Technologies). Cells are grown from an inoculation density of about 2-5 × 10 ⁵ cells to 1-2 × 10 ⁶ cells, at which point the recombinant virus stock is at 0.
It is added at a multiplicity of infection (MCI) of 1-10, more typically around 3.

Recombinant virus-Infected cells are typically recombinant 12-72 hours after infection.
Produces a zfsta2 polypeptide and secretes it into the medium with various efficiencies. Cultures are usually harvested 48 hours after infection. Centrifugation is the medium (supernatant)
Used to separate cells from The supernatant containing the zfsta2 polypeptide is filtered through a micropore, usually 0.45 μm pore size filter. The methods used are generally described in available laboratory manuals (King, LA ann.
d Possee, RD, supra; O'Reilly, DR et al., supra; Richardson, CD,
Above). Subsequent purification of the zfsta2 polypeptide from the supernatant can be achieved using the methods described herein.

[0137] Fungal cells, such as yeast cells, can also be used within the present invention. In this regard,
A yeast species of particular interest is Saccharomyces cereviae.
siae), Pichia pastoris and Pichia methanolica (pich)
ia methanolica). Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, in Kawa.
saki, 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. 5,037,74.
And Murray et al., U.S. Patent No. 4,845,075.

Transformed cells are selected for by a phenotype determined by a selectable marker, usually drug resistance, or the ability to grow in the absence of a particular nutrient (eg, leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT disclosed by Kawasaki et al. (U.S. Pat.No. 4,931,373), which allows for the selection of cells transformed by growth in a glucose-containing medium.
One vector system.

Suitable promoters and terminators for use in yeast are glycolytic enzyme genes (eg, Kawasaki, US Pat. No. 4,599,311; Kingsman et al., US Pat. No. 4,615,974; and Bitter, US Pat. No. 4,977,092). And those from the alcohol dehydrogenase gene. See also U.S. Patent Nos. 4,990,446; 5,063,154; 5,139,936; and 4,661,454.

Other enzymes, such as Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis
veromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolic
Transformation systems for a), Pichia guillermondii, and Candida maltosa are known in the art.

For example, see Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cr.
See egg, U.S. Patent No. 4,882,279. Aspergillus cells, Mckni
ght et al., US Pat. No. 4,935,349. A method for transforming Acremonium chrysogenum is disclosed by Sumino., US Patent No. 5,162,228. Neurospora (N
Eurospora) is described in Lambowitz, U.S. Pat. No. 4,486,53.
No. 3 disclosed.

For example, the use of Pichia methanolica as a host for the production of recombinant proteins has been described by Raymond, US Pat. No. 5,716,808, Raymond, US Pat. No. 5,736.
No. 383, Raymond et al., Yeast 14: 11-23, 1998, and WIPO published WO 97/17450, W.
O97 / 17451, WO98 / 02536 and WO98 / 02565. DNA molecules for use in transforming P. methanolica are usually preferably linearized prior to transformation,
It will be prepared as a double-stranded circular plasmid.

For polypeptide production in P. methanolica, the promoter and terminator in the plasmid are preferably those of a P. methanolica gene, such as the 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 a complete expression segment of the plasmid with the host DNA sequence at both ends.

A preferred selectable marker for use in Pichia methanolica encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC. 4.1.1.21), which allows the growth of ade2 host cells in the absence of adenine. P. methanolica ADE2 gene. For large-scale industrial processes where it is desired to minimize the use of methanol, it is preferable to use host cells in which both methanol utilization genes (AUG1 and AUG2) have been deleted.

For production of secreted proteins, host cells that lack the vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding the polypeptide of interest into P. methanolica cells. Having an electric field strength of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm, and a time constant (r) of 1 to 40 ms, most preferably about 20 ms;
Electroporation using a pulsed electric field that decays exponentially
Preferably, P. methanolica cells are transformed.

Prokaryotic host cells, such as bacterial strains of E. coli, Bacillus, and other genera, are also useful host cells in the present invention. Techniques for transforming those hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, eg, Sambrook et al., Supra). When expressing a zfsta2 polypeptide in bacteria, such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by bacterial secretory sequences.

In the former case, the cells are lysed and the granules are collected and denatured using, for example, guanidine isothiocyanate or urea. Next, the denatured polypeptide is regenerated and dimerized by diluting the denatured polypeptide, for example, by dialysis against a solution of a combination of urea and reduced and oxidized glutathione, followed by dialysis against a buffer solution. Can be In the latter case, the polypeptide is soluble and functional from the periplasmic space by disrupting the cells to release the contents of the periplasmic space (eg, by sonication or osmotic shock) and recovering the protein. Recovered in form, thereby avoiding the need for denaturation and regeneration.

The adenovirus system can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions such that the cells do not divide rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in a cell factory and then exposed to an adenovirus vector encoding a secreted protein of interest. The cells are then grown under serum-free conditions that allow the infected cells to survive for several weeks without significant cell division.

On the other hand, 293 cells infected with the adenovirus vector can be grown at relatively high cell densities, either as adherent cells or in suspension culture, to produce significant amounts of protein (Garnier et al., Cytotechnol. 15: 145-55, 19
94). By either protocol, the expressed and secreted heterologous protein can be repeatedly isolated from the supernatant of the cell culture. Infected29
In the three-cell production protocol, non-secreted proteins are effectively obtained.

The transformed or transfected host cells are cultured according to conventional methods in a culture medium containing the nutrients and other components required for growth of the selected host cells. A variety of suitable media, such as defined and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The medium can also include components such as growth factors or serum, if needed.

The growth medium generally comprises cells containing the exogenously added DNA, for example, in an essential nutrient supplemented with a selectable marker carried on an expression vector or co-transfected into a host cell. Will choose by drug choice or nutritional deficiency. P. methanolica cells are cultured at a temperature of about 25 ° C. to 35 ° C. in a medium comprising a suitable carbon source, nitrogen source and micronutrients. The liquid culture is provided with sufficient aeration by conventional means, such as shaking a small flask or sparging a fermenter. A preferred culture medium for P. methanolica is YEPD (2% D
-Glucose, 2% Bacto (TM) peptone (Difco Laboratories, Detroit,
MI), 1% Bacto ™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

It is preferred that the polypeptides of the present invention be purified to a purity of 80% or more, more preferably 90% or more, even more preferably 95% or more, and contaminating macromolecules, especially other proteins and Particularly preferred is a pharmaceutically pure state that is at least 99.9% pure with respect to the nucleic acid and free of infectious and pyrogenic agents. Preferably, the purified polypeptide is substantially free of other polypeptides, especially other polypeptides of animal origin.

The expressed recombinant zfsta2 polypeptide (or chimeric zfsta2 polypeptide) can be fractionated and / or purified using conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotropic agent extraction are used for sample fractionation. Typical purification steps include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. Suitable chromatography media are
Derivatized dextran, agarose, cellulose, polyacrylamide,
Includes special silicas and the like.

PEI, DEAE, QAE and Q derivatives are preferred. Typical chromatographic media are those derivatized with phenyl, butyl or octyl groups, such as phenyl-Sepharose FF (pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryvi).
lle, PA), octyl-Sepharrose (Pharmacia) and the like; or polyacrylic resins such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins, and the like, which are insoluble under the conditions in which they are used. Includes

The supports can be modified with amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or reactive groups that allow the attachment of proteins by carbohydrate moieties. Examples of coupling chemistries include carboxyl and amino derivatives for cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation and carbodiimide coupling chemistries. These and other solid media are well known in the art and are widely used, and are available from commercial suppliers.

[0156] Methods for coupling ligand or receptor polypeptides to a support medium are well known in the art. The choice of a particular method is conventional and is determined in part by the nature of the support chosen. For example, Affinity Chromatograpy: Pr
See inciples & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploiting the physical properties of the attached label or epitope. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins and those proteins comprising a polyhistidine tag. Briefly, the gel is first charged by divalent metal ions to form a chelate (Sulkow
ski, Trends in Biochem. 3: 1-7, 1985). Histidine-rich proteins are adsorbed to this matrix with different affinities, depending on the metal ions used, and are eluted by competitive elution, pH reduction, or the use of strong chelators. There will be.

Other purification methods 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). In a further aspect of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, a maltose-binding protein, a FLAG tag, a Glu-Gku tag, an immunoglobulin domain) is
It can be configured to facilitate purification.

A typical method for purifying protein structures with an N-terminal or C-terminal affinity tag is to purify the protein using chromatography known in the art, for the affinity tag epitope. Includes the use of antibodies. SDS-PAGE
Western analysis, amino acid analysis and N-terminal sequencing can be performed to confirm the identity of the purified protein.

[0160] A protein regeneration (and, optionally, reoxidation) method may be conveniently used.
It is preferred to purify the protein to a purity of 80% or more, more preferably 90% or more, even more preferably 95% or more, and 99.p. to contaminating macromolecules, especially other proteins and nucleic acids. Pharmaceutically pure forms with a purity of 9% or more and no infectious and pyrogenic agents are particularly preferred. Preferably, the purified protein is substantially free of other proteins, especially other proteins of animal origin.

Proteins / polypeptides that bind zfsta2 (eg, zfsta2-binding receptors)
Can also be used for purification of zfsta2. zfsta2-binding proteins / polypeptides can be used on solid supports such as agarose, cross-linked agarose, glass,
Identified on beads of cellulose resin, silica-based resin, polystyrene, cross-linked polyacrylamide, or similar 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 is generally formed in the form of a column, and a fluid containing the zfsta2 polypeptide is passed through the column one or more times to allow binding of the zfsta2 polypeptide to the ligand-binding or receptor polypeptide. To be. Then joined
The zfsta2 polypeptide is lysed using changes in salt concentration, chaotropic (guanidine HCl), or pH, disrupting ligand-receptor binding.

Further, according to methods described in the art, a polypeptide fusion or hybrid zfsta2 protein can be used to combine another protein, particularly another follistatin family protein (eg, FRP, SPARC, agrin or hevin), or a heterologous protein. In combination, it is constructed using the region or domain of zfsta2 of the present invention (Samb
Altschul et al., Picard, Cur. Opin. Biology, 5: 5.
11-5, 1994 and references therein. These methods allow for the determination of the biological significance of a large domain or region in a polypeptide of interest. Such hybrids alter reaction kinetics, binding, suppress or extend substrate specificity, or alter the tissue and cellular localization of the polypeptide, and are applied to polypeptides of unknown structure. You.

[0164] Fusion proteins can be prepared by methods known to those of skill in the art by preparing the individual components of the fusion protein and chemically conjugating them. On the other hand, polynucleotides encoding both components of the fusion protein, in correct reading frame alignment, can be produced using known techniques and expressed by the methods described herein. For example, some or all of the domains that confer biological function may comprise a zfsta2 of the invention and another family member, such as FRP.
From its functionally equivalent domain.

Such domains include, but are not limited to: secretory signal sequences, follistatin homologous domains, calmodulin homologous domains, I-group IG domains # 1 and # 2, N or C-terminal domain and α-helical linker. Such fusion proteins may have the same or similar biological functional profile as the polypeptides of the invention or other known follistatin family proteins described herein, depending on the fusion being constructed. Is expected to have Further, such fusion proteins can also exhibit other properties, as disclosed herein.

[0166] A zfsta2 polypeptide or a fragment thereof may also be prepared by chemical synthesis. The zfsta2 polypeptide is a monomer or multimer; may be glycosylated or unglycosylated; may be pegylated or unpegylated; and may include the starting methionine amino acid residue or You don't have to. Polypeptides, particularly polypeptides of the present invention, may also be those of Merrifield, J.
Am. Chem. Soc. 85: 2149, 1963, Stewart et al., “Sold Phase Peptide Synth
^{esis "(2 nd Edition),} (Pierce Chemical Co., Rockford, IL, 1984), Bayer &
Rapp Chem. Pept. Prot. 3: 3, 1986, and Atherton et al., Solid Phase Pepti
de Synthesis: can also be synthesized as described by A Practical APProach, IRL Press, Oxford, 1989.

As described above, the disclosed polypeptides can be used to construct zfsta2 variants and functional fragments of zfsta2. Such variants and extracellular domains are likely to be zfsta2 agonists. Another type of zfsta2 agonist is provided by anti-idiotype antibodies and fragments thereof that mimic the extracellular domain of zfsta2. In addition, recombinant antibodies comprising an anti-idiotype variable domain that mimics the zfsta2 extracellular domain can be used as an agonist (eg, Monfardini et al., Proc. Assoc. Am. Physidians 108: 4).
20, 1996).

[0168] zfsta2 agonists can also be constructed using a binding library. Methods for constructing and screening phage display and other binding libraries are described, for example, in Key et al., Phage Display of Peptides and Proteins (Academic
Press 1996), Verdine, U.S. Patent No. 5,783,384, Key, et al., U.S. Patent No. 5,7477,334 and Kauffman et al., U.S. Patent No. 5,723,323.

The invention also provides antagonists that bind to a receptor that binds to or binds to a zfsta2 polypeptide, thereby inhibiting or eliminating zfsta2 function. Such zfsta2 antagonists include antibodies;
Oligonucleotides that bind to the polypeptide or any of its receptors; including natural or synthetic analogs of the zfsta2 polypeptide that retain the ability to bind the receptor but do not result in either ligand or receptor signaling .

[0170] Such analogs can be peptides or peptidomimetic compounds. Natural or synthetic small molecules that bind to the receptor for the zfsta2 polypeptide and prevent signaling are also contemplated as antagonists. As such, zfsta2 antagonists will be useful as therapeutic agents for treating certain diseases in which blocking a signal from either the zfsta2 ligand or receptor would be beneficial.

[0171] zfsta2 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 zfsta2. In addition to those assays disclosed herein, samples are tested for inhibition of zfsta2 activity by various assays designed to measure receptor binding or stimulation / inhibition of zfsta2-dependent cellular responses. obtain. For example, a zfsta2-responsive cell line can be transfected with a reporter gene construct that responds to a zfsta2-stimulated cellular pathway.

This type of reporter gene construct is known in the art, and generally comprises a zfsta2-DNA response element operably linked to a protein that can be assayed, for example, a luciferase-encoding gene. There will be. DNA response elements include cyclic AMP response element (CRE), hormone response element (HRE), insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87: 5273-7, 1
990) and serum response element (SRE) (Shaw et al., Cell 56: 563-72, 1989), but are not limited thereto. Cyclic AMP response element is Roest
ler et al., J. Biol. Chem. 263 (19): 9063-6, 1988 and Habener, Molec. End.
ocrinol. 4 (8): 1087-94, 1990.

Hormone response elements are reviewed in Beato, Cell 56: 335-441, 1989. Candidate compounds, solutions, mixtures or extracts are tested for their ability to inhibit the activity of zfsta2 on target cells, as evidenced by reduced zfsta2 stimulation of reporter gene expression. This type of assay will detect compounds that directly block zfsta2 binding to cell surface receptors and compounds that block steps in the cellular pathway following reporter ligand binding.

On the other hand, compounds or other samples can be used to bind zfsta2 to the receptor using zfsta2 labeled with a detectable label (eg, ¹²⁵ I, biotin, horseradish peroxidase, FITC, and the like). It can be tested for direct blocking. In this type of assay, the receptor is labeled
The ability of a test sample to inhibit binding of zfsta2 is an indication of inhibitory activity that can be ascertained through secondary assays. The receptor used in the binding assay can be a cellular receptor, or an isolated, immobilized receptor.

The present invention also provides isolated and purified zfsta2 polynucleotide probes and / or primers. The probe and / or primer is RN
A or DNA. DNA can be either cDNA or genomic DNA. Polynucleotide probes and primers are single-stranded or double-stranded DNA or RNA, generally synthetic oligonucleotides, but can be generated from cloned cDNA or genomic sequences or complement thereof.

Analytical probes are generally at least 20 nucleotides, but somewhat shorter probes (14-17 nucleotides) can be used. The PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Shorter polynucleotide probes can be used if small regions of the gene are targeted for analysis.
For whole gene analysis, a polynucleotide probe comprises all exons or more.

[0177] Such probes can also be used in hybridizations to detect the presence or quantify the amount of the zfsta2 gene or mRNA transcript in a sample. The zfsta2 polynucleotide probe can be used for diagnostic purposes using techniques such as fluorescence in situ hybridization (FISH) or immunohistochemistry.
Can be used to hybridize to A or RNA targets. Polynucleotide
Probes can be used to identify the gene encoding the zfsta2-like protein.

[0178] Such probes can also be used to screen libraries for related zfsta2 sequences. Such screens are performed under low stringency conditions that allow the identification of sequences that are substantially homologous to the probe sequence, but do not require complete homology. Such methods and conditions are well known in the art and are described, for example, in Sambrook et al., Molecular Cloning.
See: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989.

[0179] Such stringent conditions are described herein. The library is genomic DNA
Alternatively, it can be produced from DNA. Polynucleotide probes are also useful for Southern, Northern, or slot blots, colony and plaque hybridizations, and in situ hybridizations. A mixture of different zfsta2 polynucleotide probes can be prepared that enhances sensitivity, or detection of low copy number targets, in a screening system.

[0180] Nucleic acid molecules can be used to detect expression of the zfsta2 gene in a biological sample. In a basic assay, a single-stranded probe molecule is combined with RNA isolated from a biological sample under conditions of temperature and ionic strength that promote base pairing between the probe and the target zfsta2 RNA species. Incubate. After separating the unbound probe from the hybridized molecule, the amount of hybrid is detected.

[0181] A method for detecting the presence of zfsta2 RNA in a biological sample is facilitated, the method comprising: a) under stringent hybridization conditions, a zfsta2 nucleic acid probe; Contacting any of the isolated test RNA molecules, or ii) a nucleic acid molecule synthesized from the isolated RNA molecule, wherein the probe has the nucleotide sequence of SEQ ID NO: 1 or 3 or their complement. And b) detecting the formation of a hybrid of the nucleic acid probe and either the test RNA molecule or the synthesized nucleic acid molecule, wherein the nucleic acid probe comprises a nucleotide sequence comprising: The presence indicates the presence of zfsta2 RNA in the biological sample.

[0182] Well-established hybridization methods for RNA detection include Northern analysis and dot / slot blot hybridization (eg, Ausub
el, p4-1 to 4-27, Wu et al. (eds.), “Analysis of Gene Express
ion at the RNA Level ”, in Methods in Gene Biotechnology, p. 225-39, CRC
Press, Inc., 1997). Nucleic acid probes may be detectably labeled with a radioisotope, eg, ³² P or ³⁵ S. On the other hand, zfsta2 RNA can be detected by non-radioactive hybridization methods (eg Isaac (ed.), P.
rotocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Pres
s, Inc., 1993). Typically, non-radioactive detection is achieved by enzymatic conversion of a chromogen or chemiluminescent substrate. Exemplary non-radioactive components include biotin, fluorescein and digoxigenin.

The zfsta2 oligonucleotide probes are also useful for in vivo diagnostics. By way of example, a ¹⁶ F-labeled oligonucleotide is administered to a subject,
And can be made visible by positron emission tomography (Tavitian et al., Nat. Med.
4: 467, 1998).

Many diagnostic methods use the polymerase chain reaction (PC) to increase the sensitivity of the detection method.
R). Standard techniques for performing PCR are well known (generally, Mathew (ed.), Protocols in Human Molecular Genetics, Humana Press, Inc.).
., 1991; White (ed.), PCR Protocols: Current Methods and Applications, H
umana Press, Inc., 1993; Cotter (ed.), Molecular Diagnosis of Cancer, Hu
mana Press, Inc., 1996; Hanausek and Walaszed (eds.), Tumor Marker Proto
cols, Humana Press, Inc., 1998; Lo (ed.), Clinical Applications of PCR,
Humana Press, Inc., 1998 and Meltzer (ed.), PCR in Bioanalysis, Humana Pr
ess, Inc., 1998). The PCR primers are designed to amplify a specific zfsta2 region, eg, a follistatin homology domain encoded by nucleotides 250-456 of SEQ ID NO: 1, and a sequence encoding calmodulin encoded by nucleotides 580-810 of SEQ ID NO: 1. Can be done.

One variant of PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is isolated from a biological sample, reverse transcribed into cDNA, and the cDNA is incubated with a zfsta2 primer (eg, Wu et al. (Eds.), “Rapid Isolation of Specific cDNA or Genes by PCR
", In Methods in Gene Biotechnology, P. 15-28, CRC Press, Inc. 1997). Next, PCR is performed and the products are analyzed using standard techniques.

As an illustration, RNA is isolated from a biological sample using, for example, the guanidinium-thiocyanate cell lysis described above. On the other hand, solid-phase techniques can be used to isolate mRNA from cell lysates. Reverse transcription reactions can be primed with isolated RNA using random oligonucleotides, short homopolymers of dT, or zfsta2 antisense oligomers. Oligo-dT primers provide the advantage that various mRNA nucleotide sequences that can provide a control target sequence are amplified. The zfsta2 sequence is amplified by the polymerase chain reaction using two flanking oligonucleotide primers, typically 20 bases in length.

[0187] PCR amplification methods can be detected using a variety of approaches. For example, PCR products are fractionated by gel electrophoresis and visualized by ethidium bromide staining. On the other hand, the fractionated PCR products can be transferred to a membrane, hybridized with a detectably labeled zfsta2 probe, and tested by autoradiography. Additional other approaches are chemiluminescence detection and
It involves the use of digoxigenin-labeled deoxyribonucleic acid trimyric acid to provide a C-TRAK colorimetric assay.

Another approach is simultaneous quantitative PCR (Perkin-Elmer Cetus, Norwal
k, CT). A fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher dye attached will specifically anneal between the forward and reverse primers. When using the 5 'endonuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye and a sequence-specific signal is generated, which rises with increasing amplification. Fluorescence intensity was measured during the PCR reaction.
It can be monitored continuously and quantified.

Another approach for the detection of zfsta2 expression is the cycling probe technique (CDT), where a single-stranded DNA target binds to an excess of DNA-RNA-DNA chimeric probe and binds the complex. Formed, the RNA portion is differentiated by RNase H, and the presence of degraded probes is detected (eg, Beggs et al., J. Clin. Microbiol
34: 2985, 1996, and Bekkuoui et al., Biotechniques 20: 240, 1996).

Another method for detection of zfsta2 SEQ ID NO: is nucleic acid sequence based amplification (NASBA)
Approaches such as cooperative amplification of templates by cross-hybridization (CATCH) and ligase chain reaction (LCR) (eg, Marshall et al., US Pat. No. 5,686,272 (1997); Dyer et al., J. Virol. Methods 60:
161, 1996; Ehricht et al., Eur. J. Biochem. 243: 358, 1997; and Chadwick.
Et al., J. Viro. Methods 70:59, 1998). Other standard methods are known to those skilled in the art.

[0191] zfsta2 probes and primers can also be used to detect and localize zfsta2 gene expression in tissue samples. Methods for such in situ hybridization are well known to those skilled in the art (eg, Choo
(ed.), In situ Hybridization Protocols, Humana Press, Inc., 1994; We et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNA by Radioacti.
ve In Situ Hybridization (RISH), “in Methods in Gene Biotechnology, pa
ges 259-278, CRC Press, Inc., 1997; and Wu et al. (eds.), “Localizaion of
DNA or Abundance of mRNA by Fluorescence In Situ Hybridization (RISH),
”In Methods in Gene Biotechnology, pages 279-289, CRC Press, Inc., 199
7).

A variety of additional diagnostic approaches are also well known to those of skill in the art (eg, Mathew (
ed.), Protocols in Human Molecular Genetics, Humana Press, Inc., 1991; C
oleman and Tsongalis, Molecular Diagnostics, Humana Press, Inc., 1996;
And Elles, molecular Diagnosis of Genetics Diseases, Humana Press Inc.,
1996).

A ligand-bound receptor (or an antibody, a member of a complement / anti-complement pair), or a binding fragment thereof, and a commercially available biosensor device (BIAcore, Pharmacia Bio)
Assay systems using sensors, Piscataway, NJ) may be conveniently used. Such receptors, antibodies, members of complement / anti-complement pairs, or fragments are immobilized on the surface of the receptor chip. Use of this device is described in Karlsson, J. Imm
unol.Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234: 554-63,1993. Receptors, antibodies, members or fragments are covalently attached to dextran fibers that are attached to gold film in flowing cells using amine or sulfhydryl chemistry.

A test sample is passed through the cells. If a ligand, epitope or opposing member of a complement / anti-complement pair is present in the sample, it binds to the immobilized receptor, antibody or member, respectively, and is detected as a change in plasmon resonance on the surface of the gold film. Cause a change in the refractive index of the medium. This system allows for the determination of on- and off-rates, from which the binding affinity is calculated and the stoichiometry of the binding is allowed.

[0195] Ligand-bound receptor polypeptides can also be used in other assay systems known in the art. Such a system is described in Scatchard analysis for the determination of binding affinity (Scatchard, Ann. NY. Acad. Sci. 51: 660-72, 194).
9) and calorimetric assay (Cunningham et al., Science 253: 545-48, 1991; Cu
nningham et al., Science 245: 821-25, 1991).

The probes and primers generated from the sequences disclosed herein are used to map the zfsta2 gene to human chromosome 4. Radial gland hybrid mapping is a somatic genetic technique that has been developed to construct a high-resolution serial map of mammalian chromosomes (Cox et al., Science 250; 245-50, 1990). Partial or complete knowledge of the gene sequence allows the design of appropriate PCR primers for use by the chromosomal radiation hybrid mapping panel. Radiation hybrid mapping panels covering the entire human genome, such as the Stanford G3 RH panel and the GeneBridge 4 RH panel, are commercially available (Research Genetics, Inc., Hud.
ntsville, AL).

[0197] These panels demonstrate rapid, PCR-based chromosomal localization and alignment of genes, sequence-labeled sites (STS), and other non-polymorphic and polymorphic markers within the region of interest. enable. This involves establishing a directly proportional physical distance between the newly discovered gene of interest and the previously mapped marker. Accurate knowledge of gene location is useful for many purposes, including: 1)
Determining whether a sequence is part of an existing contig and various forms, such as YA
Acquisition of additional surrounding gene sequences in C, BAC or cDNA clones; 2) Provision of possible candidate genes for genetic diseases showing binding to the same chromosomal region; and 3
A) Cross reference of model organisms, such as mice, to help determine the function of a particular gene.

The sequence labeled sites (STS) are also used independently for chromosome localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of chromosome. STS is defined by a pair of oligonucleotide primers used in the polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Because STSs are solely based on DNA sequences, they can be used in electronic databases, such as Dat.
abase of Sequence Tagged Sites (dbSTS), GenBank (National Center for Bio
Logical information, National Institutes of Health, Bethesda, MD, and can be searched for gene sequences of interest by mapping data contained within their short genomic landmark STS sequences.

The present invention also contemplates the use of such chromosome localization for diagnostic applications. Briefly, a probe comprising the zfsta2 gene, ie, zfsta2 DNA, or a subsequence thereof, can be used to determine if the zfsta2 gene is present on human chromosome 4 or if a mutation has occurred. . Detectable chromosomal aberrations at the zfsta2 locus include, but are not limited to:
Aneuploids, changes in gene copy number, insertions, deletions, changes in restriction sites and translocations. Such aneuploids can be identified by molecular genetic techniques, such as restriction fragment length polymorphism (RF
LP) analysis, short tandem repeat (STR) analysis using PCR techniques, and other gene linkage analysis techniques known in the art can be detected using the polynucleotides of the present invention. (Sambrook et al., Supra; Ausubel et al.,
Marian, Chest 108: 255-65, 1995).

In general, these diagnostic methods include: (a) obtaining a genetic sample from a patient; (b) combining the genetic sample with a polynucleotide probe or primer as disclosed above, Incubating the polynucleotide under conditions that will hybridize to a complementary polynucleotide sequence to produce a first reaction product; and (c) combining the first reaction product with a control reaction product. Comparing the steps;

The difference between the first reaction product and the control reaction product is an indication of the genetic abnormality in the patient. Genetic samples for use in the present invention include genomic DNA, cDNA,
And RNA. Polynucleotide Probe or primer is RNA or DN
A and can be part of SEQ ID NO: 1, the complement of SEQ ID NO: 1, or their RNA
Would comprise the equivalent. Suitable assay methods in this regard are known in the art for molecular genetics techniques, such as restriction fragment length polymorphism (RF
LP) analysis, short tandem repeat (STR) analysis using PCR techniques, ligation (Bara
ny, PCR Methods and Applications 1; 5-16, 1991), ribonucleotide protection assays, and other genetic linkage analysis techniques known in the art (Sambrook
Ausubel., Mar., Chest: 255-65, 1995).

A ribonuclease protection assay (eg, Ausubel et al., Supra, ch. 4) involves the hybridization of an RNA probe to a patient's RNA sample, followed by the reaction product (RNA-RNA hybrid) Exposure to Ase. The hybridized region of the RNA is protected from digestion. In an RCR assay, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. A change in the size or amount of product recovered is an indication of the mutation in the patient. Another PCR-based technique that can be used is single-strand conformation polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1: 34-8, 1991).

[0203] The present invention also provides anti-zfsta2 antibodies. Antibodies to zfsta2 are used as antigens.
Obtained using the product of the zfsta2 expression vector or zfsta2 isolated from a natural source.
It is. Particularly useful anti-zfsta2 antibodies “specifically bind” zfsta2. Antibodies
Ten⁶M^-1Or more, preferably 10⁷M^-1Or more, more preferably 10⁸M ^-1 Or more and most preferably 10⁹M^-1Or higher binding affinity (Ka)
Antibodies bind to zfsta2 polypeptides, peptides or epitopes.
It seems to bind differently.

The binding affinity of the antibody can be determined by one skilled in the art, for example, by Scatchard analysis (Scatchard, Ann.
NY Acad. Sci. 51: 660, 1949). Suitable antibodies include specific domains, such as the zfsta2 follistatin homology domain (amino acid residues 65 to about 133 of SEQ ID NO: 2), the calmodulin homology domain (amino acid residue 1 of SEQ ID NO: 2).
75-250), or I-group IG domain # 1 or # 2 (approximately located at amino acid residues 251-334 of SEQ ID NO: 2 and amino acid residues 335-432 of SEQ ID NO: 2)
Antibodies that bind to zfsta2.

Anti-zfsta2 antibodies can be generated using antigenic zfsta2 epitope-bearing peptides and polypeptides. The antigenic epitope-bearing peptides and polypeptides of the present invention comprise a sequence of at least 9, preferably 15 to about 30, amino acids contained within SEQ ID NO: 2. However, peptides or polypeptides comprising a majority of the amino acid sequences of the invention, including amino acids of any length up to 30-50 amino acids, or up to the entire amino acid sequence of the polypeptide of the invention, also include: Useful for inducing antibodies that bind zfsta2. It is desired that the amino acid sequence of the epitope-bearing peptide be selected to provide substantial solubility in aqueous solvents (ie, the sequence contains relatively hydrophilic residues and hydrophobic residues Are preferably avoided). Furthermore, the amino acid sequence containing the proline residue also
Desired for antibody production.

Polyclonal antibodies against recombinant zfsta2 protein, or zfsta2 isolated from natural sources, can be prepared using methods well known to those skilled in the art. For example,
Green et al., “Production of polyclonal Antisera,” in Immunochemical Pr
otecols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.
, “Expression of foreign proteins in E. coli using plasmid vectors and
purification of specific polyclonal antibodies, ”in DNA Cloning 2: Expr
ession Systems, 2 ^nd Edition, such as Glover., (eds.), page 15 (Oxford Unive
See rsity press 1995. The immunogenicity of a zfsta2 polypeptide can 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 zfsta2
Or a part thereof and an immunoglobulin polypeptide or a maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. Where the polypeptide moiety is "hapten-like", such moiety is conveniently conjugated to a macromolecular carrier (eg, limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. Can be joined or joined.

Polyclonal antibodies are typically raised in animals, such as horses, cows, dogs, chickens, rats, mice, rabbits, hamsters, rats, goats or sheep, but the anti-zfsta2 antibodies of the invention Can also be derived from near human primate antibodies. General techniques for generating diagnostically and therapeutically useful antibodies in baboons are described, for example, in Goldenberg et al., International Patent Application Nos. WO 91/11465 and Losman et al., Int. J. Cancer 46: 310, 1990 can be found. Antibodies can also be raised in transgenic animals, such as transgenic sheep, cattle, goats or pigs, and can be expressed in denatured forms in yeasts and fungi and in mammalian and insect cells.

On the other hand, monoclonal anti-zfsta2 antibodies can be generated. Rodent monoclonal antibodies against a specific antigen can be obtained by methods known to those skilled in the art (eg, Kohler et al., Nature 256: 495 (1975), Coliga et al., (Eds.), Curren
t Protocols in Immunology, Vol. 1, page 2.5.1-2.6.7 (John Wiley & Sons 1
991), Picksley et al., “Production of monoclonal antibodies against prot.
eins expressed in E. coli, "in DNA Cloning 2: Expression Systems, 2 nd E
dition, Glover et al. (eds.), page 93 (see Oxford University Press 1995).

[0210] Briefly, monoclonal antibodies were tested for the presence of antibody production by injecting a composition comprising the zfsta2 gene product into mice and removing serum samples;
The spleen is removed to obtain B-lymphocytes, the lymphocytes are fused with myeloma cells to produce a hybridoma, the hybridoma is cloned, a positive clone producing an antibody to the antigen is selected, and an antibody to the antigen is selected. Culturing the resulting clones and isolating the antibody from the hybridoma culture.

Further, the anti-zfsta2 antibodies of the present invention can be derived from human monoclonal antibodies. Human monoclonal antibodies are obtained from transgenic mice that have been constructed to produce specific human antibodies in response to antigenic challenge. In this technique,
Elements of the human heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line that contains targeted disruption of the endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to generate human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nat. Gen.
et. 7: 13, 1994, Lonberg et al., Nature 368: 856, 1994, and Taylor et al.,
Inc. Immun. 6: 576, 1994.

[0212] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, eg, Coligan, page 2.
7.1-2.7.12 and page 2.9.1 ^ 2.9.3; Baines et al., ”Purification of Imunoglob
ulin G (IgG) ”, Methods in Molecular Biology, vol, 10, p. 79-104 (The Hu
mana Press, Inc. 1992)).

For certain uses, it is desirable to prepare fragments of the anti-zfsta2 antibody. Such antibody fragments are obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments are obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be generated by enzymatic digestion of an antibody with pepsin to provide a 5S fragment designated as F (ab ') ₂ . This fragment can be further degraded using a thiol reducing agent to generate 3.5S Fab 'monovalent fragments.

Optionally, the degradation reaction can be performed using a blocking group for a sulfhydryl group resulting from the dissociation of a disulfide bond. Alternatively, oxygen degradation with pepsin produces two unit Fab fragments and Fc fragments directly. These methods are described, for example, in Goldenberg, U.S. Patent No. 4,331,647.
No., Nisonoff et al., Arcg Biochem. Biophys. 89: 230, 1960, Porter, Bioche.
m. J. 73: 119, 1959, Edelman et al., in Methods in Enzymology vol. 1, p.
422 (Academic Press 1967), and Coligan, supra.

[0215] Other methods of degrading antibodies, eg, to form monovalent L-H chain fragments
H chain degradation, additional degradation of fragments, or other enzymatic, chemical or genetic techniques can also be used, as long as the fragments bind to the antigen recognized by the intact antibody. For example, Fv fragments comprise an association of _VH and _VL chains. This meeting, In
Bar et al., Proc. Natl. Acad. Sci. USA 69: 2659, 1972, as described.
Can be non-shared. On the other hand, the variable chains can be linked by intermolecular disulfide bonds or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Crit. Rew. Biotech. 12: 437, 1992).

[0216] Fv fragments comprise V _H and V _L chains connected by a peptide linker. These single-chain antigen binding proteins (scFvs) are prepared by constructing a structural gene comprising DNA sequences encoding the _VH and _VL domains linked by an oligonucleotide. The structural gene is subsequently inserted into an expression vector that is introduced into a host cell, eg, E. coli. Recombinant host cells synthesize a single-chain polypeptide with a linker peptide bridging the two V domains. ScFv
For example, Whitlow etc., Methods: A Companion to Met
heds in Enzymology 2:97, 1991. Bird et al., Science 242: 4
23, 1988, Ladner et al., U.S. Patent No. 4,946,778, Pack et al., Bio / Techno
logy 11: 1271, 1993 and Sandhu, supra.

As an example, scFv can be used to expose lymphocytes to zfsta2 polypeptides in vitro and select an antibody display library on phage or similar vectors (eg, immobilized or labeled zfsta2). (Through the action of proteins or peptides). Genes encoding polypeptides with potential zfsta2 polypeptide binding domains can be phage (phage display) or bacteria, such as E. coli. It is obtained by screening a random peptide library displayed on E. coli. The nucleotide sequence encoding the polypeptide can be obtained through a number of means, such as random mutagenesis and random polynucleotide synthesis.

These random peptide display libraries screen for known targets, which can be proteins or polypeptides, such as ligands or receptors, biological or synthetic macromolecules, or peptides that interact with organic or inorganic substances. Can be used to Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Pat. No. 5,223,409; Ladner et al., US Pat. No. 4,946,778).
No. 5,403,484 and Ladner et al., US Patent No. 5,571,698 and Kay et al., Phage Display of Peputides and Proteins
(Academic Press, Inc. 1996)), and random peptide display libraries and kits for screening such libraries are described, for example, in Clonte
ch (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs
, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piccataway, M
It is commercially available from J). Random peptide display libraries can be screened using the zfsta2 sequences disclosed herein to identify proteins that bind to zfsta2.

[0219] Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") are the D of the antibody of interest.
Obtained by constructing the gene encoding CR. Such a gene is
For example, prepared by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (eg, Larrick et al., Methods: A Compan
ion to Methods in Enzymology 2: 106, (1991), Courtenay-Luck, “Genetic Ma
nipulation of Monoclonal Antibodies, ”in Monoclonal Antibodies: Product
ion, Engineering and Clinical Application, Ritter, etc., (eds.), page 16
6 (Cambridge University Press 1995), and Ward et al., “Genetic Manipulat
ion and Expression of Antibodies, ”in Monoclonal Antibodies: Principles
and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)
checking).

[0220] On the other hand, anti-zfsta2 antibodies can be derived from "humanized" monoclonal antibodies. Humanized monoclonal antibodies are produced by transferring the mouse complementary determining regions from the mouse immunoglobulin heavy and light variable chains into human variable domains. Next, typical residues of the human antibody are substituted in the backbone region of the murine counterpart. The use of antibody components derived from humanized monoclonal antibodies avoids possible problems related to the immunogenicity of the murine constant region. Common techniques for cloning murine immunoglobulin variable domains include, for example,
Natl. Acad. Sci. USA 86: 3833, 1989.

[0221] Techniques for generating humanized monoclonal antibodies are described, for example, in Jones.
, Nature 321: 522, 1986, Carter, etc., Proc. Natl. Acad. Sci. USA 89
: 4285, 1992, Sandhu, Crit. Rey. Biotech. 12: 437, 1992, Singer et al., J
Tmmun. 150: 2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (
Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,
”In Protein Engineering: Principles and Practice, Cleland et al. (Eds.)
, pages 399-434 (John Wiley & Sons, Inc. 1996), and Queen et al., U.S. Patent No. 5,693,762 (1997).

[0222] Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-zfsta2 antibodies or antibody fragments using standard techniques. For example, Green etc., “Production of Ployclonal Antisera” in Methods in Mole
cular Biology: Immunochemical Protocols, Manson (ed.), P. 1-2 (Humana Pr
ess 1992).

See also Coligan, supra, p. 2.4.1-2.4.7. On the other hand, monoclonal anti-idiotype antibodies can be prepared by techniques described above, using an anti-zfsta2 antibody or antibody fragment as the immunogen. Alternatively, humanized anti-idiotype antibodies or near-human primate anti-idiotype antibodies can be prepared using the above techniques. Methods for preparing anti-idiotype antibodies are described, for example, in Irie, U.S. Pat.No. 5,208,146, Greene et al., U.S. Pat.
5,637,677 and Varthakovi and Minocha, J. Gen. Virol 77: 1875, 1996.

The antibodies or polypeptides described herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and the conjugates can be used for in vivo diagnostic or therapeutic applications Can be used for For example, a polypeptide or antibody of the invention can be used to identify or treat a tissue or organ that expresses a corresponding anti-complementary molecule (eg, an integrin or antigen, respectively). More specifically, a zfsta2 polypeptide or anti-zfsta2 antibody, or biologically active fragment or portion thereof, binds a detectable or cytotoxic molecule and expresses a cell, tissue or organ that expresses an anti-complementary molecule. Can be supplied to mammals having the disease.

A suitable detectable molecule can be directly or indirectly attached to a polypeptide or antibody, and can be a radionuclide, an enzyme, a substrate, a cofactor, an inhibitor, a fluorescent marker,
Includes chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic molecules can be conjugated directly or indirectly to the polypeptide or antibody, and can be used for bacterial or phytotoxic (eg, diphtheria toxin, pseudomonas endotoxin, ricin, abrin and the like), and therapeutic Includes radionuclides such as I-131, rhenium-188 or yttrium-90, which are bound directly to a polypeptide or antibody or indirectly by a chelating moiety.

A polypeptide or antibody can also be conjugated to a cytotoxic drug, for example, adriamycin. For indirect binding of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be bound by a member of a complementary / anti-complementary pair, where the other member is bound to the polypeptide or antibody moiety. . For those purposes, biotin / streptavidin is a typical complementary / anti-complementary pair.

The expression of zfsta2 mRNA is mainly restricted to the spinal cord, brain and placenta, and low levels of expression are found in a wide variety of other tissues. This is consistent with the reported distribution of follistatin gene transcripts and transcripts of many other follistatin family members. This distribution indicates that zfsta2 can play a role in neuronal regeneration and repair in the CNS. Damage to the adult mammalian brain or spinal cord produces a series of cellular events that lead to inflammation, astrocyte expansion, angiogenesis, and the formation of glial-mesoderm scars (Logan et al., Brain Res. 587: 216). -twenty five,
1992; Wang et al., Res. Bull. 36: 607-9, 1995 and Lindholm et al., J. Cell. B.
iol. 117: 395-400, 1992).

The generation of scar tissue in the CNS provides a physical barrier for neuronal regeneration and is likely to limit the ability of adult CAS to recover after injury. Scar tissue formation in the CNS appears to depend on localized TGF-β stimulated production of extracellular matrix components, similar to those found for scar tissue formation in the periphery. In fact, TGF-β mRNA and protein are localized on the astrocyte side at the site of injury in the CNS (Logan et al., Supra, Wong et al., Supra, and Lin).
dholm et al., supra), which is a member of the follistatin family, for example
It suggests that zfsta2 promotes neuronal regeneration and establishment of novel synaptic contacts by sequencing TGF-β.

SC1, a member of the follistatin family, is expressed in brain astrocytes following injury (Mendis et al., Brain Res. 730: 95-106, 1996), and follistatin-related protein (FRP) It is secreted by glioma cells in culture (Zwijsen et al., J. Biochem. 225: 937-46, 1994).

Proteins that can block TGFβ and stimulate neuronal regeneration are useful in treating peripheral neuropathy by increasing spinal cord and sensory axon growth. Such polypeptides, agonists and antagonists can be included in therapeutic treatment to regenerate axon growth following a stroke, brain injury caused by head trauma and paralysis caused by spinal cord injury. In neurodegenerative patients such as Alzheimer's disease, Parkinson's disease and multiple sclerosis, they can also be used for treatment by stimulating neuronal growth. Additional uses include repairing torn and axons common in multiple sclerosis injuries.

The zfsta2 polypeptides, agonists or antagonists thereof, are therapeutically useful for treating brain and spinal cord injury. To confirm the presence of this ability in a zfsta2 polypeptide, agonist or antagonist of the invention, such zfst
a2 polypeptides, agonists or antagonists are evaluated for their ability to stimulate neuronal regeneration and their ability to establish new synaptic contacts according to methods known in the art.

For example, Mendis et al., Brain Res. 730: 95-106, 1996; Lindholm et al., J. C.
ell Biol. 117: 395-400, 1992; and Logan et al., Brain Res. 587: 216-25, 19
See 92. If desired, the zfsta2 polypeptide performance in this regard can be compared to other follistatins, such as SC1 and FRP, and the like. Furthermore, z
An fsta2 polypeptide, agonist or antagonist thereof, can be evaluated in combination with one or more follistatin to identify a synergistic effect. If desired, the performance of zfsta2 in this regard can be compared to other anti-inflammatory compounds such as dexamethasone and hydrocortisone, and the like.

[0233] Besides its possible role in treating damage to the CNS, zfsta2 also
It can also have a role in host defense. Human bone marrow stromal cells have been shown to react with anti-activin A antibodies, and the production of B _A -subunit mRNA is determined by many pro-inflammatory cytokines / regulators such as interleukin 1α, lipopolysaccharide. , Tumor necrosis factor-α or 12-O-tetradecanoylphorbol 1
It is enhanced in those cells by 3-acetate (Shao et al., Cytokine 1
0: 227-35, 1998). In contrast to the stimulatory effects of these agents, the anti-inflammatory compounds dexamethasone and hydrocortisone inhibited constitutive and cytokine-stimulated expression of activin B _A -mRNA (Shao et al., Supra).

The polypeptides of the present invention can be used to inhibit inflammatory responses, stimulate a decrease in the number and activity of inflammatory cells, and reduce edema and inflammation. Such anti-
Inflammatory polypeptides include acute inflammatory conditions, bursitis, chronic inflammatory demyelinating polyneuropathy, various forms of contact dermatitis, contact vulvar vaginitis, myositis, sepsis and ulcerative hydrangitis Applied to the processing of It can also be used as a therapeutic to treat acute renal failure, pancreatitis and neonatal bronchopulmonary hypoplasia. Applicable to eye damage, for example, corneal damage from burns or foreign objects, or inflammatory diseases, for example uveitis.

Dermatological conditions and diseases such as eczema, neurodermatitis, allergies, psoriasis,
It may also be applied to alleviate xerosis, insect bites and burns such as heat, chemical and radiation burns, especially chronic itch and inflammation associated with sunburn. Symptoms associated with gout, asthma, carpal tunnel syndrome, systemic lupus erythematosus, multiple sclerosis and myasthenia gravis can also be alleviated using the compounds of the present invention.

[0236] zfsta2 polypeptide, agonist or antagonist-mediated removal of bioactive activin from sites of inflammation is a useful treatment for the treatment of a wide variety of inflammatory disorders. To determine the presence of this ability in a zfsta2 polypeptide or polypeptide fragment thereof, such polypeptides and polypeptide fragments are evaluated for their ability to inhibit acute inflammation. Such methods are known in the art, and in particular, zfsta2 polypeptides can be tested for anti-inflammatory activity in a carrageenan-induced rat footpad edema model (Winter et al., J. Pharmac. Exp. Ther. 141: 369-76, 1963 and Miele
, Nature 335: 726-30, 1988).

Other models include the endotoxin-induced uveitis (EIU) model (Cha
n et al., Arch. Ophthalmol. 109: 279-81, 1991). Oxazolone-induced inflammation model (Lloret and Moreno, Biochem. Pharmocol. 44: 1437, 1992), husk oil-induced inflammation model, PMA-induced inflammation model (Micle et al., Supra), and anti- Dextran-induced edema assay for inflammatory agents (Iale
nti et al., Agents Actions 29: 48-9, 1990 and Rosa and Willoughby, J. Phar
m. Pharmac. 23: 297-8, 1971).

Efficiency for treating disease, eg, rheumatoid arthritis, is assessed using indicators including reduced inflammation and relief of pain or stiffness, and in animal models, indications include visual inspection of joints and Derived from changes in swelling of the hind legs. If desired, the zfsta2 polypeptide performance in this regard can be compared to other anti-inflammatory agents, especially dexamethasone and hydrocortisone. In addition, zfsta2 polypeptides can be evaluated in combination with one or more anti-inflammatory agents to identify synergistic effects.

[0239] erythroid differentiation factor (EDF), and sequence identity of the recent structures of B _A subunit of activin and inhibin (Single Murata like, Proc Natl Acad Sci USA 85: ..... 2434
, 1988) suggest a role for zfsta2 in regulating hematopoiesis and erythroid precursor differentiation. EDF exhibits potential differentiation-inducing activity on cultured erythroleukemia cells and enhances the growth of normal erythroid progenitor cells in vitro and in vivo (Yu et al., Nature 330: 765, 1987, Shiozaki et al. ., Biochem. Biophys. Res.
Commun. 165: 1155, 1989), and activin A / EDF is expressed in activated macrophages (Eramaa et al., J. Exp. Med. 176: 1449-52, 1992).

Continuous intraperitoneal administration of follistatin to normal mice results in a decrease in erythroid precursors in bone marrow and spleen (Shiozaki et al., Proc. Natl. Acad. Sci.
USA 89: 1553-6, 1992), indicating that follistatin regulates murine erythropoiesis. In humans, moreover, follistatin-related genes are
-A target for chromosome transfer in cellular lymphocytic leukemia (Hayette et al.
., Oncogene 16: 2949-54, 1998).

EDF-binding proteins, such as zfsta2 polypeptides, agonists or antagonists, provide useful therapeutic agents for modulating hematopoiesis and erythroid precursor differentiation. To confirm the presence of this ability, zfsta2 polypeptides and agonists of the invention were evaluated for their ability to alter erythropoiesis by lowering erythroid progenitors in bone marrow and spleen according to methods known in the art. You.

A zfsta2 antagonist is evaluated for enhancing hematopoiesis and erythroid precursor differentiation by inactivating follistatin and follistatin-like molecules. If desired, the zfsta2 performance in this regard can be compared to other follistatin or hematopoietic factors such as erythropoietin or thrombopoietin and the like. In addition, a zfsta2 polypeptide, or agonist or antagonist thereof, can be evaluated in combination with one or more follistatins to identify ameliorating effects.

Pleiotropic effects of activin and inhibin on the gonad / hypothalamic / pituitary axis indicate that follistatin and zfsta2 are useful in treating fertility disorders characterized by abnormal hormone production . For example, activin A has been shown to stimulate hypothalamic oxytocin secretion (Sawchenko et al.,
(Nature 334: 615-7, 1988). Oxytocin stimulates uterine contractions. Activin A
Act as useful therapeutic agents for delaying delivery in precocious pregnancies.

Hair follicle formation is a physiological phenomenon characterized by morphological and functional changes in vesicles. Among those phenomena, sinus formation is the pathway through which pituitary hormones
It seems to be a sign controlled by FSH. Since FSH is required for normal functioning of the ovaries and activin is required for activation of FSH synthesis and secretion,
It is not surprising that follistatin is a key regulator of ovarian function. Follistatin mRNA is present in primordial vesicles and its levels are dramatically increased in proliferating secondary or tertiary vesicle granulosa cells and then decreased in preovulatory vesicles (Shimasaki et al. ., Mol. Endocrinol. 3: 651-9, 198
9).

Recent in vitro assay systems also show that activin is not directly at maturation, but is directly folliculogenic in immature mice, and that inhibition of follicle formation in adult mice is further reversed by follistatin. (Yoko
ta et al., Endocrinology 138: 4572-6, 1997). The balance between activin and follistatin appears to be critical for normal ovarian function, as overexpression of mouse follistatin in female transgenic mice has many reproductive deficiencies (Guno et al. , Mol. Endocrinol. 12: 96-106, 1998).

Follistatin, such as zfsta2, affects vesicle cell proliferation or differentiation;
It plays a role in regulating hair follicle formation by affecting cell-cell interactions, regulating hormones involved in the process, and by doing the same. The role of sex steroids, such as FSH, on target tissues and organs, such as the uterus, breast, adipocytes, bone and liver, has produced modulators of those activities that are desired for therapeutic use. Such uses include treatment for sexual precociousness, endometriosis, uterine leiomyoma, hirsutism, infertility, premenstrual syndrome (PMS), amenorrhea and as a contraceptive.

Gonadotropin and steroid hormone levels and rates in the blood are used to assess the presence of disease-related hormonal imbalance and to determine whether normal hormonal balance has been restored following administration of a therapeutic agent. Can be used for Determination of estradiol, progesterone, LH and FSH levels from serum is known by those skilled in the art. Such assays can be used to monitor the effects on hormone levels after administration of zfsta2 in vivo or in a transgenic mouse model in which the zfsta2 gene is expressed or the murine ortholog is deleted.

The zfsta2 polypeptides, agonists and antagonists of the present invention can be used directly for treating a reproductive disorder or incorporated into a therapeutic. As hormone-modulating molecules, zfsta2 polypeptides, agonists and antagonists may be used to treat menopausal bleeding, for example, as part of a treatment regimen for maintaining pregnancy.
Or have therapeutic use to treat symptoms associated with polycystic ovary syndrome (PCOS), PMS and menopause. In addition, other in vivo rodent models are known in the art, for example, to assay the effects of zfsta2 polypeptides, agonists and antagonists on polycystic ovary syndrome (PCOS).

Activin, inhibin and follistatin are also found in testis. MRNA encoding follistatin is located in many germinal cells, such as spermatogonia type B, primary spermatocytes, and sperm cells at stages 1-11 (Meinhardt et al., J.
Reprod. Fertil. 112: 233-41, 1998). It is also found in Sertoli cells and endothelial cells, but not in Leyding cells. Immunohistochemistry for anti-follistatin antibodies showed that the protein was located on sperm cells at all stages, and it was also located on endothelial cells and Leyding cells. This broad localization, along with the ability of follistatin to neutralize the activity of activin, indicates that follistatin regulates spermatogenesis and a wide range of other testis functions.

The balance between activin and follistatin plays an important role in normal reproduction in males, which is demonstrated in mouse follistatin transgenic mice, ie, males have varying degrees of Leyding cell hyperplasia. Spermatogenesis is suppressed, and the vas deferens that induce infertility are degenerated. This indicates that reproductive disorders due to excess activin or other TGF-β family members are susceptible to treatment by members of the follistatin family. In addition, follistatin antagonists are useful in treatment regimes to enhance male fertility.

In vivo assays for evaluating the effects of zfsta2 polypeptides, agonists and antagonists on testes are well known in the art. For example,
The compound may be injected intraperitoneally for a specific period. After the treatment period, the animals are sacrificed and the testes removed and weighed. Testes are homogenized and sperm heads are counted (Meistrich et al., Exp. Cell Res. 99: 72-78, 1976).

[0252] Other activities, such as chemotactic activity associated with the proteins of the invention, can be analyzed.
For example, late stage factors in spermatogenesis have been implicated in egg-sperm interaction and sperm motility. Activities such as enhancing the viability of cryopreserved spermatozoa, stimulating the acrosome reaction, increasing sperm motility, and enhancing egg-sperm interaction are involved in the proteins of the invention.

Assays for assessing such activity are known (Rosenberger, J. Androl.
11: 89-96, 1990, fuchs, Zentralbl 11: 117-120, 1993; Neurwinger et al., A
ndrologia 22: 335-9, 1990; Harris et al., Human Reprod. 3: 856-60, 1988;
And Jockenhovel. Andrologia 22: 171-178, 1990; Lesing et al., Fertil. Ste.
ril. 44: 406-9 (1985); Zaneveld, In Male Infertility Chapter 11, Comhair
e Ed., Chapman & Hall, London 1996). Their activity is expected to result in enhanced fertility and successful reproduction.

A zfsta2 polypeptide, agonist or antagonist provides a useful therapeutic agent for modulating reproductive hormones. To confirm the presence of this ability, the zfsta2 polypeptides, agonists and antagonists of the invention are evaluated for their ability to modulate hormones associated with reproduction, according to methods known in the art. For example, Guoqetal, Mol. Endocrinol. 12: 96-106, 1998 describes RIA measurements of serum LH, FSH, testosterone, estradiol, activin and follistatin.

The zfsta2 polypeptides and agonists are useful for treating male and female reproductive disorders. zfsta2 antagonists are also useful as contraceptives. If desired, the zfsta2 performance in this regard can be compared to other follistatin and the like. Additionally, a zfsta2 polypeptide, or agonist or antagonist thereof, can be evaluated in combination with one or more follistatins to identify a synergistic effect.

The zfsta2 polypeptides, agonists and antagonists of the present invention can also be used to enhance fertilization during assisted reproduction in humans and animals. Such assisted reproductive methods are known in the art and include artificial insemination,
Includes in vitro fertilization, embryo transfer, and gamete fallopian internalization. Such methods are useful for helping people with physiological or metabolic disorders that interfere or prevent natural concepts.

Such methods are also used in animal husbandry programs, for example for livestock, racehorses, domestic and wild animals, and can be used as methods for the creation of transgenic animals. A zfsta2 polypeptide, agonist or antagonist can be used in the induction of ovulation, alone or with a gonadotropin or agent, such as clomiphene citrate or bromocriptine (Speroff et al.
, Induction of Ovulation, Clinical Gynecologic Endocrinology and Inferti
^{lity, 5 th ed., Baltimore} , Williams & Wilkins, 1994).

The zfsta2 polypeptides, agonists and antagonists may also be used, independently or together with other gonadotropins or sex steroids, such as testosterone, in stimulating spermatogenesis. As such, the proteins of the invention can be administered to a receptor before fertilization, or combined with a sperm, egg or egg-sperm mixture before in vitro or in vivo fertilization. Such proteins can also be mixed with oocytes or sperm prior to cryopreservation to increase the viability of the preserved tissue for assisted reproductive use.

[0259] Bone and tooth formation is a multistep process known to be initiated and promoted by the TGF-β superfamily, for example, the family of TGF-βs and bone morphogenetic proteins (BMPs). Accumulating events indicate that activin and follistatin play regulatory roles in both tooth and bone formation. The spatio-temporal expression of activin and follistatin in anterior gonadoblasts indicates that activin is required for the expansion of those cells, and that terminal gonadoblast inactivation of follistatin inactivates their proliferative effects. (Heikinheimo et al., J. Dent. Res. 76: 1625-36, 1997: Heikinh
eimo et al., Eur. J. Oral. Sci. 106: 167-73, 1998).

Follistatin is also expressed in bone (Inoue et al., Calcif. Tiss.
Int. 55: 396-7, 1994), and activin and follistatin have been detected by immunohistochemistry in the treatment of fractures in rats (Nagamine et al.,
J. Orthopaed. Res. 16: 314-21, 1998). Follistatin has also been detected in growing bone, and during demineralized bone matrix-induced endochondral bone growth, the expression of follistatin and activin A genes is not affected during bone formation. Show a cooperative interaction between statins and activin (Funaba, et al.
., Endocrinology 137: 4250-9, 1996).

Such therapeutic agents may be used to repair bone defects and deficiencies that occur in closed, open and non-union fractures; preventative use to reduce closed and open fractures; promote bone treatment in plastic surgery; Stimulation of bone formation into worn prosthetic joints and dental implants;
Increased peak bone mass in premenopausal women; treatment of growth deficiency; treatment of periodontal disease and defects; and other bone repair steps; increased bone formation during distractive bone formation; and other skeletal disorders, such as It is used for the treatment of age-related osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, diabetes-related osteoporosis or disuse osteoporosis and arthritis. The compounds of the present invention may also be useful in repairing congenital, trauma-induced or surgical incisions in bone (eg, for the treatment of cancer), and in cosmetic surgery. Further uses include limiting or treating ointment damage or injury, and stimulating wound healing and tissue repair.

[0262] Well-established animal models are available for testing the in vivo efficacy of modulators of bone formation. For example, a hypocalcemic rat or mouse model can be used to determine the effect of a test compound on serum calcium, and ovariectomized rats or mice can be used as a model system for osteoporosis. During the early stages of estrogen deficiency, the bone changes seen in those models and humans are similar in nature.

[0263] Molecules that can modulate the effects of members of the TGF-β family, such as zfsta2 polypeptides, agonists or antagonists, provide useful molecules for tooth and bone formation. To confirm the presence of this ability, zfsta2 polypeptides, agonists and antagonists of the invention are evaluated for their ability to stimulate tooth or bone formation according to methods known in the art. If desired,
The zfsta2 performance in this can be compared to other follistatin and the like. Additionally, a zfsta2 polypeptide, or agonist or antagonist thereof, can be evaluated in combination with one or more follistatins to identify a synergistic effect.

Follistatin and activin also likely play a role in the pathogenesis of atherosclerosis. In vessel wall cells, activin-A has been shown to inhibit endothelial cell proliferation and promote smooth muscle cell proliferation (Koji
ma et al., Exp. Cell Res. 206: 152-6, 1993; McCarthy and Bicknell, J. Biol.
Chem. 268: 23066-71, 1993), and has been shown to produce a modest impairment of scavenger receptor, SRB1, expression and foam cell formation in THP-1 macrophages (Kozaki et al., Arterioscler. Thromb. Vasc. Biol.
17: 2389-94, 1997).

The effects are antagonized by follistatin. Activin-
A, follistatin and bone morphogenetic protein-2 were produced by human atherosclerosis disorder, and the first two expressions were localized to neointima in diseased arteries (Inoue et al., Biochem. Biophys, Res. Commun. 205: 441-8, 1994).
These data indicate that the relative balance between activin and its binding protein follistatin is important in the initiation and progression of atherosclerotic trauma.

A zfsta2 polypeptide, agonist or antagonist is useful for neutralizing the activity of a TGF-β family member. Such a molecule would provide a new therapy for the treatment of restenosis after angiogenesis. In addition, TGF-β is useful for treating atherosclerosis. The use of such molecules is also applicable for the treatment of seizures.

Follistatin and activin appear to play important roles in growth. Two types of TGF-β family members appear to determine the mesoderm dorsal / ventral pattern during early growth in Xenopus. The first member is associated with activin and triggers the formation of dorsal mesoderm, which produces muscle and dorsal cord (Asashima et al., Roux's Arch. Dev. Biol. 198: 330-
And a second member, a bone morphogenetic protein (BM) that inhibits dorsal mesoderm formation and induces cells present on the ventral fate, such as blood cells.
P) (Maeno et al., Dev. Biol. 161: 522-9, 1994).

[0268] Follistatin has been identified in those systems as activin (Fukui et al., De.
v. Biol. 159: 131-9, 1993), and BMP (Iemura et al., Proc. Natl. Acad. Sci.
USA 95: 9337-42, 1998). Thinking together,
These findings indicate that as a member of the follistatin family of TGF-β binding proteins, zfsta2 is useful as a therapeutic for a wide range of growth disorders.

The effect of zfsta2 can be measured in vitro using cultured cells or in vivo by administering a molecule of the invention to a suitable animal model. The zfsta2 transfected expression host cells can be, for example, zfsta2 transfected or co-transfected expression host cells embedded in an alginate environment and injected (implanted) into a recipient animal. . Alginate-poly-L-lysine microencapsulation, permeable membrane encapsulation and diffusion chambers have been described as a means for capturing transfected or primary mammalian cells.

These types of non-immunogenic “encapsulation” or microenvironment allow nutrient transfer to the microenvironment and / or proteins and other macromolecules secreted or released by the captured cells. Allows for its spread to recipient animals through environmental barriers. Most importantly, the capsule or microenvironment masks and blocks foreign embedded cells from the immune response of the recipient animal. Such a microenvironment can extend the life span of injected cells from hours or days (naked cells) to weeks (embedded cells).

Alginate threads provide a simple and rapid means to generate embedded cells. The materials needed to produce alginate threads are readily available and relatively inexpensive. When manufactured, the alginate thread:
Relatively strong and durable in vivo, and based on data obtained with the yarn, in vivo. The alginate thread is easy to operate and its methodology can be evaluated for many preparations. In a typical method, 3% alginate is prepared in sterile water and sterile filtered. Immediately before the preparation of the alginate thread, the alginate solution is filtered again. About 50% cell suspension (1
about 5 × 10 ⁵ to about 5 × 10 ⁷ cells per ml) are mixed with a 3% alginate solution.

One ml of the alginate / cell suspension is extruded into a 100 mM sterile filtered Cacl ₂ solution for about 15 minutes to form a “Thread”. next,
The extruded yarn is transferred to a solution of 50 mM Cacl ₂ and then to a solution of 25 mM Cacl ₂ . Next, the yarn is rinsed with deionized water, after which the poly-L-
The threads are coated by incubating in a 0.01% solution of lysine. Finally, the thread is rinsed with a lactated Ringer's solution and withdrawn from the solution in a syringe (without a needle). A large bore needle is then attached to the syringe, and the thread is injected intraperitoneally into the recipient in a minimal amount of lactated Ringer's solution.

Another in vivo approach to assay the proteins of the invention involves a virus delivery system. Typical viruses for this purpose are adenovirus, herpes virus, vaccinia virus and adeno-associated virus (AAV)
Is included. Adenovirus, a double-stranded DNA virus, is currently the most studied gene transfer vector for the provision of heterologous spread (TCC).
Becker et al., Meth. Cell Bio. 43: 161-89, 1994; and JT Douglas and D.
.T. Curiel, Science & Medicine 4: 44-53, 1997).

The adenovirus system offers several advantages: (i) the adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high titers; (iii) a wide range of Infect mammalian cell types; and (iv)
It can be used with many available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

By deleting part of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. The inserts are constructed by direct ligation or by homologous recombination with a co-transfected plasmid.
A can be incorporated into A. In a typical system, the essential E1 gene is deleted from the viral vector, and the virus will not replicate unless the E1 gene is supplied by a host cell (a human 293 cell line is typical).

When administered intravenously to an intact animal, adenovirus primarily targets the liver. If the adenovirus delivery system has an E1 gene deletion, the virus cannot replicate in the host cell. However, the host tissue (eg, liver) will express and process the heterologous protein (and secrete if a secretory signal sequence is present). Secreted proteins enter the circulation in the highly vascularized liver and the effect on infected animals can be determined.

Polynucleotides encoding zfsta2 polypeptides are useful in gene therapy applications where it is desired to increase or inhibit zfsta2 activity. If the mammal has or lacks the mutated zfsta2 gene, the zfsta2 gene can be introduced into mammalian cells. In one embodiment, the gene encoding the zfsta2 polypeptide is introduced in vivo in a viral vector. Such vectors include attenuated or defective DNA viruses such as herpes simplex virus (HSV), papillary virus, Epstein-Barr virus (EBV), adenovirus, adeno-associated virus (AAV) and the like. However, it is not limited only to them.

Defective viruses that completely or almost completely lack viral genes are preferred. The defective virus is not infectious after introduction into the cell. The use of defective viral vectors allows for administration to cells in specific localized areas without having to worry about the vector infecting other cells. Examples of particular vectors include, but are not limited to: defective herpesvirus 1
(HSV1) vector (Kaplitt et al., Molc. Cell. Neurosci. 2: 320-30, 1991),
Attenuated adenovirus vectors, such as Stratford-Perricaudat.
(J. Clin. Invest. 90: 626-30, 1992) and defective adeno-associated virus vectors (Samulski et al., J. Virol. 61: 3096-101, 1987).
Samulski et al., J. Virol. 63: 3822-28,1989).

[0279] In another embodiment, the zfsta2 gene is as described in
Can be introduced into retroviral vectors: Anderson et al., US Patent 5,39.
No. 9,346; Mann et al., Cell 33: 153, 1983; Temin et al., U.S. Pat.
No. 0,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Vir
ol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty.
, WIPO Publication WO95 / 07358; and kuo, etc., Blood 82: 845-52,
1993.

On the other hand, vectors can be introduced by in vivo lipofection using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of genes encoding markers (Felg
ner et al., Proc. Natl. Acad. Sci. USA 84: 7413-17, 1987; and Mackey etc.
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.

It is clear that directing transfection to specific cells represents an advantage of one area. It is clear that directing transfection to specific cell types is particularly advantageous in tissues with cellular heterogeneity, such as pancreas, liver, kidney and brain. Lipids are scientifically derived into other molecules for targeting. Targeted peptides, such as hormones or neurotransmitters, and proteins, such as antibodies or non-peptide molecules, can be chemically attached to liposomes.

It is possible to remove cells from the body and introduce the vector as a naked DNA plasmid, and then re-transplant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by any method known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, It can be introduced by the use of a cancer or the use of a DNA vector transporter (eg, Wu et al., J. Biol. Chem. 267: 963-7, 1992; W
u et al., J. Biol. Chem. 263: 14621-24, 1988).

Antisense methods can be used to inhibit zfsta2 gene transcription, for example, to inhibit cell growth in vivo. A polynucleotide that is complementary to a segment of a zfsta2-encoding polynucleotide (eg, a polynucleotide as set forth in SEQ ID NO: 1) binds to the zfsta2-encoding mRNA and directs translation of such mRNA. It is planned to inhibit. Such antisense polynucleotides are used to inhibit expression of a gene encoding a zfsta2 polypeptide in a cell culture or subject.

Transgenic mice constructed to express the zfsta2 gene, and mice exhibiting a complete absence of zfsta2 gene function, referred to as “knockout mice” (Snouwaert et al., Science 257: 1083, 1992), also have (Lowell et al., Nature 366: 740-42, 1993). Those mice are
The zfsta2 gene fragment, and the protein encoded thereby, are used to study in vivo systems.

[0285] zfsta2 polypeptides are also designed for pharmaceutical use. A pharmaceutically effective amount of a zfsta2 polypeptide, agonist or zfsta2 antagonist of the invention is parenteral,
It may be formulated with a pharmaceutically acceptable carrier for oral, nasal, rectal, topical, transdermal administration or the like. The formulation further comprises one or more diluents,
It can include fillers, emulsifiers, preservatives, buffers, excipients and the like, and is supplied in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets .

[0286] Delayed or prolonged open systems, including any of a number of biopolymers (biological-based systems), systems using liposomes, and polymer delivery systems can also be used for continuous or extended delivery of zfsta2 polypeptides or antagonists. It can be used with the compositions described herein to provide a long-term source. Such a delay system may be applicable to the formulation for oral, topical and parenteral use. The term "pharmaceutically acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and is not toxic to the host or patient.
One skilled in the art will recognize the compounds of the invention in an appropriate manner and in acceptable practice, for example, Re.
mington's Pharmaceutical Sciences, Gennaro (ed.), Mack Publishing Co.,
It can be formulated according to their practice as disclosed in Easton, PA, 1990.

[0287] As used herein, a "pharmaceutically effective amount" of a zfsta2 polypeptide, agonist or antagonist is an amount sufficient to induce a desired biological result. The result may be the resolution of the signs, symptoms or causes of the disease, or any other desired mutation in the biological system. For example, an effective amount of a zfsta2 polypeptide is an amount of the polypeptide that provides either a subjective reduction of symptoms or an objectively identifiable improvement, as indicated by a clinician or other qualified observer. is there. An effective amount of a zfsta2 polypeptide can vary widely, depending on the disease or symptom being treated.

The amount of polypeptide to be administered, and its concentration in the formulation, will depend on the vehicle selected, the route of administration, the ability of the particular polypeptide, the clinical condition of the patient, side effects, and the compound in the formulation. Depends on the stability of Thus, the clinician will use the appropriate preparation, including the appropriate concentration in the formulation, and the amount of formulation to be administered, depending on clinical trials with the patient in question or a similar patient. Such amount will depend, in part, on the particular condition being treated, the age, weight, and general health of the patient, and other factors apparent to those skilled in the art. Typically, doses will range from 0.1 to 100 mg / kg subject.

Dosages for particular compounds may be determined from in vitro or ex vivo studies, in combination with studies based on experimental animals. The concentration of the compound that is found to be effective in vitro or ex vivo provides a guide for animal studies, where the dose is calculated to provide a similar concentration at the site of action.

The dosage of the compounds of the present invention used to practice the present invention includes those effective to produce the desired effect. Evaluation of an appropriate dosage effective for an individual patient is within the scope of a physician or other appropriate healthcare practitioner. As a guide, clinicians may refer to the Physician's Desk Reference, 48 ^th Edition, Medical E
The device from conomics Data Production Co., Montvale, New Jersey 07645-1742 (1994) can be conveniently obtained and used.

Preferably, the compositions of the present invention are provided for administration in unit dosage form.
The term "unit dosage form" refers to physically discrete units suitable for unit administration for human subjects and animals, with each unit comprising a required pharmaceutical diluent, carrier or carrier. Calculated with the vehicle to produce the desired pharmaceutical effect,
Contains a predetermined amount of active material. Examples of unit dosage forms include vials, ampoules, tablets, caplets, pills, powders, granules, ophthalmic solutions, oral or ophthalmic solutions or suspensions, ointments, and oil-in-water emulsions. The means of preparation, formulation and administration are known to those skilled in the art and are generally described in Remington: The Science and Practice of Pha
rmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19 th ed., see 1995.

The present invention is further illustrated by the following non-limiting examples. [Example] Example 1 Extension of EST Sequence A polynucleotide encoding the novel zfsta2 polypeptide of the invention was first identified by searching the EST database for follistatin homologs. EST is
It was found and predicted to be related to the follistatin family, but deleted the complete 5 'and 3' regions. To identify its corresponding full-length cDNA, a clone that probably contained the missing 3 'coding region was used for sequencing.

Invitrogen SNAP ^™ Miniprep Kit (Invitrogen, Corp., San Diego, C.)
A) was used to prepare 5 ml of an overnight culture in LB + 50 μg / ml ampicillin according to the manufacturer's instructions. The mold, ABI PRISM ^TM Dye Terminator Cycle Se
Sequencing was performed using the qnencing Ready Reaction Kit (Perkin-Elmer Corp.) on an ABIPRISM ^™ model 337 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Conn.) according to the manufacturer's instructions. The sequencing reaction was performed using the Hybaid OmniGene TemniGene Te
mperature Cycling System (National Labnet Co., Woodbridge, NY). SEQUENCHER ^™ 3.0 sequence analysis software (Gene Code Corporation, Ann
Arbor, MI) was used for data analysis. The resulting 1965 bp sequence is zfst
The 3 'end of the a2 cDNA (SEQ ID NO: 7) was provided.

Fetal brain, brain, spinal cord and retina Marathon ^™ cDNA libraries (Clontech, Palo Alto, CA) as separate templates, and oligonucleotide primers ZC18,415 (SEQ ID NO: 5) for early EST and zfsta2 from above 5′RACE was performed at 94 ° C. for 1.5 hours using oligonucleotide primer ZC14701 (SEQ ID NO: 6) for the internal sequence of
Time, followed by 5 seconds at 94 ° C and 1.5 minutes at 66 ° C (35 cycles), followed by 10 minutes at 72 ° C.
Minutes.

The approximately 1255 bp (SEQ ID NO: 8) band was resolved from individual templates by gel electrophoresis. The 5'RACE fragment from each of the above reactions was transferred to a TA vector (Invitrogen In
c., San Diego, CA) according to the manufacturer's instructions. The sequence of the 5 'end of zfsta2 was confirmed from fetal brain PCR fragments by sequence analysis as described above. The 3 ′ EST-derived sequence and the 5 ′ RACE-derived sequence are ligated together through overlapping sequences, and the complete cDNA sequence of zfsta2 is disclosed in SEQ ID NO: 1.

Example 2 Tissue distribution Human multi-tissue Northern blots (MTN I, MTN II and MTN III; Clontech) were probed to determine the tissue distribution of human zfsta2 expression. An approximately 140 bp PCR-derived probe (SEQ ID NO: 4) was used as a template for fetal brain, brain, spinal cord and retina Marathon ^™ cDNA libraries (Clontech), and oligonucleotides ZC12881 (SEQ ID NO: 9) and ZC12884 (SEQ ID NO: 9) as primers. No. 10). Amplification was performed as follows: one cycle at 94 ° C for 1.5 minutes, 35 cycles at 94 ° C for 15 seconds and 60 ° C for 30 seconds, followed by one cycle at 72 ° C for 10 minutes.

The PCR products were visualized by agarose gel electrophoresis and 140
The bp PCR product was purified using a gel extraction kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Probes were radioactively labeled using the MULTIPRIME DNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.

The probe was purified using a NUCTRAP push column (Stratagene). EXPRE
SSHYB (Clontech) solution was used for prehybridization and as a hybridization solution for Northern blots. Hybridization and washing were performed under appropriate stringent conditions. Strong transcripts of about 5 kb were found in the spinal cord and placenta, and weaker transcripts were detected in the brain. RNA, including RNA from various tissues, normalized to eight housekeeping genes
Master Dot Blot (Clontech) was also probed and hybridized as described above. Expression was found in the cerebellum, occipital lobe and pituitary.

Example 3 The chromosomal localization zfsta2 was converted to a commercially available Genebridge 4 Radiation Hybrid Panel (Research Genetics).
, Inc.). GeneBridge 4 Radiation Hy
The brid Panel contains PCRable DNA from each of the 93 radiation hybrid clones, and two control DNAs (HFL donor and A23 receptor). The public WWW server (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) is GeneBri
Whitehead Instit of human genome constructed by dge 4 Radiation Hybrid Panel
ute / MT Center for Genome Research's Enables mapping for radiation hybrid maps ("WICGR" radiation hybrid maps).

For mapping zfsta2 with the GeneBridge 4RH Panel, 20 μl of the
Provided in 6-well microtiter plates (Stratagene, La Jolla, CA)
And used in a "RoboCycler Gradien 96" thermal cycler (Stratagene). Each 95 PCR reactions consisted of 2 μl of 10 × PCR reaction buffer (Clontech), 1.6 μl of d
NTP mixture (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC15,570 (SEQ ID NO: 11), 11 μl antisense primer, ZC15,5
75 (SEQ ID NO: 12), 2 μl of RediLoad (Research Genetics, Inc.), 0.4 μl of 50 ×
Advantage KlenTaq polymerase mix (Clontech), 25 ng of DNA from individual hybrid clones or controls, and ddH ₂ O for a total volume of 20 μl.

The reaction was covered with the same amount of mineral oil and sealed. PCR cycler conditions were as follows: denaturation at 95 ° C for 5 minutes, initial cycle, denaturation at 95 ° C for 1 minute,
Anneal for 1 minute at 62 ° C and extend for 1.5 minutes at 72 ° C, 35 cycles, followed by 72 cycles.
Extended for 7 minutes at ℃, one final cycle. Reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).

[0302] The results show that zfsta2 has the skeletal marker W on the chromosome 4WICG radiation hybrid map.
It was shown to be located from I-5113 to 2.84cR_3000. The proximal and distal skeletal markers were WI-5113 and CHLC.GATA4C05.17, respectively. The use of surrounding markers localizes zfsta2 in the 4q28.3 region on the integrated LDB chromosome 4 map (The
Genetic Location Database, University of Southhampton, WWW server: http
: // cedar. genetics. soton. ac. uk / public_html /). From the foregoing, while certain features of the invention have been described for purposes of illustration, it will be apparent that various modifications can be made within the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 19/00 C07K 14/59 25/00 16/26 C07K 14/59 19/00 16/26 C12N 1 / 15 19/00 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 21/08 5/10 C12N 15/00 A C12P 21/02 5/00 A // C12P 21/08 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ , TZ, UG, ZW), EA (AM, AZ, BY, G, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventors Ellsworth, Jeff L. United States, Washington 98118, Seattle, Fifty First Avenue South 4225 F-term (Reference) 4B024 AA01 BA01 BA43 CA01 FA02 FA07 FA18 GA11 4B064 AG15 AG27 CA19 CC24 DA01 4B065 AB01 AC15 CA24 CA25 CA44 4C084 AA02 AA06 AA07 BA21 BA08 CA18 CA19 CA53 NA14 ZA01 ZA452 ZA512 ZA812 ZA962 4H045 AA10 AA11 BA10 BA41 CA40 DA45 DA76 EA20 FA74

Claims (44)

[Claims] 1. An isolated polypeptide comprising a follistatin homology domain comprising amino acid residues 65 to 133 of the amino acid sequence of SEQ ID NO: 2. 2. The polypeptide further comprises an α-helical linker region located at the carboxyl-terminal position to the follistatin homology domain, wherein the α-helical linker region is an amino acid of the amino acid sequence of SEQ ID NO: 2. 2. The isolated polypeptide of claim 1, comprising residues 134-174. 3. The polypeptide further comprises a calmodulin homology domain located at a carboxyl terminal position relative to the α-helix linker region, wherein the calmodulin homology domain is an amino acid residue of the amino acid sequence of SEQ ID NO: 2. 3. The isolated polypeptide of claim 2, comprising 175-250. 4. The polypeptide further comprises two I-group Ig domains located at the carboxyl-terminal position relative to the calmodulin homology domain, wherein the I-group Ig domain is SEQ ID NO: 2. Amino acid residue 251 in the amino acid sequence
3. The isolated polypeptide of claim 2, comprising -431. 5. The polypeptide further comprising a carboxy-terminal domain present at a carboxyl-terminal position relative to the I-group Ig domain, wherein the carboxy-terminal domain corresponds to the amino acid sequence of SEQ ID NO: 2. Amino acid residue 43
3. The isolated polypeptide of claim 2, comprising 3 to 847. 6. The polypeptide further comprises a hydrophobic linker region present at an amino terminal position with respect to the follistatin homology domain, wherein the hydrophobic linker region has the amino acid residue of the amino acid sequence of SEQ ID NO: 2. 2. The isolated polypeptide of claim 1, comprising groups 21-64. 7. The polypeptide further comprises a secretory signal sequence present at an amino terminal position relative to the hydrophobic linker region, wherein the secretory signal sequence is an amino acid residue of the amino acid sequence of SEQ ID NO: 2. 7. The isolated polypeptide of claim 6, comprising 1-20. 8. An isolate having an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, which specifically binds to an antibody to which the polypeptide having the amino acid sequence of SEQ ID NO: 2 specifically binds. Polypeptide. 9. The isolated polypeptide of claim 8, wherein said isolated polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. 10. The isolated polypeptide of claim 8, wherein said isolated polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. 11. The isolated polypeptide of claim 8, wherein any difference between said amino acid sequence and said corresponding amino acid sequence of SEQ ID NO: 2 is due to one or more conservative amino acid substitutions. 12. The% identity of the amino acids is ktup = 1, gap opening penalty = 10, gap extension penalty = 1 and substitution matrix = blosum62.
9. The isolated polypeptide of claim 8, which is determined using the FASTA program with other parameters set as errors. 13. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 14. a) a polypeptide consisting of the sequence of residues 21 to 64 of SEQ ID NO: 2; b) a polypeptide consisting of the sequence of residues 65 to 133 of SEQ ID NO: 2; c. A) a polypeptide consisting of the sequence of residues 134 to 174 of SEQ ID NO: 2; d) a polypeptide consisting of the sequence of residues 175 to 250 of SEQ ID NO: 2; e) the remainder of SEQ ID NO: 2 A polypeptide consisting of the sequence of amino acid residues of groups 251-334; f) a polypeptide consisting of the sequence of amino acid residues of residues 335-432 of SEQ ID NO: 2; g) amino acids of residues 433-847 of SEQ ID NO: 2 H) a polypeptide consisting of the sequence of amino acid residues 251 to 432 of SEQ ID NO: 2; i) a polypeptide consisting of the amino acid residues 65 to 174 of SEQ ID NO: 2 Polypeptide; j) amino acids at residues 65-250 of SEQ ID NO: 2 A) a polypeptide consisting of the sequence of amino acid residues 65-334 of SEQ ID NO. 2; l) a polypeptide consisting of the sequence of amino acid residues 65-847 of SEQ ID NO. M) a polypeptide consisting of the sequence of amino acid residues 134 to 250 of SEQ ID NO: 2; n) a polypeptide consisting of the sequence of amino acid residues 134 to 334 of SEQ ID NO: 2; A) a polypeptide consisting of the sequence of residues 134 to 432 of SEQ ID NO: 2; p) a polypeptide consisting of the sequence of residues 134 to 847 of SEQ ID NO: 2; A polypeptide consisting of the sequence of 334 amino acid residues; r) a polypeptide consisting of the sequence of amino acid residues 175-432 of SEQ ID NO: 2;
And s) an isolated polypeptide selected from the group consisting of a polypeptide consisting of the sequence of amino acid residues 175-847 of SEQ ID NO: 2. 15. The method of claim 1, further comprising an affinity label or a binding domain.
An isolated polypeptide as described. 16. SEQ ID NO: 2 operably linked to an additional polypeptide
A fusion protein comprising a secretory signal sequence having the amino acid sequence of 1 to 20 amino acid residues of 17. A fusion protein consisting essentially of a first part and a second part linked by a peptide bond, said first part comprising the polypeptide of claim 1 and said second part. A fusion protein, wherein the portion comprises another polypeptide. 18. An isolated polynucleotide molecule encoding the polypeptide of claim 1. 19. Encoding a polypeptide further comprising an α-helical linker region located at the carboxyl-terminal position to said follistatin homology domain, wherein said α-helical linker region is an amino acid of the amino acid sequence of SEQ ID NO: 2. 19. The isolated polynucleotide molecule of claim 18, comprising residues 134-174. 20. The polynucleotide encodes a polypeptide further comprising a calmodulin homology domain located at the carboxyl-terminal position to the α-helix linker region, wherein the calmodulin homology domain is an amino acid of SEQ ID NO: 2. 20. The isolated polynucleotide of claim 19, comprising amino acid residues 175-250 of the sequence. 21. The polynucleotide encoding a polypeptide, wherein the polynucleotide further comprises two I-set Ig domains located at the carboxyl-terminal position relative to the calmodulin homology domain, wherein the I-set Ig domains 21. The isolated polynucleotide of claim 20, comprising amino acid residues 251-431 of the amino acid sequence of SEQ ID NO: 2. 22. The polynucleotide encodes a polypeptide further comprising a carboxy-terminal domain present at a carboxy-terminal position to the I-group Ig domain, wherein the carboxy-terminal domain is SEQ ID NO: 22. The isolated polynucleotide according to claim 21, comprising amino acid residues 433-854 of the amino acid sequence of SEQ ID NO: 2. 23. The isolated polynucleotide of claim 22, wherein said polypeptide further comprises an affinity label or binding domain. 24. An isolated polynucleotide which is a degenerate nucleotide sequence encoding the polypeptide of claim 1. 25. A polypeptide having an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, which specifically binds to an antibody to which the polypeptide having the amino acid sequence of SEQ ID NO: 2 specifically binds An isolated polynucleotide encoding. 26. The isolated polynucleotide of claim 25, wherein said isolated polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. 27. The isolated polynucleotide of claim 26, wherein said isolated polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. 28. The method of claim 27, wherein any difference between the amino acid sequence and the corresponding amino acid sequence of SEQ ID NO: 2 is due to one or more conservative amino acid substitutions.
An isolated polynucleotide as described. 29. The% identity of the amino acids is ktup = 1, gap opening penalty = 10, gap extension penalty = 1 and substitution matrix = blosum62.
28. The isolated polynucleotide of claim 27, as determined using the FASTA program with other parameters set as errors. 30. An isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotides 58 to 3006. 31. The isolated polynucleotide molecule of SEQ ID NO: 1. 32) a polynucleotide consisting of nucleotides 58-117 of SEQ ID NO: 1; b) a polynucleotide consisting of nucleotides 118-249 of SEQ ID NO: 1; c) a polynucleotide consisting of nucleotides 250-456 of SEQ ID NO: 1; d) a polynucleotide consisting of nucleotides 457-579 of SEQ ID NO: 1; e) a polynucleotide consisting of nucleotides 580-810 of SEQ ID NO: 1; f) a polynucleotide consisting of nucleotides 811-1059 of SEQ ID NO: 1; g) SEQ ID NO: 1 H) a polynucleotide consisting of nucleotides 1354 to 3006 of SEQ ID NO: 1; i) a polynucleotide consisting of nucleotides 250 to 579 of SEQ ID NO: 1; j) a polynucleotide consisting of nucleotides 250 to 810 of SEQ ID NO: 1. A) consisting of nucleotides 250-1059 of SEQ ID NO: 1 A) a polynucleotide consisting of nucleotides 250 to 1353 of SEQ ID NO: 1; n) a polynucleotide consisting of nucleotides 457 to 810 of SEQ ID NO: 1; A) a polynucleotide consisting of nucleotides 457 to 1353 of SEQ ID NO. 1; q) a polynucleotide consisting of nucleotides 457 to 3006 of SEQ ID NO. 1; r) a nucleotide consisting of nucleotides 457 to 3006 of SEQ ID NO. S) a polynucleotide consisting of nucleotides 580 to 1353 of SEQ ID NO: 1; t) a polynucleotide consisting of nucleotides 580 to 3006 of SEQ ID NO: 1; and u) a polynucleotide consisting of nucleotides 811-1353 of SEQ ID NO: 1. An isolated polynucleotide selected from the group consisting of: Polynucleotide. 33. SEQ ID NO: 2 operably linked to an additional polypeptide
A polynucleotide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1 to 20 of 34. A polynucleotide molecule encoding a fusion protein consisting essentially of a first portion and a second portion linked by a peptide bond, said first portion comprising the polypeptide of claim 1. A polynucleotide molecule comprising, and said second portion comprising another polypeptide. 35. An expression vector comprising: an operably linked element: a transcription promoter; a polynucleotide molecule encoding the polypeptide of claim 1; and a transcription terminator. 36. The expression vector according to claim 35, further comprising a secretory signal sequence operably linked to said DNA segment. 37. The expression vector of claim 35, wherein said polynucleotide encodes a polypeptide covalently attached at the amino or carboxy terminus to an affinity label. 38. The method of claim 1, wherein the expression vector comprises: a transcription promoter; a polynucleotide molecule encoding the polypeptide of claim 1; and a transcription terminator. A cultured cell that expresses the polypeptide encoded by a nucleotide segment. 39. A method for producing a polypeptide, comprising: an operably linked element: a transcription promoter; a polynucleotide molecule encoding the polypeptide of claim 1; and a transcription terminator. Culturing cells into which the vector has been introduced, whereby said cells express said polypeptide encoded by said polynucleotide segment; and recovering said expressed potypeptide. 40. An antibody or antibody fragment that specifically binds to the polypeptide of claim 1. 41. The antibody of claim 40, wherein said antibody is selected from the group consisting of: a) a polyclonal antibody; b) a murine monoclonal antibody; c) a human adapted antibody derived from b); and d) a human monoclonal antibody. Antibodies. 42. The antibody fragment may be F (ab '), F (ab), Fab', Fab,
42. The antibody fragment according to claim 41, wherein the antibody fragment is selected from the group consisting of Fv, scFv and a minimal recognition unit. 43. An anti-idiotype antibody that specifically binds to the antibody of claim 40. 44. The polypeptide of claim 1, in combination with a pharmaceutically acceptable vehicle.