JP2001525195A - Mammalian alpha helix protein-1 - Google Patents

Abstract

  (57) [Summary] Novel mammalian alpha helical polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods, including antibodies and anti-idiotype antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The growth, survival and differentiation of cells in multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to interconnect,
Coordinates to form cells and organs, allowing repair and regeneration of damaged tissue. Examples of hormones and growth factors include steroid hormones (eg, estrogen, testosterone), parathyroid hormone, follicle-stimulating hormone, interleukins, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colonies There are stimulants (GM-CSF), erythropoietin (EPO) and calcitonin.

[0002] Hormones and growth factors bind to proteins and affect the metabolism of cells. Proteins are intramembrane proteins linked to intracellular signaling pathways, such as the second messenger system. Other proteins are soluble molecules such as transcription factors.

[0003] Of particular interest are cytokines, which are molecules that promote proliferation, maintenance, survival, or differentiation of cells. Examples of cytokines include erythropoietin (EPO), which promotes differentiation of erythrocytes; thrombopoietin (TPO), which promotes differentiation of megakaryocytic cells; and granulocyte-colony stimulation which promotes differentiation of neutrophils. Factor (G-CSF
). These cytokines are useful for restoring normal blood cell levels in anemic patients or patients undergoing cancer chemotherapy. In these cytokines
In vivo activity illustrates the great clinical potential and demand of other cytokines, cytokine agonists and cytokine antagonists.

SUMMARY OF THE INVENTION The present invention addresses this need by providing novel polypeptides and related compositions and methods. One aspect of the present invention is to provide an isolated polynucleotide that encodes a mammalian protein called "alpha helical protein-1" or Zalphal. The human sulfur polypeptide has an amino acid sequence starting at Met and having a length of 146 amino acids as shown in SEQ ID NO: 1 and SEQ ID NO: 2. Amino acid residues 1-20 are considered to be a signal sequence, and the mature zulfal polypeptide is represented by an amino acid sequence consisting of isoleucine at residue 21 and a tyrosine at amino acid residue 146. The mature sulfur polypeptide is SEQ ID NO: 45
Is also defined by In another embodiment, the polypeptide further comprises an affinity tag. In another embodiment, the polynucleotide is DNA.

[0005] Also claimed are polypeptides that are at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 45, and polynucleotides that encode the polypeptides. According to a second aspect of the present invention, there is provided a (a) transcription promoter, (b) a DNA fragment encoding a sulfur polypeptide, and (c) a transcription terminator, wherein the promoter, the DNA fragment, and the terminator are operably linked. An expression vector is provided. According to a third aspect of the present invention, there is provided a cultured eukaryotic cell or bacterial cell into which the expression vector disclosed above has been introduced, wherein the cell expresses a sulfur polypeptide encoded by a DNA fragment. You.

In another aspect of the invention, there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion linked by a peptide bond. The first portion of the chimeric polypeptide may be (a) the sulfur polypeptide shown in SEQ ID NO: 2 or SEQ ID NO: 45, or (b) a protein polypeptide that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 45. Either. The second portion of the chimeric polypeptide consists essentially of another polypeptide, such as an affinity tag. In one embodiment, the affinity tag is an immunoglobulin Fc polypeptide. The invention also provides an expression vector encoding the chimeric polypeptide and a transfected host cell producing the chimeric polypeptide.

[0007] Another embodiment of the present invention relates to a peptide or polypeptide having the amino acid sequence of the epitope-bearing portion of a sulfur polypeptide having the amino acid sequence disclosed above. The peptide or polypeptide having the amino acid sequence of the epitope-bearing portion of the sulfur polypeptide of the present invention includes a polypeptide portion having at least 9, preferably at least 15, and more preferably at least 30 to 50 amino acids as described above. Epitope-bearing polypeptides of any length up to the entire amino acid sequence of the polypeptides of the invention disclosed above are also included in the invention.

Further, any of these polypeptides fused to another polypeptide or carrier molecule are claimed. Antibodies generated from these portions bearing the epitope of sulfur can be used for purification of sulfur from cell culture medium. Examples of such epitope-bearing polypeptides are the polypeptides of SEQ ID NOs: 46-56. Similarly, proteins or polypeptides comprising sequences that are at least 90% identical to the epitope-bearing polypeptide are claimed. In another aspect of the present invention, there is also provided an antibody that specifically binds to the above-mentioned sulfur polypeptide, and an anti-idiotype antibody that neutralizes an antibody against the sulfur polypeptide. These and other aspects of the invention will become more apparent with reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION The term "allelic variation" as used herein means any of two or more variants of a gene occupying the same chromosomal locus. Allelic variation arises spontaneously through mutation, resulting in genetic and phenotypic polymorphism within a population. Gene mutations are silent (the encoded polypeptide does not change) or encode the polypeptide with an altered amino acid sequence. The term allelic variation herein also refers to a protein encoded by an allelic variation of a gene.

[0010] The term "expression vector" refers to a linear or circular fragment containing a fragment encoding the desired polypeptide operably linked to additional fragments to provide its transcript.
Used to mean a DNA molecule. Such additional fragments include promoter and terminator sequences, as well as one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

As used with polynucleotides, the term “isolated” refers to polynucleotides that have been removed from their natural genetic environment and, in other words, free from other exogenous or unwanted coding sequences. Means that it is in a form suitable for application into a genetic engineering protein production system. "Operably linked" when referred to as a DNA fragment is one in which the fragment is arranged in a manner that functions for a given purpose, such as initiation of transcription within a promoter and passage through a coding fragment to a terminator. To indicate that

“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 may be prepared by isolation from natural sources, by synthesis in vitro, or by a combination of natural and synthetic molecules. The term "promoter" is used in the art-recognized meaning to mean the portion of the gene that contains the DNA sequence that provides for the binding of DNA polymerase and initiation of transcription. The promoter sequence is generally, but not entirely, within the 5 'non-coding region of the gene.

“Soluble protein” is a protein polypeptide that is not associated with the cell membrane. In a preferred embodiment of the invention, the isolated polynucleotide hybridizes under stringent conditions to a similarly sized region of SEQ ID NO: 1, or a sequence complementary thereto. Generally, stringent conditions are selected to be about 5 ° C. below the melting point (Tm) of the sequence at a particular ionic strength and pH. Tm (specific ionic strength and
50% of the target sequence (at pH) is the temperature at which it hybridizes to a suitable compatible probe. Typical stringent conditions are a salt concentration of about 0.02M or less at pH 7, and a temperature of at least about 60 ° C.

As described above, the isolated polynucleotides of the present invention include DNA and RNA. DN
Methods for isolating A and RNA are known to those skilled in the art. Total RNA can be isolated by guanidine HCl extraction followed by centrifugation in a CsCl gradient [Chirgwin et al., Bioche.
mistry 18: 52-94 (1979)]. Poly (A) + RNA was obtained from total RNA by Aviv and Leder's method [Pr
oc.Natl.Acad.Sci.USA 69: 1408-1412 (1972)]. Complementary DNA
(CDNA) is prepared from poly (A) + RNA using a known method. Subsequently, the polynucleotide encoding the Sulfur polypeptide is identified and isolated, for example, by hybridization or PCR.

Further, the polynucleotide of the present invention can be synthesized using a DNA synthesizer. Currently, the method of choice is the phosphoramidite method. When chemically synthesized double-stranded DNA is required for the purpose of application to the synthesis of genes or gene fragments, complementary strands are separately synthesized. Short pronucleotides (60-80 bp)
) Is technically unidirectional and can be accomplished by synthesizing complementary strands and subsequently annealing them. However, when producing longer polynucleotides (> 300 bp), special methods may be used because the coupling efficiency in each cycle during chemical DNA synthesis is rarely 100%. Many.

In order to solve this problem, a synthetic gene (double-stranded) is assembled in a modular form from a single-stranded fragment having a length of 20 to 100 nucleotides. Glick and Pasternak, Mole
cular Biotechnology, Principles & Applications of Recombinant DNA, (ASM
Press, Washington, DC 1994); Itakura et al., Annu. Rev. Biochem. 53; 323-35.
6 (1984), and Climie et al., Proc. Natl. Acad. Sci. USA 87: 633-637 (1990).
checking ... One skilled in the art will recognize that the sequences disclosed in SEQ ID NOs: 1 and 2 represent a single allele of humans. Allelic variations of these sequences can be cloned by probing a cDNA or genomic library from various solids by a conventional method.

The present invention further provides proteins and polypeptides of interest from other species ("species orthologs"). Of particular interest are sulfur polypeptides from other mammalian species, including mice, pigs, goats, cows, dogs, cats, horses, and other primates. Species homologues of the human sulfur protein can be cloned by combining the information and components provided by the present invention, usually with cloning techniques.
For example, cDNA can be cloned using mRNA obtained from a tissue or cell expressing the protein. 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 a positive tissue or cell line. The cDNA encoding the protein can then be isolated by various methods, such as by probing with complete or partial human or mouse cDNA, or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA was prepared using the polymerase chain reaction or PCR (Mullis, US Pat.
No. 683,202).

In another method, host cells can be transformed or transfected with a cDNA library, and expression of the desired cDNA can be detected with an antibody against the protein.
Similar techniques can be applied to the isolation of genomic clones. The term "isolated polynucleotide encoding a polypeptide, the polynucleotide of which is defined in SEQ ID NO: 2" is used and, when claimed, all alleles of the polypeptide of SEQ ID NO: 2 Includes mutation and species orthologs.

The invention also provides a protein polypeptide of SEQ ID NO: 2 and an isolated protein polypeptide that is substantially identical to an ortholog thereof. "Isolated" refers to a protein or polypeptide that is found in conditions other than its natural environment, such as in blood and animal tissues. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, especially other polypeptides of animal origin. Highly purified form, for example, having a purity of 95% or more, more preferably a purity of 99% or more.
It is preferred to provide at least% polypeptide.

As used herein, the term “substantially identical” refers to SEQ ID NO: 2 or SEQ ID NO: 45
Or a polypeptide having 50%, preferably 60%, more preferably at least 80% sequence identity to the orthologs of that species.
Such polypeptides are more preferably at least 90% identical to SEQ ID NO: 2, or SEQ ID NO: 45, or a species ortholog thereof, most preferably 95% or more.

[0022] The percent sequence identity is determined by conventional methods. For example, Altshul et al., Bull. Math.
Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89
: 10915-10919, see 1992. In summary, the two amino acid sequences are shown in Gap Opening Penalty 10, Gap Extension Penalty 1, and Henikoff and Heniko shown in Table 1 (amino acids are indicated by standard one letter code).
ff Use the "blossom 62" scoring matrix (above) to align the alignment scores to optimize. Then calculate the identity rate as follows:

[0023]

(Equation 1)

[0024]

[Table 1]

[0025] Sequence identity of polynucleotide molecules is determined in a similar manner using the ratios disclosed above. Substantially identical proteins and polypeptides can have one or more amino acid substitutions,
It is characterized as having deletions or additions. These changes are conservative amino acid substitutions (see Table 2), and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; typically, small deletions of 1 to about 30 amino acids;
A small extension of the amino or carboxyl terminus, such as an amino-terminal methionine residue, a small linker peptide of about 25-30 residues, or a poly-histidine tract,
Protein A [Nilsson et al., EMBO J 4: 1075 (1995); Nisosson et al., Methods Enzy.
mol. 198: 3 (1991)], glutathione S transferase [Smith and Johnson, G
ene 67:31 (1988)], or other antigenic epitopes, or those of minor nature, such as binding domains, which are small extensions to facilitate purification. DNAs encoding affinity tags are commercially available (eg, Pharmacia Biotech, P
iscataway, NJ).

[0026]

[Table 2]

[0027] The essential amino acids in the polypeptides of the present invention may be site-directed mutagenesis or alanine scanning mutagenesis [Cunnigham and Wells, Science 244, 1081-1085 (
Natl. Acad. Sci. USA 88: 4498-4502 (1991)], and methods known to those skilled in the art. In the latter technique, one alanine mutation is introduced at every residue in the molecule, and the resulting mutated molecule is tested for biological activity (eg, ligand binding and signal transduction),
Identify amino acid residues that are important for the activity of the molecule.

Analysis of the crystal structure determined by techniques such as nuclear magnetic resonance, crystallography or photoaffinity labeling can also identify ligand-protein interaction sites. For example, Vos et al., Science 255: 306-312 (1992); Smith et al., J. Mol. Bio.
1, 224: 899-904 (1992); see Mlodaver et al., FEBS Lett. 309: 59-64 (1992). The identification of essential amino acids can also be inferred from analysis of abuse with related proteins.

Multiple amino acid substitutions are described in Reidhaar-Olson and Sauer, Science 241: 53-57, (1988).
Alternatively, it can be performed by the mutagenesis method and screening disclosed in Bowie and Sauer, Proc, Natl. Acad. Sci. USA 86: 2152-2156 (1989). In summary, these authors randomized simultaneously two or more sites in a polypeptide, selected a functional polypeptide, and subsequently sequenced the mutated polypeptide,
A method for determining the range of possible substitutions for each site is disclosed. Other methods that can be used include phage display (eg, Lowman et al., Biochem. 30: 10832-10837 (1.
Ladner et al., US Pat. No. 5,223,409; Huse, WIPO published WO 92/06204) and region-directed mutagenesis [Derbyshire et al., Gene 46: 145 (1986); Ner et al., DNA 7; 127.
(1988)].

The above-described mutagenesis method can be combined with a high-throughput screening method to detect the activity of a protein cloned and expressed in a host cell. Suitable assays in this regard include the cell proliferation assays and biosensor-based ligand binding assays described below. The mutated DNA molecule encoding the active protein, or a portion thereof (eg, a ligand binding fragment), can be recovered from the host cell and rapidly sequenced using modern equipment. These methods determine the importance of individual amino acid residues in the desired polypeptide and can be applied to polypeptides of unknown structure.

Using the above method, one skilled in the art can prepare various polypeptides that are substantially homologous to SEQ ID NO: 2 or an allelic variant thereof and that retain the properties of the wild-type protein. As expressed and claimed herein, the term "polypeptide as defined in SEQ ID NO: 2" includes all allelic variations and species orthologs of the polypeptide. A polynucleotide, generally a cDNA sequence, of the present invention encodes the above polypeptide. The cDNA sequence encoding the polypeptide of the present invention contains a series of codons, each amino acid residue of the polypeptide is encoded by a codon, and the codon is composed of three nucleotides. Amino acid residues are encoded by the following codons:

Alanine (Ala) is encoded by GCA, GCC, GCG or GCT; Cysteine (Cys) is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid (Glu) Is encoded by GAA or GAG; phenylalanine (Phe) is encoded by TTC or TTT; glycine (Gly) is encoded by GGA, GGC, GGG or GGT; histidine (His) is encoded by CAC or CAT;

Isoleucine (Ile) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA, CCC, CCG or CCT;

Glutamine (Gln) is encoded by CAA or CAG; Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is AGC, AGT, TCA, TCC, TCG or Threonine (Thr) is encoded by ACA, ACC, ACG or ACT; Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG and tyrosine (Trp) Tyr) is coded by TAC or TAT.

According to the present invention, when a cDNA is claimed as described above, the claimed subject matter is a two strand having a sense strand, an antisense strand, and both a sense strand and an antisense strand each annealed by hydrogen bonding. It is strand DNA. Also, a messenger RNA (mRNA) encoding the polypeptide of the present invention, and an mR encoded by the above cDNA
NA is also charged. Messenger RNA (mRNA), each thymine nucleotide (T)
Encodes a polypeptide with the same codons described above, except that is replaced by a uracil nucleotide (U).

The protein polypeptides of the present invention, including full-length proteins, protein fragments (eg, ligand binding fragments) and fusion polypeptides, can be produced in genetically engineered host cells by conventional methods. Suitable host cells are those types of cells that have been transformed or transfected with the foreign DNA and are cultivable, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, especially cultured cells of multicellular organisms, are preferred. Manipulating cloned DNA molecules and foreign DNA into various host cells
For techniques for inserting molecules, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laborator).
y Press, Cold Spring Harbor, NY, 1989). and Ausubel see above.

Generally, a DNA sequence encoding a Zalphal polypeptide is required for its expression in an expression vector and is operably linked to genetic elements, generally including a transcription promoter and terminator. One skilled in the art will recognize that in certain systems, the selectable marker is provided by a separate vector, and replication of the foreign DNA is provided by integration into the host cell genome, but in general, the vector may comprise one or more vectors. Also includes a selectable marker and one or more origins of replication. Promoters, terminators,
Selection of selectable markers, vectors and other elements is a matter of ordinary skill in the art. Many of these elements are described in the literature and are commercially available.

To direct a Zalphal polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence is that of the protein, or may be derived from another secreted protein (eg, t-PA) or de novo synthesized. The secretory signal sequence contains the DN of Sulfur in the correct reading frame.
A Joins the array. Generally, the secretory signal sequence is a DN encoding the desired polypeptide.
Located 5 ′ of the A sequence (see, eg, Welch et al., US Pat. No. 5,037,743; HOLland et al., US Pat. No. 5,143,830).

In the present invention, cultured mammalian cells are a preferred host. Foreign introduction to mammalian host cells
DNA transfection methods include calcium phosphate mediated transfection methods [Wigler et al., Ce
ll 14: 725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7: 603, (1981);
Graham and Wan der Eb, Virology 52: 456, (1973)], electroporation [
Neumann et al., EMBO J. 1: 841-845, (1982)], DEAE-dextran mediated transfection method [Ausubel et al., Ed., Currrent Protocols in Molecular Biology,
(John Wiley and Sons, Inc., NY, 1987)] and a liposome-mediated transfection method [Hawley-Nelson et al., Cocus 15:73 (1993); Ciccarone et al., Focus 15: 8.
0, (1993)].

The production of recombinant polypeptides in cultured mammalian cells is described, for example, in Levinson et al., US Pat. No. 4,713,339; Hagen et al., US Pat. No. 4,784,950, Pa; miter et al.
U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include COS-1 (ATCC number, CRL1650), CO
S-7 (ATCC number CRL1651), BHK (ATCC number CRL1632), BHK570 (ATCC number, CRL1)
0314), 293 [ATCC number CRL1573; Graham et al., J. Gen. Virol. 36: 59-72 (1977)].
And Chinese hamster ovary cell lines (eg, CHO-K1; ATCC No. CCL61).

[0041] Other suitable cell lines are known to those of skill in the art and are available from depository institutions such as the American Type Culture Collection, Rockville, Maryland. Generally, strong transcription promoters, such as the SV-40 or cytomegalovirus promoters, are preferred. See U.S. Patent No. 4,956,288. Other suitable promoters include the metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and promoters from the adenovirus late major promoter.

[0042] Drug selection is generally used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly called "transfectants." Cells cultured in a selection agent and capable of delivering the desired gene to its progeny are termed "stable transfectants." A preferred selection 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. The selection system can also be used to increase the expression level of the desired gene, a step called "amplification."

Amplification is performed by culturing the transfectants in the presence of a low concentration of the selection agent, and then increasing the concentration of the selection agent to a level that allows selection of cells that produce high levels of the introduced gene product. You. A preferred amplification selection marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (
For example, hygromycin resistance, multidrug resistance, puromycin acetyltransferase) can also be used.

Other higher eukaryotic cells, including insect cells, plant cells, and bird cells, can also be used as host cells. Transformation of insect cells and production of foreign polypeptides therein has been described by Guarino et al., US Pat. No. 5,162,222; Bang et al., US Pat. No. 4,775,624;
It is disclosed in PO Publication No. WO94 / 06463. The use of Agrobacterium rhizogenes as a vector for gene expression in plant cells has been reviewed by Sinkar et al., J. Biosci (Bangalore) 11: 47-58, (1987).

In the present invention, yeast cells and especially fungal cells including cells of the genus Saccharomyces can also be used for the production of protein fragments or polypeptide fusions. Methods for transforming yeast cells with foreign DNA and producing recombinant polypeptides therefrom are described, for example, in Kawasaki, US Pat. No. 4,931,373;
U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray.
No. 4,845,075. Transformed cells are sorted by 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 yeast is described in US Pat. No. 4,931,373 by Kawa.
A POT1 vector system disclosed by saki et al. wherein transformed cells are selected for by growth in a glucose-containing medium. Suitable promoters and terminators for yeast applications include glycolytic enzyme genes (eg, Kawasaki, US Pat.
No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974 and Bittere, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patent Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.

[0047] Hansenula polymorpha, Schizosaccharomyces
Pombe (Schizosaccharomyces pombe), Kluyve
romyces lactis), Kluyveromyces fragilis
, Ustilago maydis, Pichia pastoris
pastoris), Pichia methanolica, Pichia guillermondii, and Candida malt
Transformation systems for other yeasts, including osa) are known to those of skill in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465 (1986) and Cregg et al., U.S. Patent No. 4,882,279.

Aspergillus cells are described in McKnight et al., US Pat. No. 4,935,349.
It is used by the method of No. The method of transforming Acreniuymn chrysogenum
No. 5,162,228 to Sumino et al. A method for transforming Neurospora is disclosed by Lambowitz, U.S. Patent No. 4,486,533.

The transformed or transfected host cells are cultured in a conventional manner in a medium containing nutrients and other components necessary for the growth of the selected host cells.
Various suitable media are known to those of skill in the art, including defined and complex media, and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The medium can also optionally contain growth factors or serum. In general, the growth medium is selected for cells containing DNA using, for example, drug selection or deletion of essential nutrients, which are complemented by expression vectors or selectable markers brought into the host cell by co-transfection.

[0050] Another embodiment of the present invention provides a peptide or polypeptide comprising an epitope-bearing portion of a Zalphal polypeptide of the present invention. The epitope of the polypeptide portion is an immunogenic or antigenic epitope of the polypeptide of the present invention. The region of the portion to which the antibody can bind is defined as an "antigenic epitope." For example, Geysen, H.
See M et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984).

When selecting a peptide or polypeptide having an antigenic epitope (ie, including a region of a protein molecule to which an antibody can bind), a relatively short synthetic peptide having a similar protein sequence can be partially simulated by ordinary procedures. It is well known to those skilled in the art that it is possible to generate antisera that bind to the selected proteins. Sutcliffe, JG et al.
Science 219: 660-666 (1983). Peptides capable of giving rise to protein-reactive sera are often expressed in the primary sequence of proteins, can be characterized by a combination of simple chemical rules, and have the immunodominant regions (ie, immune epitopes) or amino acids of the intact protein. Or, it is not limited to any of the carboxyl terminals. Extremely hydrophobic peptides, and peptides of 6 or fewer residues, are generally ineffective at inducing antibodies that bind to the mimetic protein; usually longer water-soluble peptides, especially those containing proline residues, It is effective.

Accordingly, the peptides and polypeptides having an antigenic epitope of the present invention are useful for raising antibodies that specifically bind to the polypeptides of the present invention, including monoclonal antibodies. The antigenic epitope-bearing peptides and polypeptides of the present invention comprise at least 9, preferably 15 to about 30 amino acids contained in the amino acid sequence of the polypeptide of the present invention. However, peptides or polypeptides comprising a longer portion of the amino acid sequence of the invention, 30 to 50 amino acids, or comprising the entire amino acid sequence of the polypeptide of the invention are also useful for inducing antibodies that react with the protein.

Preferably, the amino acid sequence of the epitope-containing peptide is selected to provide substantial solubility in aqueous solution (ie, the sequence comprises relatively hydrophilic residues and hydrophobic residues are preferably Avoid); and sequences containing proline residues are particularly preferred.
All polypeptides found in the listed sequences include the antigenic epitopes utilized by the present invention, but specifically designed antigenic epitopes include the peptides defined in SEQ ID NO. The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zalphal polypeptide described herein. These fragments or peptides will contain an "immunogenic epitope" which is the portion of the protein that elicits an antibody response when the whole protein is used as an immunogen.

[0054] Immunogenic epitope-containing peptides can be identified by conventional methods [eg, Geys
See also en et al., supra, or US Patent No. 4,708,791 (1987), which describes methods for identifying peptides having an immunogenic epitope of the desired protein]. A specific example of an epitope-bearing polypeptide is a polypeptide comprising SEQ ID NOs: 46-56, particularly a polypeptide comprising the polypeptide defined by SEQ ID NOs: 50-56.

SEQ ID NO: 50 is a polypeptide constituting helicases A and B; SEQ ID NO: 51
Is a polypeptide constituting helicases A, B and C; SEQ ID NO: 52 is a polypeptide constituting helicases A, B, C and D; SEQ ID NO: 53 is a polypeptide constituting helicases B and C SEQ ID NO: 54 contains helicases B, C and D;
SEQ ID NO: 55 is a polypeptide that comprises helicases C and D; SEQ ID NO: 56 consists of the polypeptide of SEQ ID NO: 2 that extends from the beginning of helix C to the end of the polypeptide.

The peptides and polypeptides of the present invention having an antigenic epitope can bind to the polypeptides described herein and subsequently be used to purify the native or denatured proteins or to detect Zalpal polypeptides in Western blots. It is useful for producing usable antibodies.

Isolation of protein: The expressed recombinant polypeptide (or chimeric polypeptide) was fractionated, and / or
Alternatively, it can be purified using a conventional purification method and medium. For example, “Affinity Chromatography: Principles & Method
s "(Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988), Methods in E
nzymol., Vol.182, "Guide to Protein Purification",
M. Deutscher, (eds.), Pp.529-39 (Acad. Press. SanDiego (1990) and "Protein Purificaiton, Principles and Practice", No. 3)
See the edition, Scopes, Robert.

Structure of Zalphal Zalfa is presumed to be a 4-helical polypeptide similar to the helical cytokine family represented by growth hormone, erythropoietin, leptin and interleukin-10. Helix A of Sulfur has amino acid residue 23 of SEQ ID NO: 2.
From aspartic acid to arginine at amino acid residue 37. Helix A is also defined by SEQ ID NO: 46. Sulfur helix B is believed to contain from phenylalanine at amino acid 53 of SEQ ID NO: 2 to phenylalanine at amino acid residue 67.

Helix B is also defined by SEQ ID NO: 47. It is believed that the helix C of sulfur contains from phenylalanine at amino acid 82 of SEQ ID NO: 2 to a leucine residue at amino acid residue 96. Helix B is also defined by SEQ ID NO: 48. Helix D of Zalphal is believed to include tyrosine at amino acid residue 118 of SEQ ID NO: 2 to aspartic acid at amino acid residue 132. Helix D is also defined by SEQ ID NO: 49.

Utilization Using a radiation hybrid panel, zalfar was mapped in close proximity to X127.3, the base of FMR1, a gene linked to Fragile-X syndrome. FMR1 is an evolutionarily conserved gene that is transcribed at relatively high levels in brain, testis, heart, lung, kidney, and placenta, and has low or undetectable transcription in liver, pancreas, and smooth muscle. [Hinds et al., Nature Genet, 3: 36-43 (1993)]. The function of the FMR1 protein is unknown. Studies have shown that FMR1 binds to mRNA and has a nuclear translocation consensus sequence, suggesting that it is a nuclear protein [Ashley et al., Science 262: 563-566) 1993); Siomi et al. Cell74: 291-298 (1993)
].

[0061] The molecular basis of Fragile-X Syndrome relies on the unstable, hereditary, multi-step expansion of the polymorphic triplet repeat (CGG) n within the 5'-untranslated region of FMR1. Hagerman, ENG GB
See Men. Ret. And Devel. Dis, Res. 1: 276-280 (1995). In the normal population, the number of triplets varies, ranging from 6-53 units. Disease carriers exhibit a repeat length of 43-200 units and are said to be in the premutation stage. A perfect mutation has the characteristic that this repetition extends to more than 200. This expansion results in hypermethylation of the CGG repeat and FMR1 promoter sequence, resulting in FMR1
And possibly the transcription of nearby genes is inactivated. Injured individuals have immature ovarian failure, premature menopause, or precocious sexual maturity associated with the premutation stage.

The severity of these and other conditions varies from individual to individual and may reflect underlying dysfunction in the hypothalamus-pituitary-gonadal pathway. In men who have a complete mutation, almost all cases have moderate to severe mental illness [Rousseau et al.
Am. J. Hu. Genet. 55: 225-137 (1994)]. Approximately 50-70% of women with a complete mutation exhibit a mental disorder [Hagerman et al., Pediatrics 99: 395-400 (1993); Rousseau et al., Supra]. Fully mutated males show giant orchidism, characteristic facial features, velvety skin and hyperextensible joints. Women's symptoms are more diverse, which may be due to differences in X-inactivation.

Although the involvement of EMR1 is evident in fragile-X chromosome-complete patients with complete mutations, some of the pleiotropic expression of fragile-X chromosome syndrome may be the result of impaired expression of nearby genes Sex is also left. Methylation is known to inhibit transcription by altering the conformation of DNA and preventing direct binding of transcription factors. It has been shown that methylated DNA tends to form Z-DNA. Thus, if the CGG repeat is hypermethylated or the FMR1 promoter is abolished by other mechanisms, the expression of other genes over a considerable distance will be affected.
Am. J. Med. Genet., 43: 299-396 (1992) describe a fragile-proximal region of the X chromosome locus 1000.
It was reported that the level of iduronate sulfatase encoded by the IDS gene located at kb (Hunter syndrome) was decreased in fragile-X chromosome patients.

Zulfar used radiation panels to identify two vulnerable parts in Xq27.3, FRAXA and
Positioned during FRAXE. Due to its close proximity to the FMR1 locus, the expression of zalfar would be inhibited by hypermethylation leading to expansion of CGG repeats and loss of FMR1. Thus, the diverse phenotypes associated with Fragile-X Chromosome Syndrome may be due, at least in part, to the loss of Zalphal expression. Administration of a Zalphal polypeptide, agonist or antagonist thereof will provide a clinical treatment for these conditions.

The exact physical distance from Sulfur to FMR1 can be refined using multiple existing mapping reagents, including somatic cell hybrids, yeast artificial chromosome (YAC) clones, and bacterial artificial chromosome (BAC) clones. The existing somatic cell hybrid mapping panel describes the CGG triplet repeat of FRAXA associated with Fragile-X chromosome syndrome [Warren et al., Proc. Nat. Acad. Sci., 87: 3856 (1990)] and the base of FRAXE.
Provide an effective breakpoint at the IDS locus (Hunter syndrome) [Suthers et al., Am. J. Hum. Genet., 47: 187 (1990)].

Using these reagents, a radiation hybrid panel can confirm that Zalphal is located in the correct chromosomal region for RMR1. Including both FRAXA and FRAXE,
A YAC clone containing a BSSHII fragment that matches the pulsed field map of the genomic region is obtained; therefore, this YAC is free of deletions, in other words, is considered to be an effective mapping reagent. The mapping has one unprocessable gap, and a BAC contig that connects FRAXA and FRAXE can also be used.

Physical mapping of sulfur can also be easily performed by the polymerase chain reaction (PCR) using mapping reagents such as genetically specific primers and templates. Analysis will first be performed using a somatic cell hybrid panel; confirmation of region localization will be performed by higher resolution mapping using YAC and BAC clones. By mapping Zalphal to one or more BAC clones, the localization position can be known with an accuracy within 100 kb, and as a result, the relative distance to the fragile site can be easily determined.

The presence of detectable levels of sulfal transcripts in peripheral leukocytes means that
One can also examine readily available lymphoblastoid cell lines derived from patients with fragile-X chromosomes with expanded CGG repeats. Zalphal transcript level analysis of cell lines using Northern blot analysis or RT / PCR will confirm reduced or zero levels of Zalphal transcript in vulnerable-X chromosome patients.

Changes in Zalphal transcripts in vulnerable-X chromosome patients can be determined by Northern blot analysis or RT / PCR on pituitary, aortic, or other RNA samples isolated from vulnerable-X chromosome patients. Can be determined directly. Zalpha of the present invention
l Transcripts were found to be at high levels in the pituitary and aorta. Low levels have been found in brain, kidney, pancreas, prostate, testis, ovaries, thyroid, spinal cord, esophagus, and adrenal glands. Trace levels were observed for placenta, lung, liver, bone marrow and peripheral leukocytes. Expression in the brain and pituitary gland suggests that Zalphal plays a regulatory role in the hypothalamus-pituitary-gonadal pathway.

Recent studies have provided clinical evidence of connective tissue dysfunction in some vulnerable-X chromosome patients. Opitz et al., Am, J. Med. Genet. 27: 101-109 (1984) report a connective tissue dysplasia characterized by overextended finger joints, flat feet, and mitral prolapse. Waldstein and Hagerman, Am. J. Med. Genet., 30; 83-98 (1988) report patients with aortic hypoplasia and heart valve abnormalities. Other physical findings commonly found in vulnerable-X chromosome patients, such as macro testis, ears and hyperelastic skin, may also be associated with connective tissue dysplasia. Elastin fibers are reduced in these diseased tissues and their appearance has been reported to be abnormal, fragmented and unsequential. Such fibers may not provide adequate networking for joint stability, skin tenacity, normal arterial growth and development, heart valve structure and function.

It has been suggested that the locus at or near Xq27.3 is involved in the modulation of elastin structural integrity, or its production [Waldstein and Hagerman Am, J. et al.
Med. Cenet. 30: 83-98 (1988)]. Zulfar would be the proposed locus. That is, the Sulfur polypeptides, agonists or antagonists thereof, are therapeutically useful for the growth, differentiation, maintenance or survival of connective tissue. Specifically, connective tissue of the cardiovascular and epithelial systems. Clinical applications include vascular disease, giant testicular disease, skin disease,
It may include joint instability and other clinical connective tissue dysfunctions. Other applications include improving osmotic pressure on normal connective tissue, such as enhancing skin tone and softness.

Evaluation of Zulfal-containing fusion proteins such as Zulfal polypeptides, fragments thereof, Zalphal-Fc constructs, antibodies, agonists or antagonists for proliferation, differentiation, maintenance, or survival activity of connective tissue can be performed using cell culture or cell culture. It can be performed using an animal system. Using the expression of elastin, collagen, and other phenotypic markers,
The effect in vitro can be monitored. When administered to a mouse model, the proteins of the invention are formulated for parenteral administration, specifically, for intravenous or subcutaneous supply, by conventional methods.

Delivery to animals also includes the use of viral systems such as adenovirus, adeno-associated virus and retroviral systems. The method of administration is empirically determined considering the stability of the protein and other pharmacokinetic parameters known to those skilled in the art. The effects of the present invention on connective and other tissues, or the growth, differentiation, maintenance or survival of a period, can be determined by examination of histological sections obtained from recipient animals.

Particular attention will be paid to tissues or periods in which high levels of sulfur are expressed. Evaluation includes abnormal cell proliferation, or cell death. Masson's triple staining for collagen, Orcein and Wehoff-Vangieson staining for elastin and collagen; Colloidal iron staining of hell for acidic mucopolysaccharides. The direct effect of the present invention on skin softness, as well as other effects on skin, may be determined using transdermal delivery systems known to those skilled in the art. The role of the present invention on the hypothalamus-pituitary-gonadal pathway can be examined by measuring changes in blood levels of gonadotropin, luteinizing hormone, follicle-stimulating hormone and other hormones in recipient animals. it can.

The present invention also provides reagents with distinct therapeutic value. Sulfur polypeptide (
Natural or recombinant), fragments thereof, antibodies and anti-idiotypic antibodies thereto, when combined with a compound identified as having a binding affinity for a sulfur polypeptide, results in abnormalities such as, for example, a cancerous state or a degenerative state. Useful for treating abnormal physiology, including growth, or conditions associated with development. Diseases or disorders involving aberrant expression or signaling, eg, by Zaplhal polypeptides, would be suitable targets for agonists or antagonists of the Sulfur polypeptide.

[0076] Antibodies to the Sulfur polypeptide can be purified and subsequently administered to a patient. These reagents can be combined with pharmaceutically acceptable carriers, or diluents, together with additional active or inert additives, such as physiologically harmless stabilizers and excipients, for therapeutic applications. Such combinations can be sterile filtered, lyophilized into dosage forms in dosage vials, or stored as stabilized solution preparations. The invention also encompasses the use of single chain antibodies of antibodies, including antibodies, binding fragments thereof, or non-complement binding forms.

The amount of reagent required for effective treatment will depend on a variety of factors, including the mode of administration, the target site, the physiological condition of the patient, and the other drug being administered. That is, the therapeutic dose must be titrated to maximize safety and efficacy. Typically, in vitro
The dosages used in the above provide valuable guidance as to the beneficial amounts of these reagents for in vivo administration. Animals with an effective dose for the treatment of a particular disease will provide a more accurate indication of human dose. Methods of administration include oral, intravenous, peritoneal, intramuscular, or transdermal.

Pharmaceutically acceptable carriers will include water, saline, and a few buffers. Dosage ranges will usually be determined from 1 μg to 1000 μg / kg body weight / day. However, higher or lower dosages may be determined by a physician of ordinary skill in the art. For a complete discussion of drug composition and dosage ranges, see Reming.
ton's Pharmaceutical Sciences, 18th edition (Mack Publishing Co., Easton, Penn.
, 1996) and Goodman Gilman's; The Pharmacological Bases of Therapeutics
, 9th edition (Pergamon Press 1996).

Introduction of Nucleic Acid into Mammalian Cells When a mammal has a mutated sulfur (Zalphal) gene or is deficient in the sulfur (Zalphal) gene, the zinc (Zalphal) gene is introduced into the mammalian cells. Can be introduced. In one example, a gene encoding a Zalphal polypeptide is inserted into a viral vector and introduced in vivo. Such vectors include, but are not limited to, herpes simplex virus (HSV), papilloma virus, Epstein-Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and similar viruses. Such as attenuated or defective DNA viruses.

A defective virus in which the viral gene is completely or almost completely deleted is desirable. The defective virus does not infect after being introduced into the cells. When a defective viral vector is used, it can be administered to a specific localized cell without worrying that the vector may infect other cells.

Examples of detailed vectors include, but are not limited to, a defective herpesvirus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci., 2: 320-330.
(1991)], Stratford-Perricaudet et al., J. Clin. Invest., 90: 626-630 (1992)
And a defective adeno-associated virus vector [Samulski et al., J. Virol., 61: 3096-3101 (1987)].
Samulski et al., J. Virol., 63: 3822-3828 (1989)].

In another embodiment, for example, Anderson et al., US Pat. No. 5,399,346, Ma
nn et al., Cell 33: 153 (1983); Temin et al., U.S. Patent No. 4,650,764; Temi.
n et al., U.S. Pat.No. 4,98,0289, Markowitz et al., J. Virol. 62: 1120 (1988
), Temin et al., U.S. Patent No. 5,124,263, International Publication WO 95/07358, and
The gene can be introduced by inserting it into a retroviral vector, as described in Blood 82: 845 (1993).

[0083] Alternatively, the vector can be introduced in vivo by lipofection using liposomes. Synthetic cationic lipids can be used in the preparation of liposomes for transfecting a marker-encoding gene in vivo [Felgner et al. ,, Proc. Natl. Acad. Sci. USA 84: 7413-7417, ( 19
87); Mackey et al. ,, Proc. Natl. Acad. Sci. USA 85: 8027-8031 (1988)]. Using lipofection methods to introduce foreign genes into specific organs in vivo has several practical advantages.

Performing molecular targeting on specific cells with liposomes shows one benefit. Clearly, directed transfection of specific cells shows one benefit. Obviously, directed transfection of particular types of cells is particularly advantageous in tissues composed of xenogeneic cells such as pancreas, liver, kidney, and brain.

[0085] Lipids can be chemically attached to other molecules for targeting. For example, targeted peptides, such as hormones or neurotransmitters, and proteins, such as antibodies, or non-peptide molecules can be chemically bound to liposomes.

It is possible to remove cells from the body, introduce the vector as a naked DNA plasmid, and then retransplant the transformed cells into the body. Naked DN for gene therapy
A vector can be used for transfection, electroporation,
Transfection into desired host cells by methods known to those skilled in the art, such as microinjection, transduction, cell fusion, DEAE-dextran treatment, calcium phosphate coprecipitation, use of a gene gun, or use of a DNA vector transporter. can do. [Wu et al
., J. Biol. Chem. 267: 963-967, (1992); Wu et al., J. Biol. Chem. 263: 14621-14624.
, (1988)].

[0087] Zalphal polypeptides can also be used to prepare antibodies that specifically bind to Zalphal polypeptides. Furthermore, these antibodies can be used for preparing anti-idiotype antibodies. As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F (ab ') 2 and Fab, and antibodies prepared by genetic engineering.

Methods for preparing polyclonal and monoclonal antibodies are well known (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition).
, (Cold Spring Harbor, NY, 1989); Hurrell, JGR, Ed., Monoclonal Hybridoma
Antibodies: Techniques and Applications (CRC Press, Inc., Boca Raton, FL, 1982)
See). As will be apparent to the ordinarily skilled artisan, polyclonal antibodies can be produced from various warm-blooded animals such as, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. .

The immunogenicity of a Zalphal polypeptide can be enhanced by using an adjuvant, such as Freund's complete or incomplete adjuvant. Various assays known to those of skill in the art can be used to detect antibodies that specifically bind to Zalphal polypeptide. A typical measurement method is Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), (Cold Sprin
g Harbor Laboratory Press, 1988). Representative examples of such assays include cross immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme immunoassay (ELISA), dot blot assay, inhibition or competition assay, and sandwich assay. is there.

An antibody is determined to specifically bind if it 1) exhibits a threshold binding activity and 2) does not cross-react with a conventional polypeptide molecule. First, the antibody has a binding constant (Ka) of 10 ⁶ M ^-1 or more, preferably 10 ⁷ M ^-1 or more, more preferably 10 ⁸ M ^-1 or more, and most preferably 10 ⁹ M ^-1 or more. When binding to a Zalphal polypeptide, peptide, or epitope, an antibody is referred to herein as specifically binding. Antibody binding constants can be readily determined by one of ordinary skill in the art, for example, by Scatchard plots.

Second, it is determined that an antibody specifically binds if it does not cross-react with a conventional polypeptide. For example, standard Western blotting (Ausubel et al., Ibid.)
When an antibody detects Zalphal but does not detect a known related polypeptide, it is said that the antibody does not significantly cross-react with the related polypeptide molecule. Examples of well-known related polypeptides include orthologs, proteins that are members of a protein family (eg, IL-16) from the same species, Zalphal polypeptides, and Zalphal that is surprising to humans. .

Furthermore, antibodies may be screened against well-known related polypeptides to isolate antibodies that specifically bind to a polypeptide of the invention. For example, an antibody that binds non-specifically to Zalphal is adsorbed to a related polypeptide attached to an insoluble substrate; an antibody specific to Zalphal is prepared under appropriate buffer conditions under appropriate buffer conditions. Flow through the substrate.

[0093] Such screening allows the isolation of polyclonal or monoclonal antibodies that do not cross-react with intimately related polypeptides;
es: A Laboratory Manual, Harlow and Lane (eds.) (Cold Spring Harbor Laborat
ory Press, 1988); Current Protocols in Immunology, Cooligan, et al. (eds.)
National Institutes of Health (John Wiley and Sons, Inc., 1995).

Screening and isolation of specific antibodies are well known in the art. Fundamen
tal Immunology, Paul (eds.) (Raven Press, 1988); Cetzoff et al., Adv. in Immu
nol. 43: 1-98 (1988); Monoclonal Antibodies: Principles and Practice, Goding
, JW (eds.), (Academic Press Ltd., 1996); Benjamin et al., Ann. Rev. Immuno
See l.2: 67-101 (1984).

Various assays well known to those skilled in the art can be used to detect antibodies that specifically bind to Zalphal protein or peptide. A typical measurement method is Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold
Spring Harbor Laboratory Press, 1988). Representative examples of such assays include cross immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme immunoassay (ELISA), dot or western blot assays, inhibition or competition assays, and sandwiches. Assays and the like. In addition, the binding of the antibody to wild-type as compared to the binding to the mutated Zalphal protein or polypeptide can be screened.

[0096] Among the diagnostic methods for determining the circulating concentration of soluble polypeptide protein, for targeting of cells expressing the Zalphal protein, for affinity purification, and in vitro and in vivo Antibodies against Zalphal may be used as antagonists to block ligand binding and signal transduction. Anti-idiotypic antibodies can be used to find the receptor for Zalphal.

[0097] Radiation hybrid mapping is a somatic genetic engineering technique that has been developed to construct high-resolution continuous maps of mammalian chromosomes. [Cox et al., Science 250: 245-250 (1990)]. Knowledge of some or the complete gene sequence allows the design of appropriate PCR primers for use with chromosome radiation hybrid mapping panels.

For example, Stanford G3 RH panel and GeneBridge 4 RH Panel (Research Genetic
commercially available radiation hybrid mapping panels that cover the entire human genome, such as S. Inc., Huntsville, AL. These panels allow for rapid, PCR-based chromosomal localization and sorting of genes of interest, base sequence tag sites (STSs), and other non-polymorphic and polymorphic markers. This involves determining the relative physical distance between the newly discovered gene of interest and the marker already located.

[0099] Accurate knowledge of the location of a gene can be obtained by: 1) determining whether a sequence is part of an existing gene sequence, and further converting surrounding gene sequences into species such as YAC-, BAC-, or cDNA clones; 2) providing candidate genes for hereditary diseases that show a causal relationship to the same chromosomal region; and 3) mice that can help determine what function a particular gene has. It is useful in many ways, such as for cross-referencing such model organisms.

The present invention also provides such reagents that can be used for diagnostic applications. For example, the Zalphal gene is located on chromosome Xq27.3. X
Zalphal nucleic acid probes can be used to check for chromosomal abnormalities. For example, to determine whether the Zalfa gene is at chromosome Xq27.3, or whether a mutation has occurred, Zalfa DNA or RNA, or a subsequence thereof.
Can be used.

Detectable chromosomal abnormalities at the Zalphal locus include, but are not limited to, aneuploidy, altered gene transcript numbers, insertions, deletions, altered restriction sites, altered translocations, and the like. No. Such abnormalities include restriction fragment length polymorphism (RFLP) analysis and short tandem repeats using PCR technology (short tandem repeats).
tandem repeat (STR)) analysis and other genetic analysis techniques known in the art, such as linkage analysis techniques, can be detected using the polynucleotides of the present invention [Sambrook et al. ,, ibid .; Ausubel et al. ,, ibid .; Marian, AJ
., Chest, 108: 255-265, (1995)]. Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Pituitary cDNA Library RNA extracted from pituitary cells was purchased from Clontech, Palo Alto, Calif. And reverse-transcribed by the following method. The first strand cDNA reaction solution was 1.0 mg / ml human pituitary twice poly d
(T) -selected pol y (A) ⁺ mRNA (Clontech, Palo Alto, CA) 10 μl, 20 pmole / μ
l containing the Xho I restriction enzyme cleavage site, the first strand primer ZC6191 of SEQ ID NO: 4 (GTC TGG
GTT CGC TAC TCG AGG CGG CCG CTA TTT TTT TTT TTT TTT TTT). The mixture was heated at 70 ° C. for 2 minutes and then cooled on ice.

First-strand buffer (5 × SUPERSCRIPT ^™ buffer; Life Technologies, Gaithersburg)
, MD) 8 μl, 100 mM dithiothreitol 4 μl, 10 mM dTTP, dATP, dG
First-strand cDNA synthesis was initiated by adding a solution of deoxynucleotide triphosphate (dNTP) containing TP, 5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, NJ) to the RNA-primer mixture. The reaction mixture was incubated for 2 min at 37 ℃, 200U / μl RNase H - reverse transcriptase (SUPERSCRIPT II ○ _R; Life Tec
hnologies) was added. To label the reaction, the synthesis efficiency of first-strand cDNA in a parallel reaction was measured by adding 10 μCi of ³² P-αdCTP to 5 μl of the reaction mixture taken from one of the reaction mixtures.

After incubating the reaction at 37 ° C. for 5 minutes and 45 ° C. for 45 minutes,
Incubated for minutes. Unincorporated ³² P-αdCTP in labeled reactions
Was removed by gel chromatography using a gel filtration column with a pore size of 400 (Clontech Laboratories). Unincorporated nucleotides and primers in the unlabeled first-strand reaction are purified using a 400-pore gel filtration column (Clont
Gel chromatography (ech Laboratories). The length of the labeled first strand cDNA was determined by agarose gel electrophoresis.

The second strand cDNA reaction solution was 100 μl of unlabeled first strand cDNA, 5 × polymerase I buffer (12
5mM Tris: HCl, pH7.5, 500mM KCl, 25mM MgCl 2, 50mM (NH 4) 2 SO 4) 30μl, 100m
2.0 μl of M dithiothreitol, 3.0 μl of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 μl of 5 mM β-NAD, 10 U / μl E. coli DNA ligase (
New England Biolabs; Beverly, MA) 2.0 μl, 10 U / μl E. coli DNA polymerase I 5 μl, 10 U / μl RNase H (Life Technologies) 1.0 μl.

To monitor the efficiency of second strand cDNA synthesis, one of the second strand cDNA synthesis reactions was
10 μl of the solution was collected and labeled by adding 10 μCi of ³² P-αdCTP. After incubating the reaction at 16 ° C for 2 hours, 1 μl of 10 mM dNTP solution and T4
DNA polymerase (10 U / μl, Boehringer Mannheim, Indianapolis, IN) 5.0 μl
Was added and further incubated at 16 ° C. for 10 minutes. Prior to analysis by agarose gel electrophoresis, unincorporated ³² P-αdCTP in the labeled reaction was removed by gel chromatography using a 400 pore size gel filtration column (Clontech Laboratories).

The reaction was stopped by adding 10.0 μl of 0.5 M EDTA and further extracting with phenol / chloroform and chloroform, followed by 3.0 M sodium acetate and Pellet Paint carrier (Novagen, Madison, Wis.).
) Ethanol precipitation was performed in the presence of 2 μl. About 2 μg of cDNA was recovered from 10 μg of the first template mRNA.

To enable cloning into an expression vector, an Eco RI adapter was
It was ligated to the 5 'end of NA. cDNA solution ^(- 2.0 μg) 12.5μl, and 69 Pmole/myu
l 3 μl of Eco RI adapter (Pharmacia LKR Biotechnology Inc.) with 10x ligase buffer (660 mM Tris-HCl pH7.5, 100 mM MgCl ₂ ), 2.5 μl of 10 mM ATP, 0.1 MD
3.5 μl TT and mixed with 15 U / μl T4 DNA ligase (Promega Corp., Madison, Wis.). The reaction solution is 1 hour at 5 ° C, 2 hours at 7.5 ° C, 2 hours at 10 ° C,
Incubated at .5 ° C for 2 hours and at 10 ° C for 16 hours. 65 μl water, and 10x
The reaction was stopped by adding 10 μl of H buffer (Boehringer Mannheim) and further incubating at 70 ° C. for 20 minutes.

To facilitate directional cloning of the cDNA into an expression vector, the cDNA was cloned into Xho
C. As a result, the cDNA had a 5 ′ EcoRI sticky end and a 3 ′ XhoI sticky end. The Xho I restriction site at the 3 'end of the cDNA was previously introduced. Restriction enzyme digestion was performed using 40 U / μl Xho I (Boehringer Mannhei
m) Performed in the reaction mixture by adding 1.0 μl. Cleavage was performed at 37 ° C. for 45 minutes. The reaction was stopped by incubating at 70 ° C. for 20 minutes and further performing gel chromatography using a gel filtration column with a pore size of 400 (Clontech Laboratories).

The cDNA was precipitated with ethanol, washed with 70% ethanol, air-dried, and
.5 μl, 2 μl of 10x kinase buffer (660 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ )
0.5 μl of 0.1 M DTT, 2 μl of 10 mM ATP, T4 polynucleotide kinase (10 U / μl, L
resuspended in 2 μl ife Technologies). After incubation at 37 ° C. for 30 minutes, the cDNA was ethanol precipitated in the presence of 2.5 M ammonia acetate and electrophoresed on a 0.8% low melting point agarose gel. Mixed adapters and cDs less than 0.6 kb in length
NA was cut from the gel.

The electrodes were reversed and electrophoresed until the cDNA was concentrated near the origin. Concentrated
The range of the gel containing the cDNA was cut out and placed in a microtube, and the approximate volume of the gel slice was measured. About 3 times the volume of gel slice (300 μl) water, and
10x β-agarose I buffer (New England Biolabs) was added to the tubes and the agarose was dissolved by heating at 65 ° C for 15 minutes. After equilibrating the sample to 45 ° C., 3 μl of 1 U / μl β-agarose I (New England Biolabs) was added, and the mixture was incubated at 45 ° C. for 60 minutes to further digest the agarose.

After the incubation, 40 μl of 3M sodium acetate was added to the sample and the mixture was incubated on ice for 15 minutes. The sample was centrifuged at 14,000 xg for 15 minutes at room temperature to remove undigested agarose. The cDNA was ethanol precipitated, washed with 70% ethanol, air-dried and resuspended in 20 μl of water. After recovery from the low melting point agarose gel, the cDNA was cloned into the EcoRI and XhoI sites of the pBLUESCRIPT SK + vector (Gibco / BRL), and introduced into DH10B cells by electroporation.

[0113] hi-density colony filter array
s) By repeated probe hybridization to (Genome Systems), bacterial colonies containing ESTs of known genes were identified and excluded from sequence analysis. CDNAs of known genes were pooled into 50-100 inserts and
Labeled with ³² P using PRIME labeling kit (Amersham). Colonies that did not hybridize to the probe mix were selected for sequence analysis. Sequence analysis was performed using an ABI 377 sequencer using either T3 or a reverse primer. As a result of analyzing the data, a new EST LPI
F1021273 (SEQ ID NO: 3) could be identified. Example 2. The cDNA clone corresponding to the EST of SEQ ID NO: 3 was determined. As a result, the DNA sequence and amino acid sequence of sulfur DNA of SEQ ID NO: 1 and SEQ ID NO: 2 were obtained. Example 3 Northern Blot Analysis Human multiple tissue blots 1, 2, 3 and human RNA dot blots (Clontech) were probed to determine the tissue distribution of Sulfur. Zulfar c
The 1.2 kb PCR product representing DNA was ligated with pBLUESCRIPTSK + ™ and TBL, respectively.
It was generated using primers (SEQ ID NO: 26) and (SEQ ID NO: 27) made to match the 7 promoter and M13 reverse primer sequences. The resulting fragment was
Electrophoresed on a 7% agarose gel.

DNA was extracted from gel slabs using QIA quick gel extraction kit (Qiagen)
did. 100 ng of this DNA was transferred to a PEI using the REIPRIMER labeling system (Amersham). ³² The radioactivity that was not incorporated was labeled with a NucTrap probe purification column (
Stratagene). Multiple tissue northern blot and human RNA masterbro
Boil the mixture for 5 minutes, then cool on ice for 1 minute and add to 10 ml of ExpressHyb solution
10 ml of EXPRESSHYBR solution containing 1 mg of salmon sperm DNA (clontech)
Were prehybridized for 3 hours, mixed and added to the blot.

The hybridization was performed at 65 ° C. overnight. Initial wash conditions were as follows: 2 × SSC, 0.05% SDS RT for 40 minutes with several solution changes, then 0.1 × SSC, 0.1% at 50 ° C. for 40 minutes with one solution change. SDS. Then blot 2.
Exposed to the film at -80 ° C for 5 hours. RNA dot blots showed high salfar expression in the pituitary and aorta. Multiple tissue northern blots showed abundant expression in thyroid, spinal cord, brain, kidney and pancreas, and even lower expression in heart, spleen, prostate, testicle, stomach, trachea, and adrenal gland.

Example 3 The chromosome assignments and arrangements of sulfur were analyzed using a commercially available “Gene Bridge 4 radiation hybrid panel” (Rese
ach Genetics, Inc, Huntville, AL). The Gene Bridge 4 radiation hybrid panel contains PCRable DNA and two control DNAs (HFL donor and AFL) from each of the 93 radiation hybrid clones.
23 recipients).

A publicly available www server (http://www-genome.wi.mit.edu/cgi-bin/c
ontig / rhmapper.pl) allows the mapping of the Genome Research radiation hybrid map ("WICGR" radiation hybrid map) of the human genome, constructed with the GeneBridge4 radiation hybrid panel, relative to the Whitehead Institute / MIT center.

For mapping of sulfur in “Gene Bridge 4RH Panel”, PC
R Possible 96-well microtiter plate (Stratagene, La Jolla, CA)
A 20 μl reaction was set up in a RoboCycler Gradient 96 thermal cycler (Stratagene). Each of the 95 PCR reactions contained 2 μl of 10 × Kl
en Taq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1
6 μl dNTP mix (2.5 mM each, PERKIN-ELMER, Foster City CA),
1 μl of sense primer, (SEQ ID NO: 5) 15,869, 5′GCA GCA GTC CCA CAG ATG
3 ′, 1 μ antisense primer, (SEQ ID NO: 6) ZC15,868, 5′TGG GCT GAG
TGC TTG TTT 3 ′, 2 μl of “RediLoad” (Research Genetics, Inc, Huntville,
AL), 0.4 μl of 50 × Advantage Klen Taq polymerase mix (Clontech Lab
oratories, Inc), 25 ng of DNA from individual hybrid clones or controls
And x μl of ddH ₂ O to make the total volume 20 μl.

The reaction was overlaid with an equal volume of mineral oil and sealed. The PCR cycler conditions were as follows: one cycle of denaturation at 95 ° C. for 5 minutes, denaturation at 95 ° C. for 1 minute,
35 cycles of annealing at 64 ° C. for 1 minute and expansion at 72 ° C. for 1.5 minutes,
Thereafter, expansion at 72 ° C. for 7 minutes is finally performed for one cycle. Reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).

The results showed that Zalfar maps 4.71CR_3000 from the framework marker WI-5285 on the WICGR radiation hybrid map. By using the surrounding markers, Zalfar can be converted to Xq on the integrated LDB map of the X chromosome.
Located within the 27.3 region (locus database, Southampton University, www server: http: // cedar. Genetics, soton. Ac. Uk / public_html /)
.

Example 4 Untagged for Zarufaru, with the amino terminus the Glu-Glu tag, and the three mammalian vectors expressing mosquitoes Rubokishi terminal Glu-Glu tagged mammalian expression vector Zarufaru, it is constructed using enzyme recombinant vector I have. Three different variants of the sulfur polypeptide were generated.
(I) those without a tag, (ii) those with an amino-terminal Glu-Glu tag, and (
iii) Carboxy terminal It has Glu-Glu tag. The amino acid sequence of the Glu-Glu tag is Glu-Glu-Tyr-Met-Pro-Met-Glu (SEQ ID NO: 25).

The vector can be made as follows: Generation of Recombinant Linker Construction of an expression vector without any Glu-Glu tags attached to the sulfur can be performed as follows. It is possible to construct both 5 'and 3' linkers that do not encode a Glu-Glu tag and are ligated using homologous recombination with the digested plasmid and the Zalphal gene to produce a plasmid containing the Zalphal gene. it can.

This is done by simultaneously transfecting the two linker DNAs, the sulfur gene and the digested plasmid into yeast. The number of plasmids can be amplified in yeast, which can be isolated from yeast, transfected into E. coli, amplified, separated, and then transfected into mammalian cells to produce sulphalf.

A tagged version of the amino acid carboxy terminus is generated in the same manner, except that an appropriately tagged recombinant linker is used. The yeast plasmid PCZR199 was transformed into the vector pRS316 (Sikorski and Hieter, Genetics 122: 19-2).
7 (1989)], in which two PVu II sites are formed, and EcoRI, Xbz are formed between these two PVU II sites.

A) Generation of untagged amino terminus A DNA linker that homologously recombines with the 3 'terminus of the digested plasmid and the 5' terminus of the sulfur gene and does not add a Glu-Glu tag to the sulfur is made as follows. be able to. Sense oligonucleotide ZC16,469 5'TCG CCC AGC CAG GAA ATC CAT GCC GAG
TTC CAA CGC GGC CGT AGA 3 '(SEQ ID NO: 8) and antisense nucleotide ZC
16,465 5'CAG CAG CCG CAG CTG TTC CAT GAG CTG GCT GTT CTC GAT TCT ACG GCC
A solution containing 4 pmol of each of GCG TTG GAA CTC GG3 ′ (SEQ ID NO: 9) is amplified together by PCR.

The amplification products of these two oligonucleotides were combined with 400 pmoles of sense primer ZC16,470 5′CTG CTG TGT GGC GCC GTC TTC GTT TCG CCC AGC CAG GAA ATC CAT
3 ′ (SEQ ID NO: 7) and 400 pmoles of antisense primer ZC16,028 5′TAC C
TG GCG CAG CAG GCT GGC CCT CTC GCA CAC CAG CAG CCG CAG CTG TTC CAT 3 '(
In the same PCR reaction using SEQ ID NO: 10), it was expanded in the same reaction tube.

The PCR mixture for the reaction was composed of 40 μl of 10 × PCR buffer and 8 μl of EXTAG (both Takara
8) 2.5 mM triphosphate nucleotide mix (Takara) and water
It contained 300 μl. The PCR reaction was incubated for 1.5 minutes at 94 ° C. and then run for 10 cycles. Here, each individual cycle is performed at 94 ° C. for 30 seconds, 50 seconds.
1 minute at 72 ° C and 1 minute at 72 ° C. The reaction was terminated by incubation at 72 ° C for 10 minutes.

This resulted in the following recombinant linker:

Embedded image

[0129] B) were homologously recombinant terminus and 3 'terminus and Zarufaru gene 5' untagged carboxy terminus digested plasmid, without the addition of Glu-Glu tag to the carboxy-terminus of Zarufaru DNA
The linker can be made as follows. 4 picomoles of sense oligonucleotide ZC16,484 5'GAC GAA GAC GAC GAC GAC
GAA GAA GAG GAG GAT GAT TAT TAA TCT AGA GGA TCT GGG GTG GCA TCC CTG T3 '
(SEQ ID NO: 13) and 4 picomoles of the antisense oligonucleotide ZC14,455 5
'CAG GAG AGG CAC TGG GGA GGG GTC ACA GGG ATG CCA CCC CAG ATCC3' (SEQ ID NO: 14) was added together into the PCR reaction mixture.

Also added to the mixture was 400 pmoles of sense primer ZC16,027 5′G
AT GAG ATG AAA CAG TGC TTT GGC TGG GAT GAC GAC GAA GAC GAC GAC GAC GAA 3
'(SEQ ID NO: 12) and 400 pmoles of antisense primer ZC14,394 5'GGC
ACT GGA GTG GCA ACT TCC AGG GCC AGG AGA GGC ACT GGG GAG G3 ′ (SEQ ID NO: 1
5).

The PCR mixture for the reaction further contained 40 μl of 10 × PCR buffer, 8 μl of EXTAG (both from Takara), 8 μl of 2.5 mM nucleotide triphosphate mix (Takara) and 300 μl of water. Incubate the PCR reaction at 94 ° C for 1.5 minutes,
Next, each individual cycle was performed at 94 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute.
The reaction was run for 10 cycles per minute. The reaction was terminated by incubation at 72 ° C. for 10 minutes.

Thus, the following oligonucleotide linkers were produced that homologously recombine at the 5 ′ end of the digested plasmid and at the 3 ′ end of the Zalphal gene:

Embedded image

C) The Glu-Glu tagged amino-terminal digested plasmid will homologously recombine with the 3′-end and the 5′-end of the sulfur gene, and when the sulfur gene is expressed, the amino-terminal of the sulfur A linker that attaches a Glu-Glu tag thereon was produced as follows. 4 picomoles of sense oligonucleotide ZC14,397 5'CCG AGT TCC AAC G
CG GCC GTA GAG AGG AGT ATA TGC CTA TGG AG3 '(SEQ ID NO: 18) and 4 picomoles of antisense oligonucleotide ZC15,485 5'CAG CAG CCG CAG CTG TTC CAT
GAG CTG GCT GTT CTC GAT CTC CAT AGG CAT ATA CTC CTC TCT ACG 3 ′ (SEQ ID NO: 19) was added together during the PCR reaction mix.

Also added to the mixture was 400 pmoles of sense primer ZC14,396 5'GTT
TCG CCC AGC CAG GAA ATC CAT GCC GAG TTC CAA CGC CGC CGT3 '(SEQ ID NO: 17
) And 400 pmoles of antisense primer ZC16,028 5'TAC CTG GCG CAG CAG G
CT GGC CCT CTC GCA CAC CAG CAG CCG CAG CTG TTC CAT 3 '(SEQ ID NO: 10). The PCR mixture for the reaction was further divided into 40 μl of 10 × PCR buffer and 8 μl of EXTAG (
8 μl of 2.5 mM triphosphate nucleotide mix (Takara)
) And 300 μl of water.

The PCR reaction was incubated at 94 ° C. for 1.5 minutes and then the reaction was run for 10 cycles, each individual cycle consisting of 30 seconds at 94 ° C., 1 minute at 50 ° C., and 1 minute at 72 ° C. . The reaction was terminated by a 10 minute incubation at 72 ° C.

Embedded image

D) Glu-Glu tagged carboxy-terminal Digestion will result in homologous recombination with the 5′-end of the plasmid and the 3′-end of the sulfur gene, and the carboxy-terminal of sulfur at the time the sulfur gene is expressed. A linker that attaches a Glu-Glu tag thereon was produced as follows. 4 picomoles of sense oligonucleotide, ZC16,486 5'GAC GAA GAC
GAC GAC GAC GAA GAG GAG GAT GAT TAT GAA GAA TAC ATG CCC ATG GAA TAA3 '(
SEQ ID NO: 21) and 4 picomoles of the antisense oligonucleotide ZC14,393 5'A
T GCCACCCCAG ATCCTCTAGA TTA TTC CAT GGG CAT GTA TTC TTC 3 '(SEQ ID NO: 22
) Were added together into the PCR reaction mixture.

Similarly added to the mixture was 400 pmoles of sense primer ZC16,027 5
'GAT GAG ATG AAA CAG TGC TTT GGC TGG GAT GAC GAC GAA GAC GAC GAC GAC GAA
3 '(SEQ ID NO: 12) and 400 pmoles of antisense primer ZC14,395 5'GGCAC
TGGGGAGGGG TCACAGGGAT GCCACCCCAG ATCCTCTAGA 3 '(SEQ ID NO: 23).

The PCR mixture for the reaction was further divided into 40 μl of 10 × PCR buffer and 8 μl of EXTAG (both
It contained 8 μl of 2.5 mM triphosphate nucleotide mix (from Takara) (Takara) and 300 μl of water. Incubate the PCR reaction at 94 ° C for 1.5 minutes,
The reaction was then run in 10 cycles, each consisting of 30 seconds at 94 ° C, 1 minute at 50 ° C and 1 minute at 72 ° C. The reaction was terminated by a 10 minute incubation at 72 ° C.

Thus, the following oligonucleotide linker was produced.

Embedded image

Assembly of Plasmids in Yeast Following three procedures for pransmid assembly through recombination of homologous DNA sequences in yeast, three versions of sulfur are generated: A) Total untagged version of sulfur In 10 ml, the linear pCZR19
A mixture of 90.1 μg, 1 mg of full-length sulfur cDNA and about 1 μg of each of untagged amino-terminal and untagged carboxy-terminal recombination linkers is mixed. This mixture
Competent yeast cells Saccharomyces cerevisi
used to transform ae). Plasmid DN from successful transformation
A was transferred to E. coli and the desired construct was identified by DNA sequence analysis. At this time, the plasmid can be transfected into mammalian cells such as Chinese hamster ovary (CHO) cells.

B) Amino-Terminal of Sulfur Glu-Glu-tagged version Same as described above except that Glu-Glu-tagged amino-terminal and untagged carboxy-terminal recombination linkers were used. C) Carboxy-terminal Glu-Glu-tagged version of sulfur Same as described above, except that Glu-Glu-tagged carboxy-terminal and untagged amino-terminal recombination linkers were used.

Example 6 Expression of Sulfur in Pichia methanolica Homologous Recombination / Sulfur Expression of Sulfur in Pichia methanolica was performed using the co-assigned PCTWO. It utilizes the expression system described in 97/17450. An expression plasmid containing all or a portion of the polynucleotide encoding the sulfur is constructed via homologous recombination. The expression vector contains an AUG1 promoter followed by an αFpp leader sequence, followed by an amino-terminal peptide tag, a blunt-ended SmaI restriction site, a carboxy-terminal peptide tag, a translation stop codon, followed by an AUG1 terminator, an ADE2 selectable marker and finally Constructed from pCZR204 containing the AUG13 'untranslated region.

Included in this vector is Saccharomyces cerevisiae (S. cerev
URA3 and CEN-ARS sequences required for selection and replication in isiae) and Amp ^R and colE1 ori sequences required for selection and replication in E. coli. This baek
The sulfur sequence inserted into the amino acid sequence is at residue 27 (Al
Start with a).

In this case, three expression plasmids were produced for expression of Sulfur in P. methanolica. These plasmids are as follows: the so-called NEE-sulfur construct is a Sulfur polypeptide tagged with a Glu-Glu tag at the N-terminus (SEQ ID NO: 2).
5); what is called the CEE-sulfur construct has a Glu-Glu
Tagging produces a tagged sulfur polypeptide (SEQ ID NO: 25); and another is one in which the sulfur was not tagged. In each case,
Two linkers were created to produce each plasmid.

One linker, when transformed in the P. methanolica with the digested plasmid and Zalphal gene, is the 3 'end of the digested plasmid and the 5' end of the sulfur gene. Homologous recombination was performed. The second linker was used for digested plasmids and the sulfur gene and Pichia methanolica (P.
metnamolica) when homologously recombined with the 5 'end of the digested plasmid and the 3' end of the sulfur gene.

Construction of NEE-Sulfur Plasmid N-Terminal-NEE Producing Sulfur with N-Terminal Tagged with Glu-Glu Tag
-Sulfur plasmid construct comprises 100 ng of SmaI digested pCZR204 acceptor vector, 1 μl of EcoRI-XhoI Sulfur cDNA donor fragment.
g, 1 μg of N-terminal NEE-sulfur linker (SEQ ID NO: 35) and 1 μg of C-terminal untagged linker (SEQ ID NO: 36) were added to P. methanolica (P. methanolica).
) Made by recombination in transformation.

4 picomoles of ZC13,497 (SEQ ID NO: 28), 4 picomoles of ZC13,731 (SEQ ID NO: 2)
9) The NEE-sulfur linker was synthesized by a PCR reaction in which 4 pmoles of ZC16,023 (SEQ ID NO: 30) and 4 pmoles of ZC16,028 (SEQ ID NO: 10) were placed in a PCR reaction mixture. The PCR mixture for the reaction is further added to 10x PCR buffer
It contained 40 μl, 8 μl of EXTAG (both from Takara), 8 μl of 2.5 mM nucleotide triphosphate mix (Takara) and 300 μl of water. Run the PCR reaction at 94 ° C for 1
Incubate for 5 minutes, then each individual cycle is performed at 94 ° C for 30 seconds, 50
The reaction was run for 10 cycles consisting of 1 minute at 70 ° C and 1 minute at 72 ° C. The reaction was terminated by a 10 minute incubation at 72 ° C. Thus, the oligonucleotide linker SEQ ID NO: 35 was produced.

The C-terminal untagged sulfur linker was 4 picomoles of ZC13,734 (SEQ ID NO: 31), 4 picomoles of ZC15,633 (SEQ ID NO: 32), 400 picomoles of oligonucleotide ZC16,025 (SEQ ID NO: 33) And 400 picomoles of oligonucleotide ZC16,0
27 (SEQ ID NO: 34) was made by placing it in the same PCR mixture as the above mixture, and the PCR reaction was run under the same conditions as described above. Thus,
The linker of SEQ ID NO: 36 was produced.

Construction of CEE-Zalphal Plasmid The N-terminal untagged sulfur linker is composed of 4 picomoles of oligonucleotide (SEQ ID NO: 37), 4 picomoles of oligonucleotide (SEQ ID NO: 38), 400
The picomolar oligonucleotide (SEQ ID NO: 39) and 400 picomoles of the oligonucleotide (SEQ ID NO: 10) are mixed together in the same PCR mixture as the PCR mixture described above, and the PCR reaction is run as run above. Made by that. Thus, the linker of SEQ ID NO: 40 was produced.

The C-terminal sulfal-CEE linker comprises 4 picomoles of oligonucleotide (SEQ ID NO: 41), 4 picomoles of oligonucleotide (SEQ ID NO: 42), 400 picomoles of oligonucleotide (SEQ ID NO: 43) and 400 picomoles of oligonucleotide (SEQ ID NO: 43). No. 12) and 400 pmoles of the oligonucleotide (SEQ ID NO: 13) were made by mixing together in the same PCR mix as the above PCR mix and running the PCR reaction as run above. . Thus, SEQ ID NO: 44
Of linkers were produced.

C-terminal-CEE-Zalpha expressing Gcyto-tagged Zcyto10 at the C-terminal
l Plasmid construct contains 100 ng of SmaI digested pCZR204 acceptor vector.
, EcoRI-XhoI Zalphalc DNA donor fragment 1 μg, N-terminal untagged Zalphal linker (SEQ ID NO: 40) and C-terminal-CEE-sulfur-tagged linker (SEQ ID NO: 44) 1 μg were added to P. methanolica (P. methanol).
ica) Made by recombination in transformation.

Construction of Untagged Sulfur Expression Construct Untagged Sulfur expression construct was composed of 100 ng of Smal digested pCZR204 acceptor vector, 1 μg of EcoRI-xhoI Sulfur cDAN donor fragment, two recombinant linkers, ie N-terminal untagged Sulfur oligo. Nucleotide (SEQ ID NO: 40) and a C-terminal untagged sulfur oligonucleotide (SEQ ID NO: 36)
) Were recombined in a P. methanolica transformation.

Each PCR generated N-terminal double stranded linker segment spans 70 base pairs of the aFpp coding sequence at one end and 70 base pairs of the amino terminal coding sequence from the mature sulfur sequence at the other end. To join it. Each C-terminal linker, on the other hand, contains about 70 base pairs of the carboxy-terminal coding sequence from Sulfur at one end, with 70 base pairs of the AUG1 terminator sequence.

Ura ⁺ colonies were selected and DNA from the resulting yeast colonies was extracted and transformed into E. coli. PCR screening and subsequent
Individual clones harboring the correct expression construct were identified by restriction digestion to confirm the presence of the sulfur insert and DNA sequencing to confirm that the desired DNA sequences were joined together. For one of the correct clones, larger scale plasmid DNA is isolated and the DNA is digested with SfiI to release the Pichia-Sulfur expression cassette from the vector backbone.

Next, the DNA cut with SfiI is transformed into a Pichia methanolica expression host called PMAD16, and immobilized on an ADED plate for selection. Various clones are picked and screened for high levels of Zalphal expression via Western blot.

More particularly, small scale protein production (eg, plate or shake flask production)
Therefore, P. methanolica transformants that support an expression cassette containing a methanol-regulated promoter (eg, AUG1 promoter) can be transformed in the presence of methanol and in an amount that interferes with other carbon sources (eg, glucose). Grow in the absence of). For small-scale experiments including preliminary screening of expression levels, for example, 20 g / L Bacto-agar (Difco), 6.7 g / L amino acid-free yeast nitrogen base (Difco), 10 g / L methanol, 0.4 mg / L biotin and 0.56 g / L -Ade
Transformants can be grown at 30 ° C. on solid media containing -Thr-Trp powder.

Since methanol is a volatile carbon source, it is easily lost at the time of prolonged incubation. By placing a 50% aqueous methanol solution in an inverted flat lid, a continuous methanol supply can be provided, whereby the methanol is transferred to the growing cells by evaporative transfer. In general, 1
Less than 1 ml of methanol is used per 00 mm plate. Slightly larger scale experiments can be performed using cultures grown in shake flasks.

In a standard procedure, cells are cultured at 30 ° C. on a minimal methanol plate as disclosed above for 2 days, and then harvested at a cell density of about 1 × 10 ⁶ cells / ml. Colonies are used to inoculate the minimum methanol medium (6.7 g / L of yeast nitrogen base without amino acids, 10 g / L of methanol, 0.4 mg / L of biotin).
Cells are grown at 30 ° C. Cells growing on methanol have high oxygen requirements and require active shaking during culture. Methanol is replenished daily (
Standard is 1/100 volume of 50% methanol per day).

For production scale cultures, fresh cultures of high producing clones are prepared in shake flasks. The resulting culture is then used to inoculate the medium in a fermentor. Typically, a 500 ml culture in YEPD grown at 30 ° C. for 1-2 days with vigorous agitation is used to inoculate a 5 liter fermenter.

Cells are grown in a suitable medium containing salts, glucose, biotin and trace elements at 28 ° C., pH 5.0 and less than 30% dissolved O ₂ . After the initial glucose input has been consumed (as indicated by a decrease in oxygen consumption), a glucose / methanol feed is pumped into the vessel to trigger production of the protein of interest.
Because large-scale fermentations are performed under carbon-limited conditions, the presence of glucose in the feed does not suppress the methanol-inducible promoter.

[Sequence list]

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] December 15, 1999 (Dec. 15, 1999)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country 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, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, 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, ZW (72) Inventor Conklin, Darrellsee. United States, Washington 98102, Seattle, Minor Avenue East 2332, Apartment 2 (72) Inventor Parrish, Julia United States, Washington 98110, Bainbridge Island, Ginet Place Northwest 1641 F-term (reference) 4B024 AA01 AA11 BA80 CA04 CA09 CA10 CA12 CA20 DA02 DA12 EA04 FA07 GA11 HA13 HA14 4H045 AA10 AA11 AA20 BA10 BA41 CA40 DA75 EA20 EA50 FA71 FA74

Claims (10)

[Claims] An amino acid sequence defined by SEQ ID NO: 45 or SEQ ID NO: 4
An isolated polynucleotide that encodes a mammalian polypeptide consisting of an amino acid sequence that is at least 90% identical to 5. 2. The polynucleotide according to claim 1, wherein said polynucleotide is RNA. 3. The isolated polynucleotide according to claim 1, wherein said polynucleotide is DNA. 4. A vector comprising: a transcription promoter; a DNA segment encoding a mammalian polypeptide consisting of the amino acid sequence defined by SEQ ID NO: 45; a transcription terminator; . 5. An isolated polypeptide comprising a mammalian polypeptide consisting of the amino acid sequence defined by SEQ ID NO: 45 or a polypeptide at least 90% identical to SEQ ID NO: 45 . 6. The polypeptide according to claim 6, further comprising a second polypeptide or protein. 7. An antibody that specifically binds to a mammalian polypeptide consisting of SEQ ID NO: 45 or a polypeptide that is at least 90% identical to SEQ ID NO: 45. 8. An anti-idiotype antibody which specifically binds to a mammalian polypeptide consisting of SEQ ID NO: 45 or a polypeptide at least 90% identical to SEQ ID NO: 45. 9. having at least 30 amino acid residues of SEQ ID NO: 45;
A polypeptide comprising the amino acid sequence of the epitope-bearing portion of SEQ ID NO: 45. 10. The polypeptide as set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 5
2, selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, or wherein the polypeptide is SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 10. The epitope-bearing polypeptide of claim 9, which is at least 90% identical to a polypeptide selected from the group consisting of: SEQ ID NO: 55 and SEQ ID NO: 56.