JP3169239B2 - Proteins, DNAs and their uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to human luteinizing hormone.
Human chorionic gonadotropin receptor protein (human LH / h
D containing cDNA encoding CG receptor protein)
The present invention relates to NA, human LH / hCG receptor protein, and a method for producing the protein.

[0002]

BACKGROUND OF THE INVENTION Luteinizing hormone-human chorionic gonadotropin receptor protein (LH / hCG receptor protein)
It is present in testicular Leydig cells, ovarian capsular cells, granulosa cells, luteal cells and stromal cells, and plays a central role in reproductive physiology. In males and non-pregnant females, the LH / hCG receptor is affected only by luteinizing hormone, which is produced and secreted in the anterior pituitary gland. However, in pregnant females, the ovarian LH / hCG receptor is not converted to human chorionic gonadotropin (hC
G) is also affected. LH and hCG
Is a member of the glycoprotein hormones, of which thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH) are also members. These four hormones have molecular weights of 28- to 38-kD and are heterodimeric, with specific β subunits associated with receptor binding specificity bound to a common α subunit. It is a glycoprotein. The sugar moiety of these hormones appears to play an important role in signal transduction. Β of both LH and hCG
Subunits are closely related in their structure,
These two hormones bind to the same receptor and provoke the same biological response. In view of the similarity of these glycoprotein hormones and the nature that these hormones act on their receptors to increase G-protein mediated adenylyl cyclase activity, the structure of these receptors has a hormone-inducing activity. It can be seen that they have a common mechanism of action. This increase in adenosine 3 ', 5'-monophosphate (cyclic AMP) necessarily leads to steroid synthesis and secretion. Receptors associated with G-proteins have the common structural feature of having seven transmembrane domains known to be involved in signal transduction and binding to small ligands Has been identified. Meanwhile, TS
Comparison of H and FSH receptors with LH / hCG receptors revealed that the receptors for these pituitary glycoprotein hormones were 7
It has also been characterized among G-protein coupled receptors by the presence of a large glycosylation domain, which is presumed to be located extracellularly, grafted to a structure containing two transmembrane moieties. The elucidation of the structure of the LH / hCG receptor has not been performed very well because of the very small amount of this receptor and its susceptibility to proteolysis. LH / hCG receptors, the complementary D of these receptors
NA (cDNA) has been isolated and the amino acid sequence has been deduced from this DNA [rat: Science].
245 , 494 (1989), Pig: Science 245 , 5
25 (1989)].

[0003]

As described above, the structure of rat and pig LH / hCG receptors has been elucidated, but the structure of human LH / hCG receptor has not been elucidated. Considering the use as a human therapeutic or test agent, it was necessary to clarify the structure and properties of the human LH / hCG receptor.

[0004]

Means for Solving the Problems If the present inventors can collect LH / hCG receptor from humans and produce it by genetic recombination technology, it will have a great effect on future research and treatment. As a result of repeated studies, it was found that rat LH / hCG receptor complement DN
A using a human ovary cDNA library as a probe to encode a human LH / hCG receptor
We cloned the DNA and succeeded in elucidating its complete nucleotide sequence. Furthermore, from this cDNA, human L
The present inventors have succeeded in elucidating the amino acid sequence of the H / hCG receptor and opening the way to mass-produce it by genetic recombination technology. This receptor is very similar to that of rat and pig, but with enough discrepancy to recognize each receptor as distinct. The present invention provides (1) a human luteinizing hormone-human chorionic gonadotropin receptor protein, and (2) a human luteinizing hormone-human chorionic gonadotropin receptor protein-encoding cD.
DNA containing NA, (3) human luteinizing hormone-
C encoding human chorionic gonadotropin receptor protein
A human luteinizing hormone-human chorionic gonadotropin including (4) culturing the transformant containing the DNA, and (4) culturing the transformant of (3) above, producing and accumulating protein in the culture, and collecting The present invention relates to a method for producing a receptor protein.

The present inventors have cloned two cDNAs of human luteinizing hormone-human chorionic gonadotropin receptor protein and estimated the primary structure of the complete protein (FIG. 1). The first methionine in this sequence is considered the start codon. Following this is an amino acid sequence characteristic of a signal peptide with a cleavage site. Hydropathy analysis
And comparison with rat and porcine LH / hCG receptors (FIG. 2) suggested a possible model for protein organization. The putative extracellular domain of 335 amino acids precedes a 267 amino acid region representing the seven putative transmembrane regions (boxed in FIG. 2).
There are 72 amino acid COOH-terminal intracellular domains. This mature protein consists of 674 amino acids (75632 daltons). In addition, there are 25 signal peptides (1st to 25th amino acids in FIGS. 1 and 2), which are cleaved during receptor synthesis, and 674 amino acids (26 to 26) as a receptor mature protein. 699th). At the primary structure level, this extracellular domain has approximately 85% identity with rat and porcine LH / hCG receptors and 45% identity with TSH and FSH receptors (FIG. 2). , HLH / hC
GR: human LH / hCG receptor, rLH / hCGR: rat LH / hCG receptor, pLH / hCGR: pig LH
/ HCG receptor, hTSHR: human TSH receptor [Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Comm.) 16
6 , 394 (1990)], rFSHR: rat FSH receptor [Molecular Endocrinology (Mol. Endo.) 4 , 525].
(1990)]). Six potential carbohydrate sites are found in the putative extracellular domain (Figure 1, underline). Population of cysteine is present between the extracellular domains and transmembrane domains of putative the NH ₂ -terminal part of the protein. Since these cysteines are conserved in LH, FSH and TSH receptors, the formation of disulfide bonds may be critical for the structural stability of the large extracellular domain of the glycoprotein hormone receptor. unknown.

[0006] The domain believed to contain the transmembrane region is approximately 90% of the rat and porcine LH / hCG receptor.
% Of TSH and FSH receptors
% Identity. Serine and threonine are frequently found in a putative intracellular domain with three potential phosphorylations by protein kinase C (FIG. 1). Since phosphorylation by receptor-specific kinases plays a role in agonist-specific release of adrenergic receptors from G-proteins, at least one of these sites is phosphorylated, thereby causing the LH / hCG receptor It is important to know if any functional change will occur in the system.

In the present invention, in addition to clones having a large open reading frame, clones encoding shorter proteins were obtained. Large clone those 1-699 amino acid sequence of Figure 1, and in which truncated the 227-289 th amino acid sequence (a portion enclosed by a square) is missing. This format suggests that the cleavage mechanism required to complete the mRNA is selective. These results are very similar to the porcine LH / hCG receptor data. The role of this truncated receptor is not well understood and it is not known whether this LH / hCG receptor is physiologically active as a monomer or oligomer. In humans, this TSH
Receptors can be targets for an autoimmune response that leads to excessive or hypothyroidism of the thyroid by autoantibodies in Graves' disease and idiopathic myxedema. Thus, a better understanding of ovarian physiology, as well as a contribution to the diagnosis and management of ovarian disease, requires the isolation and characterization of the human LH / hCG receptor.

FIG. 2 shows the amino acid sequence of the novel human luteinizing hormone-human chorionic gonadotropin receptor protein obtained in the present invention, and the rat and porcine luteinizing hormone-human chorionic gonadotropin receptor protein and similar functions. Are shown in comparison with the amino acid sequences of the FSH and TSH receptors having the same amino acid sequence as that of the human luteinizing hormone-human chorionic gonadotropin receptor protein of the present invention. hC
Amino acid portions different from the G receptor are shown in a single-letter format. CONSENSUS in this figure indicates amino acids common to all of the glycoproteins listed in this figure.
Since ENSUS was provided, a missing part "-" was provided in the formula. Therefore, the numbers representing amino acids are counted excluding this missing part.

Regarding the DNA sequence, the DNA encoding the human LH / hCG receptor of the present invention contains the nucleotide sequence shown in FIG. 1 or is a part thereof.

The cDNA encoding the human LH / hCG receptor of the present invention may be any cDNA as long as it contains a nucleotide sequence encoding the amino acid sequence of the human LH / hCG receptor. It is preferably a DNA containing the base sequence of FIG. 1 or a partial DNA thereof. The base sequence shown in FIG. 1 is an example of the cDNA sequence encoding the human LH / hCG receptor obtained in the present invention.

In the method of the present invention, an expression type vector containing a cDNA having a nucleotide sequence encoding a human LH / hCG receptor includes, for example, (i) isolating messenger RNA (mRNA) from human LH / hCG receptor-producing cells; And (ii) a single-stranded complementary DNA (cDN
A), followed by synthesis of double-stranded DNA, (iii) incorporation of the complementary DNA into a phage or plasmid, (iv)
A host is transformed with the obtained recombinant phage or plasmid, and (v) after culturing the obtained transformant, use a suitable method from the transformant, for example, a DNA probe encoding a part of rat LH / hCG receptor. Or a phage or plasmid containing the target DNA is isolated by hybridization with an anti-LH / hCG receptor antibody or by an immunoassay using an anti-LH / hCG receptor antibody. (Vi) A target cloned DNA is cut out from the recombinant DNA, (Vii) the cloned DNA or a part thereof is ligated downstream of a promoter in an expression vector.

MR encoding human LH / hCG receptor
NA is derived from various human LH / hCG receptor producing cells such as testicular Leydig cells, ovarian capsular cells, granulosa cells,
It can be obtained from germ cells such as luteal cells and stromal cells. RNA from human LH / hCG receptor producing cells
Can be prepared by the guanidine thiocyanate method (JM Chirgwin et al., Bio-chemistry, 18 , 5294 (1979)). Using the mRNA as a template and reverse transcriptase, for example, the method of H. Okayama et al. [Molecular and Cellular Biology, 2, 161 (1982) and 3, 280 (1983)] and the obtained cDNA is integrated into a plasmid, such as E. coli-derived pBR322.
[Gene, 2, 95 (1977)], pBR325 [gene, 4, 12
1 (1978)], pUC12 [Gene, 19 ,, 259 (1982)], pUC13
[Gene, 19, 259 (1982)], pUB110 derived from Bacillus subtilis [Biochemical and Biophysical Research Comm.
unication), 112, 678 (1983)], and any other substances can be used as long as they are replicated and propagated in the host. As a phage vector incorporating a cDNA, for example, λgt11
(Young, R., and Davis, R.,
Proceedings of the National Academy of Science of the USA (P
Roc. Natl. Acad. Sci., USA), 80 , 1194 (1983)]. Others can be used as long as they can be propagated in a host.

[0013] Methods for integration into plasmids include, for example, T. Maniatis et al., Molecular Cloning, Cold Spring Harbour Laboratory.
or La-boratory), page 239 (1982). As a method for incorporating cDNA into a phage vector, for example, the method of Hyunh (TV) et al. [DNA Cloning, A Practical Approach 1 ,
49 (1985)]. The plasmid thus obtained can be used in a suitable host such as Escherichia
richia), Bacillus, etc. Examples of the above Escherichia bacteria include Escherichia coli K12DH1 [Proceding of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 60, 160 (1968)]. , M
103 [Nucleic Acids Research, (Nucleic Acids
Research), 9, 309 (1981)), JA221 (Journal of Molecular Biology)
Biology)], 120, 517 (1978)], HB101 [Journal
Of molecular biology 41, 459 (1969)], C6
00 [Genetics, 39, 440 (1954)] and the like.

Examples of the Bacillus genus include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry 95, 87 (198)
4)]. Methods for transforming a host with a plasmid include, for example, T. Maniatis (T.
iatis) et al., Molecular Cloning (Molecular Cloning)
ning), Cold Spring Harbor Laboratory
(Cold Spring Harbor Laboratory), page 249 (1982), the calcium chloride method or the calcium chloride / rubidium chloride method. When a phage vector is used, it can be introduced into, for example, propagated Escherichia coli using an in vitro packaging method. The human LH / hCG receptor / cDNA library containing the human LH / hCG receptor cDNA can be obtained by the above method or the like, but can also be purchased as a commercial product. cDNA libraries can be obtained from Clontech Laboratories, Inc., USA.

From a human DNA library, human LH / h
Methods for cloning the CG receptor cDNA include:
For example, the phage vector λcharon28A and rat LH / h
Plaque hybridization method using CG receptor cDNA as a probe [T.
niatis) et al., Molecular Cloning (MolecularClo
ning) Cold Spring Harbor La-boratory, (1982)]. Human LH cloned in this way
The / hCG receptor cDNA may be a plasmid, if necessary, such as pBR322, pUC12, pUC13, pUC18, pUC19, pUC118, pUC
The human LH / hCG receptor cDNA can be obtained by subcloning into 119 or the like.

[0016] The nucleotide sequence of the cDNA obtained in this manner can be determined, for example, by using Maxam-Gilbert.
Law (Maxam, AM and Gilbert, w., Proceedings of the National Academy of Sciences of the USA) (Proc. Natl. Acad.
Sci., USA), 74 , 560 (1977)) or the dideoxy method [Messing, J. et al., Nucleic Acids Research.
(Nucleic Acids Research) 9, 309 (1981)], and compared with known amino acid sequences, human LH / hCG
Confirm the presence of the receptor cDNA. As described above,
A cDNA encoding the human LH / hCG receptor protein is obtained. Human LH / hCG obtained in Example 1 described later
FIG. 1 shows the nucleotide sequence of the cDNA determined by the dideoxy method for the cDNA encoding the receptor protein and the amino acid sequence determined from the nucleotide sequence.

Human L cloned as described above
The cDNA encoding the H / hCG receptor protein can be used as it is for the purpose or digested with a restriction enzyme if desired. A region to be expressed is excised from the cloned cDNA and ligated downstream of a promoter in a vehicle (vector) suitable for expression to obtain an expression type vector. The cDNA has ATG at its 5 'end as a translation initiation codon and TAA, TGA or TAG at its 3' end as a translation stop codon.
May be provided. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. To express the DNA, a promoter is connected upstream thereof. As the vector, the above-mentioned plasmid derived from Escherichia coli (eg, pBR32
2, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC1)
94), yeast-derived plasmids (eg, pSH19, pSH
15) or bacteriophage such as λ phage and animal viruses such as retrovirus and vaccinia virus.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. When the host for transformation is Escherichia, the trp promoter, lac promoter, recA promoter,
When the host is a bacterium belonging to the genus Bacillus, the λPL promoter, the lpp promoter, etc.
When the host is yeast, such as the SPO2 promoter or penP promoter, the PHO5 promoter, PGK
Promoters, GAP promoters, ADH promoters and the like are preferred. It is particularly preferred that the host is Escherichia and the promoter is the trp promoter or the λPL promoter. When the host is an animal cell, an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, or the like can be used. The use of enhancers for expression is also effective.

The human LH / hC thus constructed
A transformant is produced using a vector containing a cDNA encoding a mature peptide of a G receptor protein. Examples of the host include Escherichia, Bacillus, yeast, animal cells and the like. Specific examples of the aforementioned Escherichia and Bacillus bacteria include those similar to those described above. As the above yeast, for example, Saccharomyces cerevisiae
iae) AH22, AH22R ~, NA87-11A, DKD
-5D and the like. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells, human FL cells, and the like.

In order to transform the genus Escherichia, for example, Proceeding of the National
Academy of Science (Proc.Natl.Acad.Sci.U
SA), 69 , 2110 (1972) and Gene, 17 , 107 (1982). For transformation of Bacillus sp., For example, Molecular & General Genetics, 168 ,
111 (1979) and the like. To transform yeast, for example, the processing of
The National Academy of Science (Pro
Natl. Acad. Sci. USA), 75 , 1929 (1978). Transformation of animal cells is performed, for example, according to the method described in Virology 52 , 456 (1973). Thus, human LH / hCG
A transformant transformed with the expression vector containing the cDNA encoding the receptor is obtained.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for culturing, and a liquid medium necessary for the growth of the transformant is contained therein. A carbon source, a nitrogen source, an inorganic substance and the like are contained. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added.
The pH of the medium is preferably about 5 to 8. As a medium for culturing Escherichia, for example, glucose, M9 medium containing casamino acids (Miller (Miller), Journal of Experiments in Molecular Genetics (Journal of Experiments in Molecular Genetics), Four
31-433, Cold Spring Harbor Laboratory, New York 19
72] is preferred. If necessary, an agent such as 3β-indolyl acrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 ° C. for about 3 to 24 hours.
It is carried out for a time, and if necessary, ventilation and stirring can be added. When the host is Bacillus, the culture is usually about 30 to 4
The reaction is carried out at 0 ° C. for about 6 to 24 hours, and if necessary, ventilation or stirring may be added. When culturing a transformant whose host is yeast, for example, a bark holder (B
urkholder) minimal medium (Bostian, KL et al., "Proceding of the National Academy of Sciences"
(Proc. Natl. Acad. Sci. USA) 77 , 4505 (1980)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.

When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science 122 , 501 (19)
52)], DMEM medium [Viro-logy, 8 , 396
(1959)], RPMI 1640 medium [Journal of
The American Medical Association (The
Jounal of the American Medical Association) 199 , 51
9 (1967)], 199 medium [Pro-ceeding of the Society for the Biological Medicine (Pro-ceeding of the Society for the Biological Medicine)]
dicine) 73 , 1 (1950)]. pH is about 6
~ 8 is preferred. Culture is usually about 30 ° C to 40 ° C
For about 15 to 60 hours, and if necessary, ventilation or stirring is added.

The human LH / hCG receptor protein can be separated and purified from the above culture by, for example, the following method. When extracting human LH / hCG receptor protein from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in a suitable buffer, and subjected to ultrasonic wave, lysozyme and / or lysozyme. After the cells or cells are destroyed by freeze-thawing or the like, a method of obtaining a crude extract of the mature peptide of BMP by centrifugation or filtration may be appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. Human L in culture
When the H / hCG receptor protein is secreted, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. The human LH / hCG receptor contained in the culture supernatant or extract thus obtained can be obtained by appropriately combining known separation and purification methods. As these known separation and purification methods, methods utilizing solubility such as salting out and solvent precipitation,
Methods that mainly use differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, methods that use differences in charge, such as ion exchange chromatography, and affinity chromatography. A method using a difference in hydrophobicity such as reversed-phase high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing, and the like. Human LH / hCG receptor complement D
A method is also conceivable in which an antibody against a fusion protein expressed by fusing NA with E. coli-derived DNAlacZ is used as an immunoaffinity column. Human L thus produced
The H / hCG receptor protein can be measured by an enzyme immunoassay or the like using a specific antibody.

[0024]

The cells or cells infected or transformed with the cDNA of the present invention can produce a large amount of human LH / hCG receptor, and can advantageously lead to the production of human LH / hCG receptor protein. . The human LH / hCG receptor protein produced here can be used for studying ovarian physiology, providing antibodies to the receptor, diagnosing and managing ovarian and testicular diseases such as ovulation abnormality and sperm reduction, and developing contraceptives. In addition, in humans, this TSH receptor may be a target of an autoimmune response that leads to hyper- or hypo-stimulation of the thyroid by autoantibodies in Graves' disease and idiopathic myxedema, so that the LH / hCG receptor May inhibit LH action in the living body or perform overstimulation instead of LH, resulting in a pathological condition in the human reproductive system. As a method for detecting this anti-receptor antibody, any of the methods described above can be used. A receptor can be produced and labeled by such a method, and it can be examined whether or not a substance (antibody) that binds to the receptor exists in the living body.
In addition, a part or all of the receptor cDNA is expressed to prepare an antibody against the receptor cDNA.
Inhibition of action, that is, application as a contraceptive is considered possible.

The human LH / hCG receptor protein of the present invention is orally or parenterally administered to a mammal (eg, human, rabbit, rat, mouse, etc.) by itself or as a pharmaceutical composition in a suitable dosage form. can do. The pharmaceutical composition used for the above administration is the human LH of the present invention.
/ HCG receptor protein and a pharmacologically acceptable carrier,
And a diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders and capsules. (Including soft capsules), syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate and the like.

Compositions for parenteral administration include, for example, injections, suppositories and the like. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Dosage forms. Such injections are prepared by a method known per se, that is, by dissolving, suspending or emulsifying the human LH / hCG receptor protein of the present invention in a sterile aqueous or oily liquid commonly used for injections. Aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, suitable dissolution aids,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, H
CO-50 (polyoxyethylene (50mol) adduct of hydrogenat
ed castor oil)]. Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in a suitable ampoule. The suppository used for rectal administration is the human LH of the present invention.
/ HCG receptor protein is mixed with a conventional suppository base.

As described above, the cloning of the cDNA encoding the human LH / hCG receptor protein, the construction of an expression vector for the human LH / hCG receptor protein, the production of a transformant using them, and the human LH using the transformant / HCG
The production of the receptor protein and its usefulness have been described in detail.

In the specification and drawings of the present invention, when bases and amino acids are indicated by abbreviations, IUPAC-IU
B This is based on the abbreviation by Commision on Biochemical Nomenclature or the abbreviation commonly used in the art, examples of which are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamate Asp or Is D: aspartic acid Lys or K: lysine Arg or R: arginine His or H: histidine Phe or F: phenylalanine Tyr or Y: tyrosine Trp or W: tryptophan Pro or P: proline Asn or N: asparagine Gln or Qn Glutamine In the human luteinizing hormone-human chorionic gonadotropin receptor protein of the present invention, a part of the amino acid sequence thereof may be modified (addition, removal, substitution with another amino acid, etc.).

[0030]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
The transformant Escherichia coli JM109 / pUC18 obtained in Example 1 described below was deposited on October 9, 1990 with the Ministry of International Trade and Industry, Ministry of Industry and Industry's Institute of Industrial Technology, Microbial Industry Research Institute (FRI), under the accession number FERM BP. -3127
The microorganism has been deposited with the Fermentation Research Institute (IFO) on October 11, 1990 under the accession number IFO.
Deposited as 15096.

The transformant Escherichia coli DH1 / pHLH obtained in Example 2 described later.
R (UEX2) and Escherichi
a. coli) JM109 / pHLHR (GEX-3X) was deposited on August 29, 1991 by the Ministry of International Trade and Industry with the Ministry of International Trade and Industry's Institute of Industrial Technology (FRI) under accession number FERM BP-3
545 and FERM BP-3544, and the microorganism has been deposited with the Fermentation Research Institute (IFO) on August 30, 1991 under the accession number IFO 1521.
9, deposited as IFO 15218.

[0032]

Example 1 1) Preparation of cDNA library derived from human ovary Total R was obtained from human ovary by guanidine thiocyanate method.
After extracting NA, mRNA was purified using an oligo (dT) cellulose column (Pharmacia, type 7). c
Purified mRN using DNA synthesis kit (Pharmacia)
CDNA was synthesized from about 2 μg of A. After the end of this cDNA was blunted with T4 DNA polymerase, EcoRI was used.
Adapter was added. This cDNA was ligated to a λgt10 vector, and in vitro packaging was performed using Gigapack Gold (Stratagene). This library contained 1 × 10 ⁶ independent recombinants and was propagated.

2) Purification of probe A cDNA library was prepared from rat ovaries in the same manner as described above, and a rat LH / hCG receptor was cloned from a vector inserted into a λZaPII vector (Stratagene), thereby obtaining clone Zap3-5. -1 (2.8 kb), which was isolated by the random primer method (Amer
sham), (α- ³² P) -dNTP (deoxynucleoside tr
Labeled with iphosphate) and used as a probe.

3) Screening 5 × 10 ⁴ pfu (plaque forming unit)
Λgt10 cDNA library phage solution and 5 min
Mix with 00 μl of C600hfl (overnight culture),
After incubation for 8 minutes, 8 ml of 0.75% agarose (Nippon Gene) LB was added and a 1.5% agar LB plate (15 cm) was added.
Dish). A nitrocellulose filter (Hybond-N) (Amersham) was placed on the plate on which the plaque was formed to fix the DNA. Then the filter is 6 × S
SC (0.15 M NaCl, 0.015 M sodium citrate,
0.1% BSA (bovine serum albumin) in pH 7.0),
Polyvinylpyrrolidone, Ficoll 400 (Pharmacia) and 5%
In a solution containing pyrophosphoric acid and 0.1% SDS, 65 ° C, 1 to 1
Prehybridized for 2 hours. For hybridization, a probe was added at approximately 200,000 cpm / ml. Washing was performed at 42 ° C. for 15 minutes in 6 × SSC, 0.1% SDS, followed by 0.1%.
× SSC, 0.1% SDS at 65 ° C. for 10 minutes, −70
Autoradiography was performed at 0 ° C.

4) DNA sequence analysis Several clones were identified, the longest of which was selected for sequence analysis. This clone is called pUC
18 (Takara), Escherichia coli JM109 was transformed with this plasmid, and the transformant Escherichia coli JM109 / pUC1 was transformed.
8 was obtained. The transformant was further cut in stages by exonuclease digestion, and single-stranded DNA was prepared from large to short. Sequence analysis was performed by the dideoxy chain end method using a 7DEAZA sequencing kit. LKB2010 Macrophor Sequencing Syst
Perform electrophoresis using em and analyze data by SDC G
enetyx was used. FIG. 1 shows the DNA base sequence of the human LH / hCG receptor protein and the amino acid sequence deduced therefrom. In addition, the DNA obtained here
Is obtained by adding eight DNAs of -8 to -1 to the N-terminal of SEQ ID NO: 1.

[0036]

Example 2 Expression of Human LH / hCG Receptor (HLHR) Protein (1) Using the HLHR cDNA clone obtained in Example 1, 140 encoding the extracellular portion of this HLHR
The 0 bp EcoRI-Xba fragment was replaced with the Bam of pUEX2.
Insertion into the HI site and cloning
A Z-HLHR fusion gene was obtained. This lacZ-HLH
Escherichia coli E. coli using the R fusion gene. E. coli DH1 was transformed and the transformant E. coli was transformed. coli DH1 / pHLHR
(UEX2) (FERM BP-3545) was obtained. The transformant was cultured in LB at 30 ° C. overnight, and this culture 5
0 μl was inoculated into 5 ml of LB medium. After incubating at 30 ° C. with air stirring for 2 hours and further at 42 ° C. for 2 hours, the cells were pelleted by centrifugation. The pelleted cells were immediately dissolved in SDS-PAGE buffer and separated by 5% SDS-PAGE. At the same time, Escherichia coli incorporating only the vector pUEX2 was also 5% SDS-PA.
GE was attached. After electrophoresis, the gel was stained with Coomassie blue. The result is shown in FIG. Lane 1 is a molecular weight marker, lane 2 is Escherichia coli incorporating only the vector pUEX2, and lane 3 is of the transformant of the present invention. Band 110Kd incorporating vector only
It was confirmed that a disappeared and that a protein of 159 Kda could be newly detected. The result of the electrophoresis and the result of the nucleotide sequence analysis indicate the expression of the HLHR protein.

(2) HLHR cD obtained in Example 1
Using a NA clone, a 1400 bp EcoRI-Xba fragment encoding the extracellular portion of the HLHR was
A glutathione S-transferase (GST) -HLHR fusion gene was obtained by inserting and cloning into the BamHI site of X-3X (Pharmacia). Using this GST-HLHR fusion gene, E. coli E. coli was used. co
li JM109, and the transformant E. col
i JM109 / pHLHR (GEX-3X) (FER
MBP-3544). The transformants are cultured overnight, the culture is diluted 1:10 with 500 ml of fresh medium, cultured at 37 ° C. for 1 hour, and then IPTG is added to 0.
Add until 1 mM. After culturing for an additional 7 hours, the cells were pelleted by centrifugation. The pelleted cells were immediately dissolved in SDS-PAGE buffer and 10%
Separated by SDS-PAGE and at the same time, vector pGEX
E. coli containing only -3X is also 10% SDS-PAG.
E. After electrophoresis, the gel was stained with Coomassie blue. FIG. 4 shows the results. Lane 1 is a molecular weight marker, lane 2 is E. coli incorporating only the vector pGEX-3X, and lane 3 is of the transformant of the present invention. Band 26Kd incorporating vector only
It was confirmed that a disappeared and that a protein of 75 Kda could be newly detected. The result of the electrophoresis and the result of the nucleotide sequence analysis indicate the expression of the HLHR protein.

(3) HLHR cD obtained in Example 1
The entire coding region on the EcoRI fragment (2995 bp) of the NA clone and an additional flanking region (2995 b
The expression vector pCHLHR was constructed by inserting p) into the pCDNA1 vector. Human kidney 293
Cells (ATTC CRL 1573) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in humidified air containing 5% carbon dioxide. The cells were transfected temporarily by transfection with calcium phosphate using pCHLHR, an expression vector encoding the full length human LH / hCG receptor.
ently) transfected. These cells were tested for responsiveness to hCG by looking at elevated cAMP levels. The result is shown in FIG. In FIG. 5, points indicate average values, and lines indicate ranges of data. The intracellular cAMP level was increased in response to the hCG hormone concentration, indicating that the transformed cells expressed the HLHR protein and reacted with hCG to produce cAMP.

[0039]

[0040]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2987 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to genomic DNA Origin: Human ovary Sequence: ATG AAG CAG CGG TTC TCG GCG CTG CAG CTG CTG AAG CTG CTG CTG CTG 48 Met Lys Gln Arg Phe Ser Ala Leu Gln Leu Leu Lys Leu Leu Leu Leu 1 5 10 15 CTG CAG CCG CCG CTG CCA CGA GCG CTG CGC GAG GCG CTC TGC CCT GAG 96 Leu Gln Pro Pro Leu Pro Arg Ala Leu Arg Glu Ala Leu Cys Pro Glu 20 25 30 CCC TGC AAC TGC GTG CCC GAC GGC GCC CTG CGC TGC CCC GGC CCC ACG 144 Pro Cys Asn Cys Val Pro Asp Gly Ala Leu Arg Cys Pro Gly Pro Thr 35 40 45 GCC GGT CTC ACT CGA CTA TCA CTT GCC TAC CTC CCT GTC AAA GTG ATC 192 Ala Gly Leu Thr Arg Leu Ser Leu Ala Tyr Leu Pro Val Lys Val Ile 50 55 60 CCA TCT CAA GCT TTC AGA GGA CTT AAT GAG GTC ATA AAA ATT GAA ATC 240 Pro Ser Gln Ala Phe Arg Gly Leu Asn Glu Val Ile Lys Ile Glu Ile 65 70 75 80 TCT CAG ATT GAT TCC CTG GAA AGG ATA GAA GCT AAT GCC TTT GAC AAC 288 Ser Gln Ile Asp Ser Leu Glu Arg Ile Glu Ala Asn Ala Phe Asp Asn 85 90 95 CTC CTC AAT TTG TCT GAA ATA CTG ATC CAG AAC ACC AAA AAT CTG AGA 336 Leu Leu Asn Leu Ser Glu Ile Leu Ile Gln Asn Thr Lys Asn Leu Arg 100 105 110 TAC ATT GAG CCC GGA GCA TTT ATA AAT CTT CCC GGA TTA AAA TAC TTG 384 Tyr Ile Glu Pro Gly Ala Phe Ile Asn Leu Pro Gly Leu Lys Tyr Leu 115 120 125 AGC ATC TGT AAC ACA GGC ATC AGA AAG TTT CCA GAT GTT ACG AAG GTC 432 Ser Ile Cys Asn Thr Gly Ile Arg Lys Phe Pro Asp Val Thr Lys Val 130 135 140 TTC TCC TCT GAA TCA AAT TTC ATT CTG GAA ATT TGT GAT AAC TTA CAC 480 Phe Ser Ser Glu Ser Asn Phe Ile Leu Glu Ile Cys Asp Asn Leu His 145 150 155 160 ATA ACC ACC ATA CCA GGA AAT GCT TTT CAA GGG ATG AAT AAT GAA TCT 528 Ile Thr Thr Ile Pro Gly Asn Ala Phe Gln Gly Met Asn Asn Glu Ser 165 170 175 GTA ACA CTC AAA CTA TAT GGA AAT GGA TTT GAA GAA GTA CAA AGT CAT 576 Val Thr Leu Lys Leu Tyr Gly Asn Gly Phe Glu Glu Val Gln Ser His 180 185 190 GCA TTC AAT GGG ACG ACA CTG ACT TCA CTG GAG CTA AAG GAA AAC GTA 624 Ala Phe Asn Gly Thr Thr Leu Thr Ser Leu Glu Leu Ly s Glu Asn Val 195 200 205 CAT CTG GAG AAG ATG CAC AAT GGA GCC TTC CGT GGG GCC ACA GGG CCG 672 His Leu Glu Lys Met His Asn Gly Ala Phe Arg Gly Ala Thr Gly Pro 210 215 220 AAA ACC TTG GAT ATT TCT TCC ACC AAA TTG CAG GCC CTG CCG AGC TAT 720 Lys Thr Leu Asp Ile Ser Ser Thr Lys Leu Gln Ala Leu Pro Ser Tyr 225 230 235 240 GGC CTA GAG TCC ATT CAG AGG CTA ATT GCC ACG TCA TCC TAT TCT CTA 768 Gly Leu Glu Ser Ile Gln Arg Leu Ile Ala Thr Ser Ser Tyr Ser Leu 245 250 255 AAA AAA TTG CCA TCA AGA GAA ACA TTT GTC AAT CTC CTG GAG GCC ACG 816 Lys Lys Leu Pro Ser Arg Glu Thr Phe Val Asn Leu Leu Glu Ala Thr 260 265 270 TTG ACT TAC CCC AGC CAC TGC TGT GCT TTT AGA AAC TTG CCA ACA AAA 864 Leu Thr Tyr Pro Ser His Cys Cys Ala Phe Arg Asn Leu Pro Thr Lys 275 280 285 GAA CAG AAT TTT TCA CAT TCC ATT TCT GAA AAC TTT TCC AAA CAA TGT 912 Glu Gln Asn Phe Ser His Ser Ile Ser Glu Asn Phe Ser Lys Gln Cys 290 295 300 GAA AGC ACA GTA AGG AAA GTG AGT AAC AAA ACA CTT TAT TCT TCC ATG 960 Glu Ser Thr Val Arg Lys Val Ser Asn Ly s Thr Leu Tyr Ser Ser Met 305 310 315 320 CTT GCT GAG AGT GAA CTG AGT GGC TGG GAC TAT GAA TAT GGT TTC TGC 1008 Leu Ala Glu Ser Glu Leu Ser Gly Trp Asp Tyr Glu Tyr Gly Phe Cys 325 330 335 TTA CCC AAG ACA CCC CGA TGT GCT CCT GAA CCA GAT GCT TTT AAT CCC 1056 Leu Pro Lys Thr Pro Arg Cys Ala Pro Glu Pro Asp Ala Phe Asn Pro 340 345 350 TGT GAA GAC ATT ATG GGC TAT GAC TTC CTT AGG GTC CTG ATT TGG CTG 1104 Cys Glu Asp Ile Met Gly Tyr Asp Phe Leu Arg Val Leu Ile Trp Leu 355 360 365 ATT AAT ATT CTA GCC ATC ATG GGA AAC ATG ACT GTT CTT TTT GTT CTC 1152 Ile Asn Ile Leu Ala Ile Met Gly Asn Met Thr Val Leu Phe Val Leu 370 375 380 CTG ACA AGT CGT TAC AAA CTT ACA GTG CCT CGT TTT CTC ATG TGC AAT 1200 Leu Thr Ser Arg Tyr Lys Leu Thr Val Pro Arg Phe Leu Met Cys Asn 385 390 395 400 400 CTC TCC TTT GCA GAC TTT TGC ATG GGG CTC TAT CTG CTG CTC ATA GCC 1248 Leu Ser Phe Ala Asp Phe Cys Met Gly Leu Tyr Leu Leu Leu Ile Ala 405 410 415 TCA GTT GAT TCC CAA ACC AAG GGC CAG TAC TAT AAC CAT GCC ATA GAC 1296 Ser Val Asp Ser Gln Thr Lys Gly Gln Tyr Tyr Asn His Ala Ile Asp 420 425 430 TGG CAG ACA GGG AGT GGG TGC AGC ACT GCT GGC TTT TTC ACT GTA TTC 1344 Trp Gln Thr Gly Ser Gly Cys Ser Thr Ala Gly Phe Phe Thr Val Phe 435 440 445 GCA AGT GAA CTT TCT GTC TAC ACC CTC ACC GTC ATC ACT CTA GAA AGA 1392 Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Val Ile Thr Leu Glu Arg 450 455 460 TGG CAC ACC ATC ACC TAT GCT ATT CAC CTG GAC CAA AAG CTG CGA TTA 1440 Trp His Thr Ile Thr Tyr Ala Ile His Leu Asp Gln Lys Leu Arg Leu 465 470 475 480 AGA CAT GCC ATT CTG ATT ATG CTT GGA GGA TGG CTC TTT TCT TCT CTA 1488 Arg His Ala Ile Leu Ile Met Leu Gly Gly Trp Leu Phe Ser Ser Leu 485 490 495 ATT GCT ATG TTG CCC CTT GTC GGT GTC AGC AAT TAC ATG AAG GTC AGT 1536 Ile Ala Met Leu Pro Leu Val Gly Val Ser Asn Tyr Met Lys Val Ser 500 505 510 ATT TGC TTC CCC ATG GAT GTG GAA ACC ACT CTC TCA CAA GTC TAT ATA 1584 Ile Cys Phe Pro Met Asp Val Glu Thr Thr Leu Ser Gln Val Tyr Ile 515 520 525 TTA ACC ATC CTG ATT CTC AAT GTG GTG GCC TTC TTC ATA ATT TGT GCT 1 632 Leu Thr Ile Leu Ile Leu Asn Val Val Ala Phe Phe Ile Ile Cys Ala 530 535 540 TGC TAC ATT AAA ATT TAT TTT GCA GTT CGA AAC CCA GAA TTA ATG GCT 1680 Cys Tyr Ile Lys Ile Tyr Phe Ala Val Arg Asn Pro Glu Leu Met Ala 545 550 555 560 ACC AAT AAA GAT ACA AAG ATT GCT AAG AAA ATG GCA ATC CTC ATC TTC 1728 Thr Asn Lys Asp Thr Lys Ile Ala Lys Lys Met Ala Ile Leu Ile Phe 565 570 575 ACC GAT TTC ACC TGC ATG GCA CCT ATC TCT TTT TTT GCC ATC TCA GCT 1776 Thr Asp Phe Thr Cys Met Ala Pro Ile Ser Phe Phe Ala Ile Ser Ala 580 585 590 GCC TTC AAA GTA CCT CTT ATC ACA GTA ACC AAC TCT AAA GTT TTA CTG 1824 Ala Phe Lys Val Pro Leu Ile Thr Val Thr Asn Ser Lys Val Leu Leu 595 600 605 GTT CTT TTT TAT CCC ATC AAT TCT TGT GCC AAT CCA TTT CTG TAT GCA 1872 Val Leu Phe Tyr Pro Ile Asn Ser Cys Ala Asn Pro Phe Leu Tyr Ala 610 615 620 ATA TTC ACT AAG ACA TTC CAA AGA GAT TTC TTT CTT TTG CTG AGC AAA 1920 Ile Phe Thr Lys Thr Phe Gln Arg Asp Phe Phe Leu Leu Leu Ser Lys 625 630 635 640 TTT GGC TGC TGT AAA CGT CGG GCT GAA CTT TA T AGA AGG AAA GAT TTT 1968 Phe Gly Cys Cys Lys Arg Arg Ala Glu Leu Tyr Arg Arg Lys Asp Phe 645 650 655 TCA GCT TAC ACC TCC AAC TGC AAA AAT GGC TTC ACT GGA TCA AAT AAG 2016 Ser Ala Tyr Thr Ser Asn Cys Lys Asn Gly Phe Thr Gly Ser Asn Lys 660 665 670 CCT TCT CAA TCC ACC TTG AAG TTG TCC ACA TTG CAC TGT CAA GGT ACA 2064 Pro Ser Gln Ser Thr Leu Lys Leu Ser Thr Leu His Cys Gln Gly Thr 675 680 685 GCT CTC CTA GAC AAG ACT CGC TAC ACA GAG TGT TAACTGTTAC ATCAGTAACT 2117 Ala Leu Leu Asp Lys Thr Arg Tyr Thr Glu Cys 690 695 699 GCATTATTGA ATTGTTCTTA AACCTGTAAA AAAAAATTAC CTGTACCAGT AATTTTAACA 2177 TAAAGGGTTG GATTTAGGAA ATTATTTATT TTTAGGTACA TTAGGCAAGA GACCTCTACC 2237 TAGTAGAAAG TGTAGTCTAT GACCACTGCC ACACGTAAAA ACTATTTGTC ATTGTTACAT 2297 GGCATAAATA TGAAGTTGAG AGTGTTTAGA AATTTTTATA GAAATTTTGA CACAGTAATT 2357 TTGTTTGATG AATCTTTTAA AAAACAGAGG AGGTATTTTG CATATCTTTT TTTCATTTTC 2417 GTAATTTGTA TTGCATTCTA TAAAAATATT AGTTCATAAC AGATCAGAAA TTTAAAATAA 2477 GGGGCTTTCC CCTCAAGGTAGACTGA TCAATTCGGT 2537 ACGCACTAGC CACATGTGGC TAAATTAAAA TTAAATAAAA TGAGAAATGT AGTTTCTCAG 2597 TTGCACTACG TTTCAAGTTC TCAATGGCTA CGTCAAGTTC TCAATGGCTA CGTGTGACTA 2657 GTGCTTACCA TACTGGACAG CACAGACACA GAATATTTTC ATCACCACAG AAAGTTCTAT 2717 CTGTTCTATT ATAGAGACTT TTATGTATGC CCTATCTGGA TTCTACTTAT TTATAATTTA 2777 AGGTAAACAT CTGAAAGCAC ATTTCAGCCT ATTTGCTTAG TGAAACATTA AGCTGTAGAC 2837 TGTAAACTCC TCGTGAGTAG GAACCCTGTC TCAGTGCATT TTGTTTTCCT GCTTCCTACC 2897 TCAAGATCTT GGCAATGGTA CACTACAAAT GTGCTGAGTT AGAATTACTC TGAAGTTATG 2957 AAACATATAA TGAAAACAAT TTTTCCGGCC 2987

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the DNA of the human LH / hCG receptor protein and the amino acid sequence deduced therefrom.

FIG. 2 is a diagram showing a comparison between the amino acid sequence of human LH / hCG receptor protein and the amino acid sequences of other known LH / hCG receptor proteins or proteins having a similar action.

FIG. 3 is a schematic diagram showing the result of SDS-PAGE showing that the human LH / hCG receptor protein was expressed in Example 2.

FIG. 4 is a schematic diagram showing the results of SDS-PAGE showing that the human LH / hCG receptor protein was expressed in Example 2.

FIG. 5 is a graph showing the reactivity of human LH / hCG receptor protein obtained in Example 3 with hCG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:19) (C12P 21/02 C12R 1:19) (56) Reference MOL. ENDO. (1990) Vol. 8, p. 1264-1276 Science (1989) Vol. 245, no. 4917, p. 525-528 Science (1989) Vol. 245, no. 4917, p. 494-499 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 14/72 C12N 15/12-15/28 C12P 21/00-21/02 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq

Claims (4)

(57) [Claims] 1. A human luteinizing hormone-human chorionic gonadotropin receptor protein comprising the amino acid sequence represented by SEQ ID NO: 1. 2. A cD encoding the human luteinizing hormone-human chorionic gonadotropin receptor protein according to claim 1.
DNA containing NA. 3. A transformant carrying the DNA according to claim 2 . (4) culturing the transformant according to ( 3 ), producing and accumulating human luteinizing hormone-human chorionic gonadotropin receptor protein in the culture, and collecting the protein; A method for producing a human luteinizing hormone-human chorionic gonadotropin receptor protein comprising the amino acid sequence represented by 1.