Patent Application: US-75734291-A

Abstract:
disclosed are a human luteinizing hormone - human chorionic gonadotropin receptor protein , a dna comprising a cdna segment coding for a human luteinizing hormone - human chorionic gonadotropin receptor protein , a transformant carrying a dna comprising a cdna segment coding for a human luteinizing hormone - human chorionic gonadotropin receptor protein , and a method for preparing a human luteinizing hormone - human chorionic gonadotropin receptor protein which comprises cultivating the transformant described in , accumulating a protein in a culture broth , and collecting the same , whereby the structure and properties of the receptor protein are made clear and the mass production thereof by recombinant technology is pioneered .

Description:
the present inventors cloned two kinds of cdnas of the human luteinizing hormone - human chorionic gonadotropin receptor protein to deduce a primary structure of the complete protein ( fig1 ). the first methionine in this sequence is considered to be an initiator codon . this is followed by an amino acid sequence having the characteristics of a signal peptide with a cleavage site present . a possible model for construction of the protein was suggested by hydropathy analysis and comparison with the rat and porcine lh / hcg receptors seq id no : 3 and 4 respectively ( fig2 ). a putative extracellular domain of 335 amino acids precedes a region of 267 amino acids that displays seven possible transmembrane segments ( regions surrounded by rectangles in fig2 and labeled i to ii ). there is a 72 amino acid cooh - terminal intracellular domain . the mature protein may consist of 674 amino acids ( 75632 daltons ). in addition to this protein , 25 signal peptides ( the 1st to 25th amino acids in fig1 and 2 ) exist . however , these peptides are cut off during synthesis of the receptor , and therefore the mature protein of the receptor is considered to consist of 674 amino acids ( the 26th to 699th amino acids , seq id no : 10 is the amino acid sequence of seq id no : 9 ). at the primary structure level , this extracellular domain has about 85 % homology with the rat and porcine lh / hcg receptors and 45 % homology with tsh and fsh receptors ( in fig2 hlh / hcgr indicates the human lh / hcg receptor ; rlh / hcgr indicates the rat lh / hcg receptor ; plh / hcgr indicates the porcine lh / hcg receptor ; htshr indicates the human tsh receptor [ biochem . biophys . res . comm . 166 , 394 ( 1990 )]; and rfshr indicates the rat fsh receptor [ mol . endo . 4 , 525 ( 1990 )]). six potential glycosylation sites are found in the putative extracellular domain ( underlined portions in fig1 ). clusters of cysteine residues are present in the nh 2 - terminal portion and between the putative extracellular and transmembrane domains of the above protein . since these cysteine residues are conserved in the lh , fsh and tsh receptors , while not wishing to be bound by theory , it may be said that the formation of disulfide bonds is crucial for the conformational integrity of the large extracellular domains of glycoprotein hormone receptors . the domain considered to contain the transmembrane domains has about 90 % homology with the rat and porcine lh / hcg receptors , and 70 % homology with the tsh and fsh receptors . serine and threonine residues are found with high frequency in a putative intercellular domain having three sites which is possibly phosphorylated by protein kinase c ( fig1 ). since the phosphorylation by protein kinase specific to the receptors play a role in agonist specific decoupling of adrenergic receptors from the g proteins , it is important to know whether the phosphorylation in at least one of these sites causes any functional changes of the lh / hcg receptors . in the present invention , in addition to a clone having a large open reading frame , a clone coding for a shorter protein was obtained . the large clone is the 1st to 699th amino acid residues ( seq id no : 2 ) in fig1 ( seq id no : 1 ), and the truncated type is one from which a region of the 227th to 289th amino acid residues surrounded by a rectangle ( seq id no : 7 ) is lacking . this pattern suggests that the cleavage mechanism necessary to complete mrna has selectivity . these results are very similar to the data of the porcine lh / hcg receptor . the role of this truncated type receptor is not understood well , and it is not known either whether this lh / hcg receptor is physiologically active as a monomer or an oligomer . in humans , this tsh receptor can be a target of autoimmune reaction which leads to hyper - or hypo - stimulation of the thyroid gland by autoantibodies in grave &# 39 ; s disease and idiopathic myxedema . thus , not only for contributions to diagnosis and management of ovarian diseases , but also for better understanding of ovarian physiology , it is necessary to isolate the human lh / hcg receptor and to know its characteristics . fig2 shows the amino acid sequence of the novel human luteinizing hormone - human chorionic gonadotropin receptor protein ( seq id no : 2 ) obtained in the present invention , and compares this amino acid sequence with the amino acid sequences of the rat and porcine luteinizing hormone - human chorionic gonadotropin receptor proteins ( seq id no : 3 and 4 respectively ) and the fsh and tsh receptors ( seq id no : 6 and 5 respectively ) having similar action . the same amino acid residue as appears in the human luteinizing hormone - human chorionic gonadotropin receptor protein of the present invention , is represented by “.”, and an amino acid residue different from that of the human lh / hcg receptor is represented by the appropriate symbol as defined herein . consensus shown in fig2 indicates amino acid residues common to all the glycoproteins shown in fig2 . the illustration of consensus results in introduction of lacking portions “-” into the formulae in fig2 . accordingly , the number representing the amino acids is counted excluding these lacking portions . for a dna sequence , the dna coding for the human lh / hcg receptor of the present invention contains the nucleotide sequence ( seq id no : 1 ) shown in fig1 or a portion thereof . as the cdna coding for the human lh / hcg receptor of the present invention , any cdna may be used as long as it contains a nucleotide sequence coding for an amino acid sequence of the human lh / hcg receptor . for example , dna containing the nucleotide sequence ( seq id no : 1 ) shown in fig1 or a portion thereof is preferably used . the nucleotide sequence ( seq id no : 1 ) shown in fig1 is an example of cdna sequences coding for the human lh / hcg receptor obtained in the present invention . in the present invention , for example , an expression vector having the cdna containing the nucleotide sequence coding for the human lh / hcg receptor can be prepared by the following process : ( a ) messenger rna ( mrna ) is isolated from human lh / hcg receptor - producing cells . ( b ) single stranded complementary dna ( cdna ) is synthesized from the mrna , followed by synthesis of double stranded dna . ( c ) the complementary dna is introduced into a phage or a plasmid . ( d ) host cells are transformed with the recombinant phage or plasmid thus obtained . ( e ) after cultivation of the transformants thus obtained , plasmids or phages containing the desired dna are isolated from the transformants by an appropriate method such as hybridization with a dna probe coding for a portion of the rat lh / hcg receptor or immunoassay using an anti - lh / hcg receptor antibody . ( f ) the desired cloned dna is cut out from the recombinant dna . ( g ) the cloned dna or a portion thereof is ligated downstream from a promoter in the expression vector . the mrna coding for the human lh / hcg receptor can be obtained from various human lh / hcg receptor - producing cells , for example , germ cells such as the leydig cells in the testis , the capsular cells in the ovary , the granulosa cells , the corpus luteum cells and the interstitial cells . methods for preparing the mrna from the human lh / hcg receptor - producing cells include the guanidine thiocyanate method [ j . m . chirgwin et al ., biochemistry 18 , 5294 ( 1979 )] and the like . using the mrna thus obtained as a template , cdna is synthesized by use of reverse transcriptase , for example , in accordance with the method of h . okayama et al . [ molecular and cellular biology 2 , 161 ( 1979 ); and ibid . 3 , 280 ( 1983 )]. the cdna thus obtained is introduced into the plasmid . the plasmids into which the cdna may be introduced include , for example , pbr322 [ gene 2 , 95 ( 1977 )], pbr325 [ gene 4 , 121 ( 1978 )], puc12 [ gene 19 , 259 ( 1982 )] and puc13 [ gene 19 , 259 , each derived from escherichia coli , and pub110 derived from bacillus subtilis [ biochemical and biophysical research communication 112 , 678 ( 1983 )]. however , any other plasmid can be used as long as it is replicable and viable in the host cell . examples of the phage vectors into which the cdna may be introduced include λgt11 [ r . young and r . davis , proc . natl . acad . sci . u . s . a . 80 , 1194 ( 1983 )]. however , any other phage vector can be used as long as it is viable in the host cell . methods for introducing the cdna into the plasmid include , for example , the method described in t . maniatis et al ., molecular cloning , cold spring harbor laboratory , p . 239 ( 1982 ). methods for introducing the cdna into the phage vector include , for example , the method of t . v . hyunh et al . [ dna cloning , a practical approach 1 , 49 ( 1985 )]. the plasmid thus obtained is introduced into an appropriate host cell such as escherichia and bacillus . examples of escherichia described above include e . coli k12dh1 [ proc . natl . acad . sci . u . s . a . 60 , 160 ( 1968 )], m103 [ nucleic acids research 9 , 309 ( 1981 )], ja221 [ journal of molecular biology 120 , 517 ( 1978 )], hb101 [ journal of molecular biology 41 , 459 ( 1969 )] and c600 [ genetics 39 , 440 ( 1954 )]. examples of bacillus described above include bacillus subtilis mi114 [ gene 24 , 255 ( 1983 )] and 207 - 21 [ journal of biochemistry 95 , 87 ( 1984 )]. methods for transforming the host cell with the plasmid include , for example , the calcium chloride method or the calcium chloride / rubidium chloride method described in t . maniatis et al ., molecular cloning , cold spring harbor laboratory , p . 249 ( 1982 ). when the phage vector is used , for example , it can be transduced into proliferated e . coli , using the in vitro packaging method . human lh / hcg receptor - cdna libraries containing human lh / hcg receptor cdna can be purchased from the market , though obtainable by the methods described above . for example , a cdna library of the lh / cg receptor is available from clontech laboratories , inc ., u . s . a . methods for cloning human lh / hcg receptor cdna from the human dna library include , for example , the plaque hybridization method using phage vector λcharon 28a and rat lh / hcg receptor cdna as a probe [ t . maniatis et al ., molecular cloning , cold spring harbor laboratory , ( 1982 )]. the human lh / hcg receptor cdna thus cloned may be subcloned , for example , in pbr322 , puc12 , puc13 , puc18 , puc19 , puc118 and puc119 to obtain the human lh / hcg receptor cdna , if necessary . the nucleotide sequence of the cdna thus obtained is determined , for example , by the maxam - gilbert method [ a . m . maxam and w . gilbert , proc . natl . acad . sci . u . s . a . 74 , 560 ( 1977 )] or the dideoxy method [ j . messing et al ., nucleic acids research 9 , 309 ( 1981 )], and the existence of the human lh / hcg receptor cdna is confirmed in comparison with the known amino acid sequence . as described above , the cdna coding for the human lh / hcg receptor protein is obtained . fig1 shows the nucleotide sequence of the cdna ( seq id no : 1 ) determined by the dideoxy method for the cdna coding for the human lh / hcg receptor protein obtained in example 1 described below , and the amino acid sequence proved from that nucleotide sequence . the cdna coding for the human lh / hcg . receptor protein ( seq id no : 2 ) cloned as described above can be used as is , or after digestion with a restriction enzyme if desired , depending on the intended use . a region intended to be expressed is cut out from the cloned cdna and ligated downstream from a promoter in a vehicle ( vector ) suitable for expression , whereby the expression vector can be obtained . the cdna has atg as a translation initiating codon at the 5 ′- terminus thereof and may have taa , tga or tag as a translation terminating codon at the 3 ′- terminus . the translation initiating codon and translation terminating codon may be added by use of an appropriate synthetic cdna adaptor . a promoter is further ligated upstream therefrom for the purpose of expressing the cdna . examples of the vectors include the above plasmids derived from e . coli such as pbr322 , pbr325 , puc12 and puc13 , the plasmids derived from bacillus subtilis such as pub110 , ptp5 and pc194 , plasmids derived from yeast such as psh19 and psh15 , bacteriophages such as λ phage , and animal viruses such as retroviruses and vaccinia viruses . as the promoter used in the present invention , any promoter is available as long as it is suitable for expression in the host cell selected for the gene expression . when the host cell used for transformation is escherichia , it is preferable that a trp promoter , a lac promoter , a reca promoter , a λp l promoter , a lpp promoter , etc . are used . when the host cell is bacillus , it is preferable that a spo1 promoter , a spo2 promoter , a penp promoter , etc . are used . when the host cell is yeast , it is preferable that a pho5 promoter , a pgk promoter , a gap promoter , an adh promoter , etc . are used . in particular , it is preferable that the host cell is escherichia and the promoter is the trp promoter or the λp l promoter . when the host cell is an animal cell , a sv - 40 derived promoter , a retrovirus promoter , a metallothionein promoter , a heat shock promoter , etc . are each usable . using a vector containing the cdna coding for the mature peptide of the human lh / hcg receptor protein thus constructed , transformants are prepared . the host cells include , for example , escherichia , bacillus , yeast and animal cells . specific examples of the above escherichia and bacillus include strains similar to those described above . examples of the above yeast include saccharomyces cerevisiae ah22 , ah22r − , na87 - 11a and dkd - 5d . examples of the animal cells include monkey cell cos - 7 , vero , chinese hamster cell ( cho ), mouse l cell and human fl cell . the transformation of the above escherichia is carried out , for example , according to the method described in proc . natl . acad . sci . u . s . a . 69 , 2110 ( 1972 ) or gene 17 , 107 ( 1982 ). the transformation of the above bacillus is conducted , for example , according to the method described in molecular & amp ; general genetics 168 , 111 ( 1979 ). the transformation of the yeast is carried out , for example , according to the method described in proc . natl . acad . sci . u . s . a . 75 , 1929 ( 1978 ). the transformation of the animal cells is carried out , for example , according to the method described in virology 52 , 456 ( 1973 ). thus , transformants are obtained which have been transformed with the expression vector containing the cdna coding for the human lh / hcg receptor . when bacterial transformants are cultured , a liquid medium is particularly suitable as a medium used for culture . carbon sources , nitrogen sources , inorganic compounds and others necessary for growth of the transformants are contained therein . examples of the carbon sources include glucose , dextrin , soluble starch and sucrose . examples of the nitrogen sources include inorganic or organic materials such as ammonium salts , nitrates , corn steep liquor , peptone , casein , meat extracts , soybean meal and potato extract solution . the inorganic compounds include , for example , calcium chloride , sodium dihydrogenphosphate and magnesium chloride . yeast , vitamins , growth promoting factors and so on may be further added thereto . the ph of the medium is preferably about 5 to 8 . as the medium used for cultivation of escherichia , for example , m9 medium containing glucose and casamino acids ( miller , journal of experiments in molecular genetics 431 - 433 , cold spring harbor laboratory , new york , 1972 ) is preferably used . in order to make the promoter act efficiently , a drug such as 3 - β - indolylacrylic acid may be added thereto if necessary . when the host cell is escherichia , the cultivation is usually carried out at about 15 to 43 ° c . for about 3 to 24 hours , with aeration or agitation if necessary . when the host cell is bacillus , the cultivation is usually carried out at about 30 to 40 ° c . for about 6 to 24 hours , with aeration or agitation if necessary . when yeast transformants are cultured , for example , burkholder minimum medium [ k . l . bostian et al ., proc . natl . acad . sci . u . s . a . 77 , 4505 ( 1980 )] is used as the medium . the ph of the medium is preferably adjusted to about 5 to 8 . the cultivation is usually carried out at about 20 to 35 ° c . for about 24 to 72 hours , with aeration or agitation if necessary . when animal cell transformants are cultured , examples of the mediums include mem medium containing about 5 to 20 % fetal calf serum [ science 122 , 501 ( 1952 )], dmem medium [ virology 8 , 396 ( 1959 )], rpmi1640 medium ( the journal of the american medical association 199 , 519 ( 1967 )] and 199 medium [ proceeding of the society for the biological medicine 73 , 1 ( 1950 ). the ph is preferably about 6 to 8 . the cultivation is usually carried out at about 30 to 40 ° c . for about 15 to 60 hours , with aeration or agitation if necessary . the human lh / hcg receptor protein can be isolated and purified from the culture described above , for example , by the following method . when the human lh / hcg receptor protein is extracted from the cultured cells , the cells are collected by methods known in the art after cultivation . then , the collected cells are suspended in an appropriate buffer solution and disrupted by ultrasonic treatment , lysozyme and / or freeze - thawing . thereafter , a crude extracted solution of the human lh / hcg receptor mature peptide is obtained by centrifugation or filtration . the buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride , or a surface - active agent such as triton x - 100 . when the human lh / hcg receptor protein is secreted in the culture solution , a supernatant is separated from the cells by methods known in the art after the conclusion of cultivation , and then collected . the separation and purification of the human lh / hcg receptor contained in the culture supernatant or the extracted solution thus obtained can be performed by an appropriate combination of known separating and purifying methods . the known separating and purifying methods include methods utilizing solubility such as salt precipitation and solvent precipitation , methods mainly utilizing a difference in molecular weight such as dialysis , ultrafiltration , gel filtration and sds - polyacrylamide gel electrophoresis , methods utilizing a difference in electric charge such as ion - exchange column chromatography , methods utilizing specific affinity such as affinity chromatography , methods utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography and methods utilizing a difference in isoelectric point such as isoelectro - focussing electrophoresis . a method may also be used in which an antibody to a fused protein expressed by fusing the human lh / hcg receptor complimentary dna together with e . coli - derived dna lacz is used as an immunoaffinity column . the activity of the human lh / hcg receptor protein thus formed can be measured by an enzyme immunoassay using a specific antibody . the cells transfected or transformed with the cdna of the present invention can allow the human lh / hcg receptor protein to be produced in large amounts . the human lh / hcg receptor protein produced here is channeled into the study of ovarian physiology , the supply of antibodies to the receptor , the diagnosis and management of ovarian or testicular diseases such as ovulation aberration or oligospermia , and the development of contraceptives . in humans , this tsh receptor can be a target of autoimmune reaction which leads to hyper - or hypo - stimulation of the thyroid gland by autoantibodies in grave &# 39 ; s disease and idiopathic myxedema . the lh / hcg receptor might therefore suppress the lh action in vivo or can conduct hyperstimulation in stead of lh to cause morbidity in the human genital system . the anti - receptor antibody can be detected by producing the receptor by any of the above - described methods , labeling it and examining whether one binding to it ( antibody ) is present in vivo or not . in addition , it is considered that inhibition of the lh action by an antibody obtained by expressing a portion or all of the receptor cdna , namely the application of the antibody as a contraceptive , is possible . there have been described above in detail the cloning of the cdna coding for the human lh / hcg receptor protein , the preparation of the expression vectors for the human lh / hcg receptor protein , the production of the transformants thereby , the production of the human lh / hcg receptor protein by using the transformants and utility thereof . when nucleotides , amino acids and so on are indicated by abbreviations in this specification and drawings , the abbreviations adopted by the iupac - iub commission on biochemical nomenclature or commonly used in the art are employed . for example , the following abbreviations are used . when the amino acids are capable of existing as optical isomers , it is understood that the l - forms are represented unless otherwise specified . the precise chemical structure of the human luteinizing hormone - human chorionic gonadotropin receptor proteins of the present invention will depend on a number of factors . because ionizable amino and carboxyl groups are present in these proteins , a particular protein may be obtained as an acidic or basic salt , or in neutral form . all such preparations which retain their bioactivity when placed in suitable environmental conditions are included in the definition of the receptor proteins of the present invention . further , the primary amino acid sequence of such proteins may be argumented by derivation using sugar moieties or by other supplementary molecules such as lipids , phosphate , acetyl groups and the like . such modifications are included in the definition of the receptor proteins of the present invention so long as the bioactivity of the protein is not destroyed . it is expected , of course , that such modifications may quantitatively or qualitatively affect the bioactivity by either enhancing or diminishing the activity of the protein . further , individual amino acid residues in the chain may be modified by oxidation , reduction , or other derivatization , and the receptor proteins of the present invention may be cleaved to obtain fragments which retain bioactivity . such alterations which do not destroy bioactivity do not remove such receptor proteins from the definition . finally modifications to the primary structure itself by deletion , addition , or alteration of the amino acids incorporated into the sequence during translation can be made without destroying the activity of the receptor proteins of the present invention . the present invention will hereinafter be described in more detail with the following examples . it is understood of course that these examples are not intended to limit the scope of the invention . transformant e . coli jm109 / puc18 obtained in example 1 described below was deposited with the fermentation research institute , the agency of industrial science and technology , the ministry of international trade and industry , japan ( fri ) under the accession number ferm bp - 3127 on oct . 9 , 1990 . this microorganism was deposited with the institute for fermentation , osaka , japan ( ifo ) under the accession number ifo 15096 on oct . 11 , 1990 . transformants e . coli dh1 / phlhr ( uex2 ) and e . coli jm109phlhr ( gex - 3x ) obtained in example 2 described below were deposited with the fermentation research institute , the agency of industrial science and technology , the ministry of international trade and industry , japan ( fri ) under the accession number ferm bp - 3545 and ferm bp - 3544 respectively on aug . 29 , 1991 . total rna was extracted from the human ovary by the guanidine thiocyanate method , and then mrna was purified by use of an oligo ( dt ) cellulose column ( type 7 , pharmacia ). using a cdna synthesizing kit ( pharmacia ), cdna was synthesized from about 2 μg of purified mrna . the terminus of this cdna was rendered flush with t4 dna polymerase , followed by addition of an ecori adapter . this cdna was bound to a λgt10 vector , and in vitro packaging was carried out by use of a packaging kit ( gigapack gold , stratagene ). this library contained 1 × 10 6 independent recombinants , and was proliferated . a cdna library was prepared from the rat ovary in a manner similar to that described above , and inserted into a λzapii vector ( stratagene ). a rat lh / hcg receptor was cloned therefrom to isolate clones zap3 - 5 - 1 ( 2 . 8 kb ). the clones were labeled using the random primer method ( amersham ), and used as a probe . a λgt10 cdna library phage solution of 5 × 10 4 plaque forming units ( pfu ) was mixed with 500 μl of c600hfl ( cultivated overnight ), and the mixture was incubated at 37 ° c . for 15 minutes . then , 8 ml of 0 . 75 % agarose ( nippon gene ) lb was added thereto , and the mixture was inoculated on a 1 . 5 % agar lb plate ( 15 cm dish ). a nitrocellulose filter ( hybond - n , amersham ) was placed on the plate on which plaques were formed , and dna was fixed . subsequently , the filter was prehybridized at 65 ° c . for 1 to 2 hours in a solution prepared by adding 0 . 1 % bovine serum albumin ( bsa ), polyvinylpyrrolidone , ficoll 400 ( pharmacia ), 5 % pyrophosphoric acid and 0 . 1 % sds to 6 × ssc ( 0 . 15 m nacl , 0 . 015 m sodium citrate , ph 7 . 0 ). on hybridization , the probe was added to 200 , 000 cpm / ml as a guide . the filter was washed with 6 × ssc at 42 ° c . for 15 minutes , and subsequently with 0 . 1 × ssc at 65 ° c . for 10 minutes . then , the filter was subjected to autoradiography at − 70 ° c . some clones were identified , and the longest was selected from these clones for sequence analysis . this clone was subcloned into puc18 ( takara ), and e . coli jm109 was transformed with the resulting plasmid to yield transformant e . coli jm109 / puc18 ( ferm bp - 3127 ). this transformant was further shaved off stepwise by exonuclease digestion to prepare long to short single stranded dna fragments . sequence analysis was carried out by the dideoxy chain terminal method using a 7deaza sequencing kit . electrophoresis was carried out by use of a lkb2010 macrophor sequencing system . the sdc genetyx software was used for data analysis . fig1 shows the nucleotide sequence ( seq id no : 1 ) of the dna of the human lh / hcg receptor protein , as well as the amino acid sequence deduced therefrom . the nucleotide sequence obtained in the present invention has additional 8 dnas (− 8 to − 1 ) prior to n - terminus of the nucleotide sequence of seq id no : 1 . expression of human lh / hcg receptor protein ( sometimes referred to herein as hlhr protein ) ( 1 ) the hlhr cdna clones obtained in example 1 were used . the lac z - hlhr fusion gene was obtained by cloning the 1400 bp ecori - xba fragment coding for extracellular segment of the hlhr into the bamhi site of puex2 . the lac z - hlhr fusion construction was transformed into e . coli dh1 host to yield transformant e . coli dh1 / phlhr ( uex2 ) ( ferm bp - 3545 ). for preparation of lacz - hlhr fusion protein , the transformant was cultivated in lb overnight at 30 ° c . 5 ml of the lb medium was innoculated with 50 μl of the overnight culture . after incubation of 2 hr at 30 ° c . with aeration and further incubation of 2 hr at 42 ° c ., the cells were pelleted . the pellets were dissolved in a sds - polyacrylamide gel electrophoresis ( page ) sample buffer . the solution was subjected to 5 % sds - page . e . coli transformed with puex2 vector was similarlly subjected to 5 % sds - page . after electrophoresis , the gel was stained with coomassie blue . the result is shown in fig3 . lane 1 shows a molecular weight marker , lane 2 shows the case of puex2 vector and lane 3 shows the present transformant . a band at 110 kda of lane 2 disappears and a new band at 159 kda appears . the result of the electrophoresis and analysis of the nucleotide sequence show the expression of hlhr protein . ( 2 ) the gst ( glutathion s - transferase ) - hlhr fusion gene was obtained by cloning the 1400 bp ecori - xba fragment coding for extracellular segment of the hlhr into the bamhi site of pgex - 3x ( pharmacia ). the gst - hlhr fusion construction was transformed into e . coli jm 109 host to yield e . coli jm109 / phlhr ( gex - 3x ) ( ferm bp - 3544 ). the transformant was cultivated in lb overnight at 30 ° c . the overnight culture of jm 109 was diluted 1 : 10 in 500 ml of fresh medium and cultivated for 1 hr at 37 ° c . before adding iptg to 0 . 1 mm . after further 7 hr culture , the cells were pelleted . the pellets were dissolved in a sds - polyacrylamide gel electrophoresis ( page ) sample buffer . the solution was subjected to 10 % sds - page . e . coli transformed with pgex - 3x vector was similarily subjected to 10 % sds - page . after electrophoresis , the gel was stained with coomassie blue . the result is shown in fig4 . lane 1 shows a molecular weight marker , lane 2 shows the case of pgex - 3x vector and lane 3 shows the present transformant . a band at 26 kda of lane 2 disappears and a new band at 75 kda appears . the result of the electrophoresis and analysis of the nucleotide sequence show the expression of hlhr protein . the expression vector pchlhr was constructed by introducing the entire coding region of the cloned cdna and additional flunking regions contained on an rcori fragment ( 2995 bp ) into the pcdna 1 vector . human kidney 293 cells ( atcc crl 1573 ) were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium containing 10 % fetal calf serum in a humidified atmosphere containing 5 % co 2 . these cells were transiently transfected with pchlhr , an expression vector encoding for the full - length human lh / hcg receptor , according to the procedure of calcium phosphate - mediated transfection . these cells were tested for their response ability to hcg with an increase in camp levels . the result is shown in fig5 . in fig5 the points indicate the mean and the bars indicate the range of the data . the present invention has been described in detail , including the preferred embodiments thereof . however , it will be appreciated that those skilled in the art , upon consideration of the present disclosure , may make modifications and improvements on this invention and still be within the scope and spirit of this invention as set forth in the following claims . ctg cag ccg ccg ctg cca cga gcg ctg cgc gag gcg ctc tgc cct gag 96 ccc tgc aac tgc gtg ccc gac ggc gcc ctg cgc tgc ccc ggc ccc acg 144 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 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 tct cag att gat tcc ctg gaa agg ata gaa gct aat gcc ttt gac aac 288 ctc ctc aat ttg tct gaa ata ctg atc cag aac acc aaa aat ctg aga 336 tac att gag ccc gga gca ttt ata aat ctt ccc gga tta aaa tac ttg 384 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 ttc tcc tct gaa tca aat ttc att ctg gaa att tgt gat aac tta cac 480 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 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 gca ttc aat ggg acg aca ctg act tca ctg gag cta aag gaa aac gta 624 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 aaa acc ttg gat att tct tcc acc aaa ttg cag gcc ctg ccg agc tat 720 ggc cta gag tcc att cag agg cta att gcc acg tca tcc tat tct cta 768 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 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 gaa cag aat ttt tca cat tcc att tct gaa aac ttt tcc aaa caa tgt 912 gaa agc aca gta agg aaa gtg agt aac aaa aca ctt tat tct tcc atg 960 ctt gct gag agt gaa ctg agt ggc tgg gac tat gaa tat ggt ttc tgc 1008 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 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 att aat att cta gcc atc atg gga aac atg act gtt ctt ttt gtt ctc 1152 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 ctc tcc ttt gca gac ttt tgc atg ggg ctc tat ctg ctg ctc ata gcc 1248 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 tgg cag aca ggg agt ggg tgc agc act gct ggc ttt ttc act gta ttc 1344 gca agt gaa ctt tct gtc tac acc ctc acc gtc atc act cta gaa aga 1392 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 aga cat gcc att ctg att atg ctt gga gga tgg ctc ttt tct tct cta 1488 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 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 tta acc atc ctg att ctc aat gtg gtg gcc ttc ttc ata att tgt gct 1632 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 acc aat aaa gat aca aag att gct aag aaa atg gca atc ctc atc ttc 1728 acc gat ttc acc tgc atg gca cct atc tct ttt ttt gcc atc tca gct 1776 gcc ttc aaa gta cct ctt atc aca gta acc aac tct aaa gtt tta ctg 1824 gtt ctt ttt tat ccc atc aat tct tgt gcc aat cca ttt ctg tat gca 1872 ata ttc act aag aca ttc caa aga gat ttc ttt ctt ttg ctg agc aaa 1920 ttt ggc tgc tgt aaa cgt cgg gct gaa ctt tat aga agg aaa gat ttt 1968 tca gct tac acc tcc aac tgc aaa aat ggc ttc act gga tca aat aag 2016 cct tct caa tcc acc ttg aag ttg tcc aca ttg cac tgt caa ggt aca 2064 gct ctc cta gac aag act cgc tac aca gag tgt taactgttac atcagtaa 2117 ala gly leu thr arg leu ser leu ala tyr leu pro val lys val ile pro ser gln ala phe arg gly leu asn glu val ile lys ile glu ile ser ile cys asn thr gly ile arg lys phe pro asp val thr lys val ile thr thr ile pro gly asn ala phe gln gly met asn asn glu ser val thr leu lys leu tyr gly asn gly phe glu glu val gln ser his his leu glu lys met his asn gly ala phe arg gly ala thr gly pro lys lys leu pro ser arg glu thr phe val asn leu leu glu ala thr leu thr tyr pro ser his cys cys ala phe arg asn leu pro thr lys leu pro lys thr pro arg cys ala pro glu pro asp ala phe asn pro cys glu asp ile met gly tyr asp phe leu arg val leu ile trp leu leu thr ser arg tyr lys leu thr val pro arg phe leu met cys asn ser val asp ser gln thr lys gly gln tyr tyr asn his ala ile asp trp his thr ile thr tyr ala ile his leu asp gln lys leu arg leu ile ala met leu pro leu val gly val ser asn tyr met lys val ser ile cys phe pro met asp val glu thr thr leu ser gln val tyr ile cys tyr ile lys ile tyr phe ala val arg asn pro glu leu met ala val lys val ile pro ser gln ala phe arg gly leu asn glu val val lys asn leu leu tyr ile glu pro gly ala phe thr asn leu pro arg leu lys tyr leu ser ile cys asn thr gly ile arg thr leu pro asp asp asn leu his ile thr thr ile pro gly asn ala phe gln gly met val gln ser his ala phe asn gly thr thr leu ile ser leu glu leu lys glu asn ile tyr leu glu lys met his ser gly ala phe gln gly leu val ala thr leu thr tyr pro ser his cys cys ala phe arg asn ser lys gln cys glu ser thr val arg lys ala asp asn glu thr leu tyr ser ala ile phe glu glu asn glu leu ser gly trp asp tyr asp tyr gly phe cys ser pro lys thr leu gln cys ala pro glu pro asp ala phe asn pro cys glu asp ile met gly tyr ala phe leu arg val leu leu ile ala ser val asp ser gln thr lys gly gln tyr tyr asn his ala ile asp trp gln thr gly ser gly cys gly ala ala gly phe thr leu glu arg trp his thr ile thr tyr ala val gln leu asp gln phe ser thr leu ile ala thr met pro leu val gly ile ser asn tyr met lys val ser ile cys leu pro met asp val glu ser thr leu ser lys ile leu leu val leu phe tyr pro val asn ser cys ala asn pro arg lys glu phe ser ala tyr thr ser asn cys lys asn gly phe pro pro ser gln ala phe arg gly leu asn glu val val lys ile glu ile tyr ile glu pro gly ala phe thr asn leu pro arg leu lys tyr leu ser ile cys asn thr gly ile arg lys leu pro asp val thr lys ile ile thr thr val pro ala asn ala phe gln gly met asn asn glu ser ile thr leu lys leu tyr gly asn gly phe glu glu ile gln ser his his leu lys lys met his asn asp ala phe arg gly ala arg gly pro lys lys leu pro ser arg glu lys phe thr asn leu leu asp ala thr leu thr tyr pro ser his cys cys ala phe arg asn leu pro thr lys ser pro lys thr leu gln cys ala pro glu pro asp ala phe asn pro cys glu asp ile met gly tyr asp phe leu arg val leu ile trp leu leu thr ser his tyr lys leu thr val pro arg phe leu met cys asn ser val asp ala gln thr lys gly gln tyr tyr asn his ala ile asp trp his thr ile thr tyr ala ile gln leu asp gln lys leu arg leu arg his ala ile pro ile met leu gly gly trp leu phe ser thr leu ile cys leu pro met asp val glu thr thr leu ser gln val tyr ile cys tyr ile lys ile tyr phe ala val gln asn pro glu leu met ala ser gly cys cys lys his gln ala glu leu tyr arg arg lys asp phe ser ala tyr cys lys asn gly phe thr gly ser asn lys pro ser gln gln glu glu asp phe arg val thr cys lys asp ile gln arg ile pro phe tyr asn leu ser lys val thr his ile glu ile arg asn thr arg asn pro tyr met thr ser ile pro val asn ala phe gln gly leu cys gln gly tyr ala phe asn gly thr lys leu asp ala val tyr leu asn lys asn lys tyr leu thr val ile tyr lys asp ala phe gly gly val leu pro ser lys gly leu glu his leu lys glu leu ile ala arg asn thr arg ala asp leu ser tyr pro ser his cys cys ala phe lys asn gln lys lys ile arg gly ile leu glu ser leu met cys asn glu ser pro leu his gln glu tyr glu glu asn leu gly asp ser ile val gly tyr lys glu lys ser lys phe gln asp thr his asn asn ala his tyr glu leu lys asn pro gln glu glu thr leu gln ala phe asp ser his tyr asp tyr thr ile cys gly asp ser glu asp met val cys thr pro lys ser asp glu phe asn pro cys glu asp ile met gly tyr lys phe met tyr leu leu leu ile ala ser val asp leu tyr thr his ser glu tyr tyr asn his ala ile asp trp gln thr gly pro gly cys asn thr thr val ile thr leu glu arg trp tyr ala ile thr phe ala met arg leu asp arg lys met arg leu arg his ala cys ala ile met val gly ser ser tyr ala lys val ser ile cys leu pro met asp thr glu thr arg asn pro gln tyr asn pro gly asp lys asp thr lys ile ala lys arg met ala val leu ile phe thr asp phe 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phe val thr leu met glu ala ser leu thr tyr pro ser his cys cys ala phe ala asn leu lys arg gln ile ser glu leu his pro ile cys asn lys ser ile leu arg gln asp ile asp asp met thr gln ile gly asp gln arg val ser leu ile asp asp glu pro ser cys asn glu val val asp val thr cys ser pro lys pro asp ala phe asn pro cys glu asp ile met gly tyr asn ile leu arg val leu ile val leu thr thr ser gln tyr lys leu thr val pro arg phe leu met ile ala ser val asp ile his thr lys ser gln tyr his asn tyr ala glu arg trp his thr ile thr his ala met gln leu glu cys lys val gln leu arg his ala ala ser val met val leu gly trp thr phe ala cys gly cys tyr thr his ile tyr leu thr val arg asn pro thr ile ser lys phe gly cys tyr glu met gln ala gln ile tyr arg thr glu ala gly leu thr arg leu ser leu ala tyr leu pro val lys val ile pro ser gln ala phe arg gly leu asn glu val ile lys ile glu ile ser ile cys asn thr gly ile arg lys phe pro asp val thr lys val ile thr thr ile pro gly asn ala phe gln gly met asn asn glu ser val thr leu lys leu tyr gly asn gly phe glu glu val gln ser his his leu glu lys met his asn gly ala phe arg gly ala thr gly pro cys leu pro lys thr pro arg cys ala pro glu pro asp ala phe asn pro cys glu asp ile met gly tyr asp phe leu arg val leu ile trp leu leu thr ser arg tyr lys leu thr val pro arg phe leu met cys asn leu ser phe ala asp phe cys met gly leu tyr leu leu leu ile ala ser val asp ser gln thr lys gly gln tyr tyr asn his ala ile arg trp his thr ile thr tyr ala ile his leu asp gln lys leu arg leu ile ala met leu pro leu val gly val ser asn tyr met lys val ser ile cys phe pro met asp val glu thr thr leu ser gln val tyr ala cys tyr ile lys ile tyr phe ala val arg asn pro glu leu met tyr leu pro val lys val ile pro ser gln ala phe arg gly leu asn gln asn thr lys asn leu arg tyr ile glu pro gly ala phe ile asn leu pro gly leu lys tyr leu ser ile cys asn thr gly ile arg lys phe pro asp val thr lys val phe ser ser glu ser asn phe ile leu glu ile cys asp asn leu his ile thr thr ile pro gly asn ala phe gln gly met asn asn glu ser val thr leu lys leu tyr gly asn gly phe glu glu val gln ser his ala phe asn gly thr thr leu thr ser phe arg gly ala thr gly pro lys thr gln asn phe ser his ser ile trp asp tyr glu tyr gly phe cys leu pro lys thr pro arg cys ala pro glu pro asp ala phe asn pro cys glu asp ile met gly tyr asp val pro arg phe leu met cys asn leu ser phe ala asp phe cys met gly leu tyr leu leu leu ile ala ser val asp ser gln thr lys gly gln tyr tyr asn his ala ile asp trp gln thr gly ser gly cys ser val ser asn tyr met lys val ser ile cys phe pro met asp val glu val arg asn pro glu leu met ala thr asn lys asp thr lys ile ala cys ala asn pro phe leu tyr ala ile phe thr lys thr phe gln arg glu leu tyr arg arg lys asp phe ser ala tyr thr ser asn cys lys ser thr leu his cys gln gly thr ala leu leu asp lys thr arg tyr cgc gag gcg ctc tgc cct gag ccc tgc aac tgc gtg ccc gac ggc gcc 48 ctg cgc tgc ccc ggc ccc acg gcc ggt ctc act cga cta tca ctt gcc 96 tac ctc cct gtc aaa gtg atc cca tct caa gct ttc aga gga ctt aat 144 tyr leu pro val lys val ile pro ser gln ala phe arg gly leu asn gag gtc ata aaa att gaa atc tct cag att gat tcc ctg gaa agg ata 192 gaa gct aat gcc ttt gac aac ctc ctc aat ttg tct gaa ata ctg atc 240 cag aac acc aaa aat ctg aga tac att gag ccc gga gca ttt ata aat 288 gln asn thr lys asn leu arg tyr ile glu pro gly ala phe ile asn ctt ccc gga tta aaa tac ttg agc atc tgt aac aca ggc atc aga aag 336 leu pro gly leu lys tyr leu ser ile cys asn thr gly ile arg lys ttt cca gat gtt acg aag gtc ttc tcc tct gaa tca aat ttc att ctg 384 phe pro asp val thr lys val phe ser ser glu ser asn phe ile leu gaa att tgt gat aac tta cac ata acc acc ata cca gga aat gct ttt 432 glu ile cys asp asn leu his ile thr thr ile pro gly asn ala phe caa ggg atg aat aat gaa tct gta aca ctc aaa cta tat gga aat gga 480 gln gly met asn asn glu ser val thr leu lys leu tyr gly asn gly ttt gaa gaa gta caa agt cat gca ttc aat ggg acg aca ctg act tca 528 phe glu glu val gln ser his ala phe asn gly thr thr leu thr ser ctg gag cta aag gaa aac gta cat ctg gag aag atg cac aat gga gcc 576 ttc cgt ggg gcc aca ggg ccg aaa acc ttg gat att tct tcc acc aaa 624 phe arg gly ala thr gly pro lys thr leu asp ile ser ser thr lys ttg cag gcc ctg ccg agc tat ggc cta gag tcc att cag agg cta att 672 gcc acg tca tcc tat tct cta aaa aaa ttg cca tca aga gaa aca ttt 720 gtc aat ctc ctg gag gcc acg ttg act tac ccc agc cac tgc tgt gct 768 val asn leu leu glu ala thr leu thr tyr pro ser his cys cys ala ttt aga aac ttg cca aca aaa gaa cag aat ttt tca cat tcc att tct 816 phe arg asn leu pro thr lys glu gln asn phe ser his ser ile ser gaa aac ttt tcc aaa caa tgt gaa agc aca gta agg aaa gtg agt aac 864 aaa aca ctt tat tct tcc atg ctt gct gag agt gaa ctg agt ggc tgg 912 gac tat gaa tat ggt ttc tgc tta ccc aag aca ccc cga tgt gct cct 960 asp tyr glu tyr gly phe cys leu pro lys thr pro arg cys ala pro gaa cca gat gct ttt aat ccc tgt gaa gac att atg ggc tat gac ttc 1008 glu pro asp ala phe asn pro cys glu asp ile met gly tyr asp phe ctt agg gtc ctg att tgg ctg att aat att cta gcc atc atg gga aac 1056 atg act gtt ctt ttt gtt ctc ctg aca agt cgt tac aaa ctt aca gtg 1104 cct cgt ttt ctc atg tgc aat ctc tcc ttt gca gac ttt tgc atg ggg 1152 pro arg phe leu met cys asn leu ser phe ala asp phe cys met gly ctc tat ctg ctg ctc ata gcc tca gtt gat tcc caa acc aag ggc cag 1200 leu tyr leu leu leu ile ala ser val asp ser gln thr lys gly gln tac tat aac cat gcc ata gac tgg cag aca ggg agt ggg tgc agc act 1248 tyr tyr asn his ala ile asp trp gln thr gly ser gly cys ser thr gct ggc ttt ttc act gta ttc gca agt gaa ctt tct gtc tac acc ctc 1296 acc gtc atc act cta gaa aga tgg cac acc atc acc tat gct att cac 1344 ctg gac caa aag ctg cga tta aga cat gcc att ctg att atg ctt gga 1392 gga tgg ctc ttt tct tct cta att gct atg ttg ccc ctt gtc ggt gtc 1440 agc aat tac atg aag gtc agt att tgc ttc ccc atg gat gtg gaa acc 1488 ser asn tyr met lys val ser ile cys phe pro met asp val glu thr act ctc tca caa gtc tat ata tta acc atc ctg att ctc aat gtg gtg 1536 gcc ttc ttc ata att tgt gct tgc tac att aaa att tat ttt gca gtt 1584 cga aac cca gaa tta atg gct acc aat aaa gat aca aag att gct aag 1632 arg asn pro glu leu met ala thr asn lys asp thr lys ile ala lys aaa atg gca atc ctc atc ttc acc gat ttc acc tgc atg gca cct atc 1680 tct ttt ttt gcc atc tca gct gcc ttc aaa gta cct ctt atc aca gta 1728 acc aac tct aaa gtt tta ctg gtt ctt ttt tat ccc atc aat tct tgt 1776 thr asn ser lys val leu leu val leu phe tyr pro ile asn ser cys gcc aat cca ttt ctg tat gca ata ttc act aag aca ttc caa aga gat 1824 ala asn pro phe leu tyr ala ile phe thr lys thr phe gln arg asp ttc ttt ctt ttg ctg agc aaa ttt ggc tgc tgt aaa cgt cgg gct gaa 1872 ctt tat aga agg aaa gat ttt tca gct tac acc tcc aac tgc aaa aat 1920 leu tyr arg arg lys asp phe ser ala tyr thr ser asn cys lys asn ggc ttc act gga tca aat aag cct tct caa tcc acc ttg aag ttg tcc 1968 aca ttg cac tgt caa ggt aca gct ctc cta gac aag act cgc tac aca 2016 thr leu his cys gln gly thr ala leu leu asp lys thr arg tyr thr tyr leu pro val lys val ile pro ser gln ala phe arg gly leu asn gln asn thr lys asn leu arg tyr ile glu pro gly ala phe ile asn leu pro gly leu lys tyr leu ser ile cys asn thr gly ile arg lys phe pro asp val thr lys val phe ser ser glu ser asn phe ile leu glu ile cys asp asn leu his ile thr thr ile pro gly asn ala phe gln gly met asn asn glu ser val thr leu lys leu tyr gly asn gly phe glu glu val gln ser his ala phe asn gly thr thr leu thr ser phe arg gly ala thr gly pro lys thr leu asp ile ser ser thr lys val asn leu leu glu ala thr leu thr tyr pro ser his cys cys ala phe arg asn leu pro thr lys glu gln asn phe ser his ser ile ser asp tyr glu tyr gly phe cys leu pro lys thr pro arg cys ala pro glu pro asp ala phe asn pro cys glu asp ile met gly tyr asp phe pro arg phe leu met cys asn leu ser phe ala asp phe cys met gly leu tyr leu leu leu ile ala ser val asp ser gln thr lys gly gln tyr tyr asn his ala ile asp trp gln thr gly ser gly cys ser thr ser asn tyr met lys val ser ile cys phe pro met asp val glu thr arg asn pro glu leu met ala thr asn lys asp thr lys ile ala lys thr asn ser lys val leu leu val leu phe tyr pro ile asn ser cys ala asn pro phe leu tyr ala ile phe thr lys thr phe gln arg asp leu tyr arg arg lys asp phe ser ala tyr thr ser asn cys lys asn thr leu his cys gln gly thr ala leu leu asp lys thr arg tyr thr