Fsh
BACKGROUND OF THE INVENTION 
     This application is a continuation-in-part of copending application Ser. No. 548,228 entitled &#34;Heteropolymeric Protein&#34; filed Nov. 2, 1983, now pending. 
    
    
     This invention relates to the use of recombinant DNA techniques to produce heteropolymeric proteins. 
     Various polypeptide chains have been expressed, via recombinant DNA technology, in host cells such as bacteria, yeast, and cultured mammalian cells. Fiddes, J. C. and Goodman, H. M. (1979) Nature Vol. 281, pg. 351-356 and Fiddes, J. C. and Goodman, H. M. (1980) Nature Vol. 286, pg. 684-687 describe the cloning of, respectively, the alpha and beta subunits of human choriogonadotropin (hCG). 
     Kaname U.S. Pat. No. 4,383,036 describes a process for producing hCG in which human lymphoblastoid cells are implanted into a laboratory animal, harvested from the animal, and cultured in vitro; accumulated hCG is then harvested from the culture. 
     SUMMARY OF THE INVENTION 
     In general the invention features the biologically active heterodimeric human fertility hormone follicle stimulating hormone (&#34;FSH&#34;) which includes an alpha subunit and a beta subunit, each subunit being synthesized by a cell having an expression vector containing heterologuous DNA encoding the subunit. 
     The term &#34;expression vector&#34; refers to a cloning vector which includes heterologous (to the vector) DNA under the control of sequences which permit expression in a host cell. Such vectors include replicating viruses, plasmids, and phages. Preferred vectors are those containing at least the 69% transforming region, and most preferably all, of the bovine papilloma virus genome. 
     The invention permits the production of biologically active heterodimeric FSH from a single culture of transformed cells. The production of both subunits of FSH in the same cell eliminates the necessity of recombining subunits from separate cultures to assemble an active heterodimeric molecule. The system also allows production of FSH, in a single culture, which undergoes, in the culture, post-translational modification, e.g. glycosylation and proteolytic processing, for activity or stability. 
     In preferred embodiments, each expression vector is autonomously replicating, i.e., not integrated into the chromosome of the host cell. The use of autonomously replicating expression vectors prevents undesirable influence of the desired coding regions by control sequences in the host chromosome. 
     Other advantages and features of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     We turn now to the preferred embodiments of the invention, first briefly describing the drawings thereof. 
    
    
     DRAWINGS 
     FIG. 1 is a diagrammatic illustration of the construction of the plasmid pRF375. 
     FIG. 2 is a partial restriction map of the lambda clone 15B and the beta FSH-containing 6.8 kb EcoRI-BamHI fragment that is inserted into pBR322. 
     FIG. 3 is a partial restriction map of the beta FSH coding region and the BamHI fragment that is inserted into a BPV based expression vector. 
     FIG. 4 is a diagrammatic illustration of the construction of the BPV-containing plasmid CL28FSH2.8BPV, encoding the beta subunit of FSH. 
    
    
     STRUCTURE 
     The cloning vectors of the invention have the general structure recited in the Summary of the Invention, above. Preferred vectors have the structures shown in the Figures, and are described in more detail below. 
     CONSTRUCTION OF CLONING VECTORS 
     Isolation of cDNA Clones Encoding the Common Alpha Subunit 
     In order to produce the heterodimeric FSH of the invention, the alpha subunit of human chorionic gonadotropin (hCG) first is isolated; the alpha subunit is common to the fertility hormones hCG, luteinizing hormone (LH), and FSH. 
     All of the techniques used herein are described in detail in Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory), hereby incorporated by reference. 
     RNA is extracted from placental tissue by the following method. Homogenization of the tissue is carried out in a 1:1 mixture of phenol:100 mM Na-acetate (pH 5.5) containing 1 mM EDTA, that has been warmed to 60° C. for 20 min. After cooling on ice for 10 min., the phases are separated by centrifugation. The hot phenol extraction is repeated twice more followed by two extractions with chloroform. 
     RNA is precipitated from the final aqueous phase by the addition of 2.5 volumes of ethanol. 
     In order to enrich for poly A+mRNA, placental RNA is passed over oligo (dT)-cellulose in 0.5M NaCl buffered with 10 mM Tris-HCl, pH 7.5, and washed with the same solution. Poly A+mRNA is eluted with 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05% SDS and precipitated twice with ethanol. Typical initial yields are 1.5-2.0 mg of total RNA per g of tissue, of which about 2% is poly A+mRNA. 
     Placental cDNA libraries are constructed by reverse transcription of placental mRNA, second strand synthesis using E. coli DNA polymerase I (large fragment), treatment with SI nuclease, and homopolymer tailing (dC) with terminal deoxynucleotidyl transferase; all such procedures are by conventional techniques. 
     In a typical preparation, 20-30% conversion of mRNA to single strand (ss) cDNA; 70% resistance to digestion with nuclease S1 after second strand synthesis; and dC &#34;tails&#34; of ten to twenty-five bases in length, are obtained. These cDNA molecules are then annealed to DNA fragments of the plasmid pBR 322, which has been digested with PstI, and to which dG &#34;tails&#34; have been added. These recombinant plasmids are then used to transform E. coli cells to generate a cDNA library (transformed cells are selected on the basis of tetracycline resistance). 
     In order to identify the human alpha hCG clone, a 219 bp fragment of a mouse alpha thyroid stimulating hormone (TSH) clone is used as a hybridization probe. This probe has 77% sequence homology with the human clone. It is radioactively labeled by nick translation and hybridized to the cDNA library under conditions that take into account the extent of homology. Strongly hybridizing clones are analyzed by restriction mapping and clones containing the complete coding sequence of alpha hCG are verified by DNA sequencing. 
     CONSTRUCTION OF PLASMID pRF375 
     Referring to FIG. 1, the plasmid CL28 (identical to plasmid JYMMT(E); Hamer et al. (1983) J. Mol. Applied Gen. 1, 273-288), containing the murine metallothionein promoter, SV40 DNA, and pBR322 sequences, is cut with the restriction endonuclease BglII. At this site is inserted the cDNA clone of alpha hCG, containing untranslated regions of about 10 and 220 b at its 5&#39; and 3&#39; ends, respectively. This clone has been genetically engineered by the addition of synthetic BamHI linkers at its termini. 
     The resulting plasmid pRF302 is digested with restriction enzymes BamHI and SalI to release the SV40 DNA sequence. 
     Plasmid pB2-2, which contains the entire BPV genome, and some pBR322 sequences, is digested with BamHI and SalI to yield the BPV genome with BamHI/SalI ends; this fragment is ligated into pRF302 containing the metallothionein-hCG sequences. 
     Following transformation of E. coli, plasmid pRF375 is identified and isolated. It encodes the common alpha subunit under the control of the mouse metallothionein promoter. 
     Isolation of the Human beta FSH Gene 
     A human genomic library in phage lambda (Lawn et al., 1978, Cell 15, p. 1157-1174) is screened using &#34;guessed&#34; long probes. The idea behind such probes, set forth in Jaye et al. (1983) Nucleic Acids Research 11(8), 2325, is that if the amino acid sequence of a desired protein is at least partially known, a long probe can be constructed in which educated guesses are made as to the triplet encoding any amino acid which can be encoded by more than one, and not more than four, different triplets. Any correct guesses increase the amount of homology, and improve the specificity, of the results. 
     To isolate desired regions of DNA, two labeled 45-mer probes are used. TB36, homologous with amino acids 56-70 of human beta FSH; and TB21, homologous with amino acids 73-87. These probes have the following nucleotide compositions (corresponding amino acids are also given): 
     
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TB36:        Val--Tyr--Glu--Thr--Val--Lys--Val--                          
(AA56- 3&#39;    CAC ATG CTC TGG CAC TCT CAC                                  
70)                                                                       
             Pro--Gly--Cys--Ala--His--His--Ala--Asp                       
             GGT CCG ACG CGG GTG GTG CGA CTG 5&#39;                           
TB21:        Tyr--Thr--Tyr--Pro--Val--Ala--Thr--                          
(AA73- 3&#39;    ATG TGC ATG GGT CAC CGA TGT                                  
87)                                                                       
             Glu--Cys--His--Cys--Gly--Lys--Cys--Asp                       
             CTC ACA GTG ACG CCG TTT ACG CTG 5&#39;                           
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     The above probes are used to screen the human genomic library as follows. TB21 is labeled with  32  P and used to screen approximately 5×10 5  lambda plaques on duplicate filters by the in situ plaque hybridization technique of Benton and Davis (1977) Science 196, 180-182. The prehybridization solution is maintained at 55° C. for several hours and has the following composition: 0.75M NaCl; 0.15M Tris/HCl, pH 8.0; 10 mM EDTA; 5×Denhardt&#39;s Solution; 0.1% sodium pyrophosphate; 0.1% SDS; 100 microgram/ml E. coli t-RNA. The hybridization solution has the same composition except that it is maintained overnight at 45° C., and contains labeled probe in a concentration of about 0.5×10 6  cpm/ml. After hybridization, the filters are washed four times in 1×SSC (=0.15M NaCl, 0.015M Na 3  -citrate) and exposed to x-ray film. 
     This screening procedure yields 50 plaques which hybridize to TB21 on both sets of filters. These 50 individual plaques are picked and combined into 10 culture pools containing 5 plaques each. The 10 cultures are grown and DNA is isolated from 50 ml phage lysates. This DNA is then digested with EcoRI and fractionated on two identical 1% agarose gels, after which it is transferred to nitrocellulose paper according to the method of Southern (1975) J. Mol. Biol. 98, 503-517. 
     The DNAs on the two filters are hybridized to  32  P labeled TB21 and TB36, respectively. Individual plaques from the pool containing a restriction fragment which strongly hybridizes to both probes are then screened according to the above procedure, except that the DNAs are digested with EcoRI, BamHI, and EcoRI plus BamHI. In this way the 6.8 kb EcoRI-BamHI fragment containing human beta FSH is isolated. 
     A partial restriction map of clone 15B, containing the 6.8 kb EcoRI-BamHI fragment, is shown in FIG. 2. In order to locate the position of the beta FSH sequences within the clone, the 6.8 kb EcoRI-BamHI fragment of clone 15B is subcloned into pBR322 to yield plasmid p15B6.8R/B (FIG. 2). p15B6.8R/B is then digested with various restriction enzymes and the products are fractionated by agarose gel electrophoresis using conventional methods. The DNA is blotted to nitrocellulose paper and hybridized to fragments of a porcine beta FSH cDNA clone labelled with  32  P by nick translation. 
     As shown in FIG. 2, the porcine beta FSH probe hybridizes to only two fragments of the human DNA, namely a 1.1 kb HindIII-KpnI and a 1.4 kb PstI fragment. Partial DNA sequencing of these two fragments shows that this DNA indeed codes for human beta FSH and that the entire coding region for beta FSH is contained in these two fragments. 
     As shown by the restriction map of FIG. 3, the beta FSH coding sequence is interrupted by an intervening sequence of approximately 1.6 kb between amino acids 35 and 36 of mature beta FSH. The nucleotide sequence of the entire human beta FSH coding region and some of the flanking and intervening sequences are given below. The amino acid sequence deduced from the nucleotide sequence is given for the coding region. ##STR1## 
     Still referring to the above sequence, there is a box around the ATG initiation codon of the 18 amino acid signal peptide, which is cleaved post-translationally. The mature protein begins with the amino acid Asn encoded by the circled triplet AAT. The exon-intron boundaries are marked by arrows; they are flanked by the concensus sequence GT for the splice donor and AG for the splice acceptor site. There is a box around the stop codon TAA, the end of the coding region. 
     Below is a reproduction of the above sequence not broken into triplets, showing restriction sites; the ATG beginning and the TAA ending the coding region are boxed and the exon-intron junctions are marked by arrows. ##STR2## 
     Insertion of the Beta FSH DNA into a BPV-Based Expression Vector 
     Referring to FIG. 3, a synthetic BamHI linker is inserted at the DdeI site of p15B6.8R/B, which is located 42 nucleotides 5&#39; of the ATG initiation codon. Referring to FIG. 4, p15B6.8R/B is digested with DdeI and treated with E. coli DNA polymerase (Klenow), after which it is ligated to synthetic BamHI linkers and digested with BamHI. The 295 bp fragment containing the first exon of FSH is isolated and cloned into the BamHI site of pBR322. The resulting plasmid pBR295Bam is digested with KpnI plus EcoRI plus AvaI and ligated to p15B6.8R/B which has been digested with KpnI plus EcoRI plus SmaI. The ligation mix is then used to transform E. coli, and the plasmid pBR2.8Bam containing the human beta FSH DNA sequence as a BamHI fragment is identified from among the transformants by restriction mapping. 
     As shown in FIG. 4, expression plasmid CL28FSH2.8BPV is prepared according to the same method used to prepare pRF375 (FIG. 1), except that the 2.8 kb BamHI fragment of pBR2.8Bam is used in place of the alpha hCG cDNA clone. Plasmid CL28FSH2.8BPV can be used to transform mammalian host cells using conventional methods, and human beta FSH can be isolated and purified. 
     Transfection of Mouse Cells 
     To produce heterodimeric FSH using a mixed transfection, five ug of each BPV plasmid, i.e., pRF375 (alpha subunit) and CL28FSH2.8BPV (beta FSH), are mixed and added to 0.5 ml of a 250 mM CaCl 2  solution containing 10 ug of salmon sperm DNA as carrier. This mixture is bubbled into 0.5 ml of 280 mM NaCl, 50 mM Hepes and 1.5 mM sodium phosphate. The calcium phosphate precipitate is allowed to form for 30-40 minutes at room temperature. 
     24 hours prior to transfection, 5×10 5  cells of mouse C127 cells (available from Dr. Dean Hamer, National Cancer Institute, NIH, Bethesda, MD) are placed in a 100 mm dish or T-75 flask. Immediately before adding the exogenous DNA, the cells are fed with fresh medium (Dulbecco&#39;s Modified Medium, 10% fetal calf serum). One ml of calcium phosphate precipitate is added to each dish (10 ml), and the cells are incubated for 6-8 hours at 37° C. 
     The medium is aspirated and replaced with 5 ml of 20% glycerol in phosphate buffered saline, pH 7.0 (PBS) for 2 minutes at room temperature. The cells are washed with PBS, fed with 10 ml of medium, and incubated at 37° C. After 20-24 hours, the medium is changed and subsequent refeeding of the cells is carried out every 3-4 days. Individual clones are grown in T-25 flasks. After 7-21 days, cell clones can be transferred to larger flasks for analysis. 
     Deposits 
     The following, described above, has been deposited in the Agricultural Research Culture Collection (NRRL), Peoria, IL 61604:  CL28FSH2.8BPV in E. coli, NRRL B-15923 
     The following, described above, has been deposited in the American Type Culture Collection, Rockville, MD: 
     pRF375 in C127 cells, ATCC CRL 8401; 
     Applicants&#39; assignee, Integrated Genetics, Inc., acknowledges its responsibility to replace these cultures should they die before the end of the term of a patent issued hereon, and its responsibility to notify the ATCC and NRRL of the issuance of such a patent, at which time the deposits will be made available to the public. Until that time the deposits will be made available to the Commissioner of Patents under the terms of 37 CFR §1.14 and 35 USC §112. 
     Use 
     The transformed cell lines of the invention are used to produce glycosylated, biologically active heterodimeric human FSH, which is purified from the cells and/or their culture media using conventional purification techniques. FSH has a number of well-known medical uses associated with human fertility. For example, FSH can be used, alone or in conjunction with hCG or LH, to induce ovulation, or superovulation for in vitro fertilization. 
     In addition, heterodimeric FSH, or the beta subunit alone, can be used in diagnostic tests for fertility and pituitary functions. 
     FSH produced by recombinant cells has the advantage, compared to FSH obtained from natural sources, of being free from contamination by other human proteins, in particular other fertility hormones. 
     Other embodiments are within the following claims. For example, rather than producing heterodimeric FSH by culturing cells containing two separate expression vectors, one encoding the alpha subunit and the other encoding the beta subunit, DNA encoding both subunits can be included in the same expression vector.