FR2621324A1 - NUCLEOTIDE SEQUENCE ENCODING B2 TRANSFORMER GROWTH FACTOR PRECURSOR, B2 TRANSFORMANT GROWTH FACTOR PRECURSOR, AND B2 TRANSFORMANT GROWTH FACTOR - Google Patents

Abstract

The present invention essentially relates to a nucleotide sequence encoding the precursor of the transforming growth factor beta2. According to the invention, this nucleotide sequence comprises the coding sequence of nucleotides substantially as represented in FIG. 1a from approximately the residue of nucleotide number 1 to approximately the residue of nucleotide number 1326. The invention finds application in particular in the production in large amounts of transforming growth factor beta2.

Description

The present invention generally relates to cloning and expression of

growth factor

human transforming beta2.

  In this technical field, transforming growth factor beta (TGF-R) is known to be a member of a recently described family of polypeptides that regulate cell differentiation and proliferation. Other members of this family include the Muller inhibitory substance (Cate et al., 1986, Cell 45: 685-698), inhibins (Mason et al., 1985, Nature 318: 659-663) and a predicted protein

  from a transcript of the decapentaploid gene complex

  of Drosophila (Padgett et al, 1987, Nature

325: 81-84)

  Transforming Growth Factor Beta (TGF-B) consists of two identical disulfide bonded subunits with molecular weights of 13,000 (Assoian et al., 1983, J. Biol Chem 258: 7155-7160, Frolik et al. 1983, Proc Natl Acad Sci USA 80: 3676-3680, Frolik et al 1984, J. Biol Chem 260: 10995-11000). It has been purified from several tissue sources including placenta (Frolik et al., 1983, Nature 325: 81-84), platelets (Childs et al., 1982, Proc Natl Acad Sci. USA 79: 5312-5316, Assoian et al., 1983, J. Biol Chem 258: 7155-7160), the kidney (Roberts et al., 1983, Biochemistry 22: 5692-5698), and demineralized bones. (Seyedin et al., 1985, Proc Natl Acad Sci USA 82: 119-123). In the presence of 10% serum and epidermal growth factor, TGF-B promotes growth independent of fibroblast anchorage of normal rat kidneys (Roberts et al., 1981, Proc Natl Acad Sci USA 78: 5339-5343, Roberts et al., 1982, Nature 295: 417-419, Twardzik et al., 1985, J. Cell Biochem 28: 289-297); in the presence of 10% serum alone, it is capable of inducing the formation of AKR-2B fibroblast colonies (Tucker et al 1983, Cancer Res 43: 1518-1586). TGFB has also been shown to cause differentiation of muscle mesenchyme cells

  rat fetus to produce specific macromolecules

  cartilage (Seyedin et al., 1986, J. Biol Chem.

261: 5693-5695).

  In contrast to its effect on the proliferation

  cell ration, TGF-3 purified from human platelets as well as a functionally related protein isolated from African Callikric Cells (BSC-1) was found to inhibit the growth of certain cells in culture ( Tucker et al., 1984, Science 226: 705-707). TGF-B has also been shown to inhibit the growth of several human cancer cell lines (Roberts et al., 1985, Proc Natl Acad Sci USA 82: 119-123). This inhibitory / stimulating effect of TGF-B may depend on several factors including cell type and physiological state of cells (for review see Sporn et al., 1986, Science).

233: 532-534).

  CDNA clones encoding human TGF-B (Derynck et al., 1985, Nature 316: 701-705), murine (Derinck et al., 1986, J. Biol Chem 261: 4377-4379) and simian (Sharples et al., 1987, DNA 6: 239-244) were isolated. Analysis of the DNA sequences of these clones indicates that TGF-B is synthesized as a large precursor polypeptide, the carboxy terminus of which is cleaved to form the mature TGF-B monomer. Strong sequence homology was found in all TGF-B precursor protein from

all sources above.

  Very recently, protein isolated from demineralized bovine bone has been identified as being related to TGF-B (Seyedin et al., 1987, J. Biol Chem 262: 1946-1949). The protein was also isolated from pork platelets (Cheifetz et al., 1987, Cell 48: 409-415), from a cell adenocarcinoma cell line.

  the human prostate, PC-3 (Ikeda et al., 1987, Biochemis-

  try 26: 2406-2410), and a glio cell line.

  human blastoma (Wrann et al., 1987, EMBO 6: 1633-1636). A partial amino acid sequence of this protein indicated that it was homologous to TGF-B and was referred to as TGF-B2. Human TGF-B (Derynck et al., 1985, Nature 316: 701-705), murine (Derynck et al., 1986, J. Biol Chem 261: 4377-4379) and simian (Sharpies et al. 1987, DNA 6: 239-244) previously described has been referred to

TGF-B1.

  The present invention relates to the production of large amounts of TGF-B2 by eukaryotic host cells transfected with recombinant DNA vectors containing a sequence encoding TGF-B2 controlled by expression regulatory elements. In a specific embodiment, cDNA clones encoding the human TGF-B2 precursor were obtained from a cDNA library made from a human prostate adenocarcinoma cell line treated with tamoxifen, PC-3. The cDNA sequence of such a clone predicts that TGF-B2 is synthesized as a 442 amino acid polypeptide precursor which is derived from the mature 112-amino acid TGF-B2 subunit by proteolytic cleavage. This TGF-B2 precursor, called TGF-B2-442, shares a 41% homology with the TGF-B1 precursor. In another embodiment, cDNA clones encoding the simian TGFB2 precursor were obtained from a cDNA library made from an African callikric kidney cell line, BCS-40. The cDNA sequence of such a clone predicts that TGF-B2 is also synthesized as a 414 amino acid polypeptide precursor which is derived from the mature 112-amino acid TGF-B2 subunit by proteolytic cleavage. This TGF-B2 precursor, designated TGF-B2-414, has an amino acid sequence of 414 amino acid residues and is identical to the amino acid sequence of TGF-B2-442 except that it contains a single residue of asparagine instead of the 29 amino acid sequence of residues No. 116 to 135 of

the human TGF-B2-442 sequence.

  Clones from the BSC-40 cDNA library that encode a simian TGF-B2-442 precursor as well as clones from the human PC-3 cDNA library that encode a TGFB2-precursor. 414 human have also been identified. The human and simian TGF-B2-442 precursors appear to be perfectly homologous at the amino acid level, as are the precursors of

TGF-B2-414 human and simian.

  The mature monomers of 112 amino acids of

  TGF-B1 and TGF-B2 show 71% homology.

  Expression vectors containing the TGF-B2 encoding mature sequence joined in phase with the TGF-B1 signal and precursor sequences (US Patent Application Pending and Co-owned No. 189,984) were constructed and used to transfect Chinese hamster ovary cells (CH0 cells). The resulting CHO transfectants produce and secrete

  mature, biologically active TGF-B2.

  The following terms as used here at

  singular or plural have the meanings indicated.

  TGF-B2: a transforming growth factor B2 of human or simian origin comprising the amino acid sequence substantially as shown in Figure la from about amino acid residue No. 331 to about

amino acid residue 442.

  TGF-B2 Precursor: A family of transformant growth factor molecules B2 of human or simian origin comprising an amino acid sequence substantially as shown in Figure la from about amino acid residue No. 1 to to about amino acid residue No.442 in which the amino acid sequence from amino acid residue No. 116 to amino acid residue No. 144 is deleted and replaced

by a single residue of asparagine.

  The term means a TGF-B2 precursor designated TGF-B2-442 or TGF-B2414 of human or simian origin. Precursor of TGF-B1 /

TGF-B2

  hybrid: New precursor molecule

transformed growth factor

  mantle B comprising the amino acid sequence substantially as shown in FIG. 1b from about amino acid residue No. 1 to about

amino acid residue No. 390.

  Precursor of TGF-B1: Growth Factor Precursor and Signal Sequences

transforming B1 simian sensitive-

  as shown in FIG. 1b from about amino acid residue No.1 to about

amino acid residue No. 278.

  The characteristics and advantages of the invention

  will appear better in the detailed description

  will follow and which refers to the accompanying figures given by way of example, in which: - Figure la represents the nucleotide sequence of human TGF-B2-442 cDNA and the deduced amino acid sequence; the PC-21 bp 2597 insertional sequence was subcloned into pEMBL (Dante et al., 1983, Nucleic Acids Res., 11: 1645-1654) and sequenced on both strands using the dideoxy nucleotide termination (Sanger et al., 1977, Proc Natl Acad Sci USA 74: 5463-5467); the coding sequence is shown and the deduced amino acid sequence is presented directly above; the mature TGF-B2 sequence is flanked and the signal peptide is surmounted by a line; potential glycosylation sites are indicated by asterisks; the arrow indicates the presumed site of cleavage of the signal sequence; the nucleotide sequence of the simian TGF-B2-414 cDNA is identical to the human TGF-B2-442 cDNA sequence except that nucleotides 346 to 432 (in square brackets) are deleted and replaced by the AAT sequence, and except that several silent changes of nucleotides occur elsewhere in the structure (this is indicated by isolated letters directly beneath the modified nucleotide); the deduced amino acid sequence for the simian TGF-B2-414 precursor is identical to the human TGF-B2-442 precursor amino acid sequence except that asparagine replaces the amino acid residues 116 to 144 in the structure of human TGF-B2-442; the nucleotide sequence of a human TGF-B2-414 cDNA was sequenced in the dashed underlined region and was found to be fully homologous to the human TGF-B2-442 cDNA sequence except for the nucleotides 346 to 432 are deleted and replaced by the AAT sequence;

  FIG. 1b represents the nucleotide sequence

  TGF-B1 / TGF-B2 precursor DNA precursor DNA hybrid and deduced amino acid sequence; the coding sequence is shown and the deduced amino acid sequence is presented directly above; the mature TGF-B2 sequence is flanked and the precursor signal peptide is surmounted by a line. The glycosylation sites are indicated by asterisks; the arrow indicates the presumed site of cleavage of the signal sequence. The mature TGF-B2 coding sequence described is of human origin. The simian TGF-B2 mature coding sequence is approximately identical to the human sequence: only three base silencing changes take place and are indicated by single letters directly beneath the modified nucleotide; the details of the cDNA cloning of TGF-B2 and the construction of the TGF-B1 / TGF-B2 hybrid gene are given in the text; FIG. 1c is a schematic diagram of the hybrid TGF-B1 / TGF-B2 precursor gene; Figure 1d shows the sites of the restriction endonuclease of pPC-14 (2.2 kb) and pPC-21 (2.3 kb), the boxed regions indicate the coding sequences for the TGF-B2 monomer; ATG represents the initiation methionine codon; the

262 1,324

  distance between ATG and the KpnI site in pPC-21 (2.34 kb) is approximately 420 bp; the blackened area indicates the position of the 84 bp insertion in pPC-21 (2.3 kb); FIG. 1a shows a partial analysis of DNA sequences of pPC-14 (2.2 kb); a synthetic oligonucleotide 5'-AGGAGCGACGAAGAGTACTA-3 'which hybridized approximately 140 bp upstream of the KpnI site within the insertion sequence in pPC-21 (2.3 kb) was used to prime the DNA sequencing; in this region, the pPC-14 (2.2 kb) sequence (upper line) is identical to pPC-21 (2.3 kb) to the nucleotides encoding Asn-116; the insertion of 84 bp within the Asn-116 codon of pPC-14 (2.2 kb) that was found in pPC-21 (2.3 kb) is shown; the KpnI site within the -sertion sequence is shown; FIG. 2 represents homologies of TGF-B1 and human TGF-B2-442 precursor sequences;

  a) primary homology of sequences: the residues identified

  are framed; asterisks refer to potential glycosylation sites in TGF-B2; the potential cleavage site of the signal sequence and the site of

  cleavage of the mature polypeptide are indicated; b) comr = -

  because of dot matrices using the GO.e Pro software; each point locates a point where 5 amino acids are identical among 10 amino acids; the diagonal lines indicate the regions of homology; FIG. 3 represents a Northern blot analysis of the polyadenylated RNA of BSC-40 and PC-3; polyadenylated RNA was isolated from

  BSC-40 and PC-3, fractionated on an agarose gel-

  formaldehyde, transferred to Hybond-N filters, and hybridized with a [32p] -labeled TGF-B2 specific probe, with pPC-21 (Part A) or a mixture of [TG] -labeled TGF-B1 and TGF-B2 specific probes [ 32P] (Part B) (Sharples et al., 1987) as described in the Materials and Methods section; lane 1, polyadenylated BSC-40 RNA (5 micrograms); band 2, polyadenylated PC-3 RNA (5 micrograms); FIG. 4 represents a Northern blot analysis of polyadenylated RNA from different sources; polyadenylated RNA was isolated from MCF-7 cells (human breast carcinoma), SK-MEL 28 (human melanoma), KB (nasopharyngeal carcinoma) and HBL-100 (human mammary epithelium) and analyzed by Northern blot hybridization with a TGF-B2 specific probe (pPC-21) as described in the materials and methods part; each band contains 5 micrograms of polyadenylated RNA from cells of SK-MEL 28 (lane 1), MCF-7 (lane 2), HBL-100 (lane 3) or KB (lane 4); FIG. 5 corresponds to a recombinant TGF-B2 bioactivity test; 1B9 clone 36 Chinese hamster ovary cells bearing pSV / beta 1-beta 2 / dhfr whose cell line was deposited with the ATCC (American Type Culture Collection) under access number CRL 9800, were cultured to confluence in 100 mm tissue culture trays; the cells were washed three times with serum-free medium and incubated for 24 hours in 5 ml of serum-free medium; the medium was collected, dialyzed against 0.2M acetic acid, and tested for inhibition of DNA synthesis in CCL64 cells as described (Gentry et al., 1987, Mol Cell Biol. 7: 3418). In this test, 3.3 μg of standard natural purified TGF-B1 gave 50% inhibition; the specific activity of natural purified TGF-B2 was calculated: it was about half that of TGF-B1; and FIG. 6 represents a Western blot analysis of recombinant proteins secreted by 1B9 clone 36 Chinese hamster ovary cells bearing pSV / beta 1 -beta 2 / dhfr whose cell line has been deposited with ATCC (FIG. American Type Culture Collection) under accession number CRL 9800; acid-dialysed serum-free conditioned medium from clone 36 cells of IB9 was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot with antiserum prepared against synthetic peptide NH2-YNTINPEASASPC- COOH as described (Gentry et al., 1987,

Mol. Cell. Biol. 7: 3418).

  The present invention relates to the production of a biologically active mature form of TGF-B32 from a coding sequence of the TGF-B precursor gene and its product. The mature biologically active TGF-B2 can be produced by cloning and expressing the entire length of the TGF-B2 precursor nucleotide encoding sequence or its functional equivalent in a host cell which correctly transforms the precursor so that mature TGF-B32 is produced with biological activity that virtually can not

  to be distinguished from that of genuine natural TGF-B2.

  Functional equivalents of the total length of the TGF-B2 precursor nucleotide coding sequence include any DNA sequence which, when expressed within a suitable host cell, is capable of directing synthesis. , transformation and export of mature TGF-B2. In this regard, hybrid precursor coding sequences including, for example, the TGFB1 precursor sequence in reading frame of the mature TGF-B2 sequence, can be constructed and used to produce a biologically active TGF-B2. The process according to the invention can be divided into

  the following steps for the purpose of description

  only: (a) isolation or production of the coding sequence for a precursor form of TGF-1z2; (b) constructing an expression vector that will direct the expression of a TGF-B2 coding sequence; (c) transfection of appropriate host cells that are capable of replicating and expressing the gene and transforming the gene product to produce the biologically active mature form of TGF-B2; (d) identification and purification of mature biologically active TGF-B2. Since the transfectant which expresses high levels of mature bicactive TGF-B2 is identified, the practice of the invention includes the expansion of this clone.

  and isolating the expressed gene product.

  The method of the invention is demonstrated herein by means of examples in which cDNAs of the TGF-B2 precursor coding region have been prepared, cloned, sequenced and used to construct expression vectors that direct expression. high level of TGF-B2 in CHO cells. In a specific embodiment, the complete amino acid sequence of the mature form of human TGF-B2 has been determined and has an overall homology of 71% with TGF-B1. Using synthetic oligonucleotide probes we identified clones from

  a PC 3 cDNA library encoding TGF-B2.

  DNA sequence analysis of one of these clones revealed that TGF-B2, like TGF-B1, is synthesized as a larger precursor protein, the carboxy is cleaved to produce the mature TGF-B2 monomer. While there is 71% homology between TGF-B1 and TGF-B2 across mature portions of these molecules, there is only a maximum of 31% homology in the rest of the precursor , suggesting that the amine end regions of TGF-B1 and TGF-B2 may be

  distinct from the functional point of view.

  In a specific embodiment of the invention, expression of a novel TGF-B1 / TGF-B2 hybrid gene in CHO cells is used to produce large amounts of biologically active TGF-B2. The different aspects of the process of the invention are described in more detail in the subsections

  following examples and in the following examples.

  The nucleotide coding sequence for TGF-B2 is shown in Figure la. In the practice of the method of the invention, the nucleotide sequence shown herein, or fragments or functional equivalents thereof, can be used to produce the recombinant molecules that will direct the expression of TGF-B2 product in appropriate host cells. In a specific embodiment, a TGF-B1 / TGF-B2 hybrid gene (Figure 1b) was constructed and used to transfect CHO cells. Transfectants producing as much as 500 μg of biologically active mature TGF-B2 per ml

  of culture medium have been isolated.

  Due to the degeneracy of the nucleotide coding sequences, other DNA sequences that substantially encode the same amino acid sequences as those shown in Figure 1a and Figure 1b can be used in the practice of present invention for the cloning and expression of TGF-B2. Such modifications include deletions, additions, or substitutions of different nucleotide residues resulting in a sequence that encodes the same gene product or a functionally equivalent gene product. The gene product may contain deletions, additions, or substitutions of amino acid residues in the sequence, resulting in a silent change thereby producing a bioactive product. Such amino acid substitutions may be made on the basis of similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with groups of

  unloaded polar heads or non-head groups

  Polar compounds with similar hydrophilicity values include: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine,

  threonine; phenylalanine, tyrosine.

  the nucleotide coding sequence for TGF-B2 can be obtained from cell sources that produce TGF-B2-like activity. The coding sequence can be obtained by cloning cDNA of RNA isolated and purified from such cell sources or by genomic cloning. The cDNA or genomic libraries of clones can be prepared from DNA fragments produced using methods well known in the art including but not limited to the use of restriction enzymes. Fragments that encode TGF-B2 can be identified by screening such libraries with a nucleotide probe that is substantially complementary to any portion of the sequence shown in Figure la. Total length clones, i.e. those containing the total coding region for the TGF-B2 precursor, may be

chosen for the purpose of expression.

  In another embodiment of the invention, the coding sequence of Figure la may be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res.

  Symp. Ser. 7: 215-223; Crea and Horn, 1980, Nuc Acids.

  Res. 9 (10): 2331; Matteucci and Carruthers, 1980,

  Tetrahedron Letters 21: 719 and Chow and Kempe, 1981, Nuc.

  Acids. Res. 9 (12): 2807-2917. Alternatively, the protein could be produced using chemical methods to synthesize the amino acid sequence shown in Figure la in whole or in part. For example, peptides can be synthesized by solid phase methods on a Beckman 990 instrument, and cleaved from the resin as previously described (Gentry, LE, et al, 1983, J. Biol Chem 258: 11219- 11228;

  Gentry, L.E. and Lawton, A., 1986, Virology 152: 421-431).

  The purification can be carried out by high performance preparative liquid chromatography. Peptide composition was confirmed by amino acid analysis. In a specific embodiment, described in the examples herein, the TGF-M2 coding sequence was obtained by cDNA cloning of human TGF-B2 precursor coding sequences derived from polyadenylated RNA isolated from a human prostate adenocarcinoma cell line treated with tamoxifen, PC3, which has previously been shown to produce TGF-B2. The entire coding region of a cDNA clone was sequenced and compared to the published sequence

human TGF-B1 (see Figure 2).

  DNA sequence analysis of TGF-B2 cDNA clones indicates that TGF-B2 as well as TGF-B1 is synthesized as a large precursor protein, the carboxy terminus of which is cleaved to produce the TGF-B2 mature monomer of 112 amino acids. TGF-B2 has been shown to have a molecular weight of 24,000 composed of two disulfide bonded 13,000 dalton subunits (Ikeda et al., 1987, Biochemistry).

  26: 2406-2410; Cheifetz et al., 1987, Cell 48: 409-415).

  Thus, the production of TGF-32 requires appropriate proteolytic cleavage as well as the formation of intra- and intermolecular disulfide bridges. A hydrophobic leader sequence at the amine end (residue 3-19) is present in the precursor and may be responsible for directing the protein out of the cell. The mature TGF-B2 can still be associated with the remaining part of the

  precursor during this process.

  TGF-B2 exhibits 71% homology with TGF-B1 throughout the mature portion of the precursor, implying a functional similarity that is supported by experimental evidence (Seyedin et al., 1987, J. Biol Chem 262: 1946-1949, Cheifetz et al., 1987, Cell 48: 409-415). The amine portion of the TGF-B1 precursor region from human sources, rodents, or simian sources (Derynck et al., 1985, Nature 316: 701705, Derynck et al., 1986, J. Biol Chem 261: 4377-4379, Sharples et al., 1987, DNA 6: 239-244) is highly conserved and suggests that this part of the

  molecule can have an important biological function.

  In contrast, there is no more than 31% homology between the N-terminal precursor regions of TGF-B1 and TGF-B2. After cleavage of the supposed signal peptide, the TGF-B2 precursor would also contain more amino acids than the TGF-B1 precursor. Primary structural differences within the amine end region of TGF-B1 / TGF-B2 precursor proteins may reflect functional differences. However, significantly homologous regions within the precursors are found in isolated blocks suggesting the retention of important functional domains even within

  the N-terminal precursor region.

  Northern blot analysis revealed two large size classes of 4.1 and 6.5 kb TGF-B2 specific mRNA in BSC-40 cells. Tamoxifen-treated PC-3 cells contain three 4.1 kb, 5.1 kb and 6.5 kb TGF-B2 transcribed sequences. These messages of different sizes could be the result of differential RNA splicing, polyadenylation, or both as has been described for

  other genes (Helfman et al., 1986, Mol Cell Biol.

  6: 3582-3595; Sayre et al., 1987, Proc. Natl. Acad. Sci. USA 84: 2941-2945). Preliminary analysis of another TGF-B2 cDNA clone shows that it contains a 3 'untranslated region about 1 kb larger than pPC-21 and pPC-14 and contains a different polyadenylation site suggesting that another polyadenylation is a factor responsible for the production of multiple TGF-B2 mRNA observed on

Northern blots.

  BSC-40 cells contain comparable levels of transcribed sequences specific for TGF-B1 and TGF-B2: Tamoxifen-treated PC-3 cells contain more TGF-B1 mRNA than TGF-B2 (Figure 3B). This latter result is unexpected since these cells produce more TGF-B2 protein than TGF-B1 (Ikeda et al., 1987, Biochemistry 26: 2406-2410) and suggest a level of regulatory post-transcription concerning the synthesis of this modulator. growth. Experiments to understand transcription and translation control mechanisms leading to the production of TGF-B1 and TGF-B2 are underway. The production of adequate amounts of TGF-B2 by recombinant DNA techniques, as has been done for TGF-B1, should further assist in designing experiments to explore the different effects of

this protein.

  In another embodiment, the TGF-B2 coding sequence was obtained by cDNA cloning of simian TGFB2 precursor coding sequences derived from polyadenylated RNA isolated from an African callikric cell line, BSC- 40. The total coding region of a cDNA clone was sequenced. Human and simian TGF-B2 precursors appear to have identical amino acid sequences, and their sequences

  nucleotides are about the same.

  In order to express a biologically active mature form of TGF-B2 an expression / hte vector system should be chosen which provides not only for high levels of transcription and translation but also for the correct elaboration of the produced gene. This is particularly important when using the entire coding sequence of a TGF-B2 precursor in the expression vectors because the mature form of TGF-B2 appears to be derived from the precursor product via cellular transformation phenomena. . In addition, a system expression / cell h8te

  which provides for the secretion of the product may be chosen.

  In particular, it appears that the mature TGF-B2, a disulfide bonded homodimer of 112 amino acids per subunit can be formed by cell transformation involving proteolytic cleavage between the amino acids Arg-Ala of the

  precursor (residues numbers 330 and 331 in Figure la).

  In addition, the TGF-B2 precursor contains three potential sites of Nglycosylation not found in the mature form. Proper glycosylation of the precursor may be important for cell synthesis and release or secretion of the mature molecule. In addition, the mature form of TGF-B2 comprises a disulfide-linked dimer involving 9 residues of cysteine per subunit. Some of these are involved in interchain disulfide bridges and others in intracellular disulfide bridges that modify the tertiary structure and configuration of the mature molecule and thus its biological activity. Thus, the ability of a host cell used in the expression system to express and correctly transform the TGF-B32 gene product is important for the

  production of mature biologically active TGF-B2.

  A variety of animal host systems / expression vectors (i.e. vectors that contain the elements necessary to direct the replication, transcription and translation of the TGF-B2 coding sequence into a suitable host cell ) can be used both by the skilled person. These include, but are not limited to, mammalian virus / host cell expression vector systems (e.g., cytomegalovirus, vaccinia virus, adenovirus, and the like); insect virus / insect cell expression vector systems (e.g., baculovirus); or non-viral promoter expression systems derived from mammalian cell genomes (e.g.

mouse metallothionine).

  The expression elements of these vectors vary in strength and specificity. Depending on the host / vector system used, any of a number of suitable transcription and translation elements may be used. For example, when cloning into mammalian cell systems, promoters isolated from the mammalian cell genome, (for example the mouse metallothionin promoter) or from viruses that grow in these cells, (e.g. the 7.5K promoter of vaccinia virus) can be used. Promoters produced by recombinant DNA techniques or by synthetic techniques can also be used to provide the

  transcription of inserted sequences.

  Specific initiation signals are also needed for sufficient translation of the inserted protein coding sequences. These signals include the initiation ATG codon and the adjacent sequences. In cases where the entire TGF-B2 gene including its own initiation codon and the adjacent sequences is inserted into the appropriate expression vectors, translation control signals

  additional information may not be necessary.

  However, in cases where only a portion of the coding sequence is inserted, exogenous translation control signals, including the ATG initiation codon, must be provided. In addition, the initiation codon must be in phase with the reading frame of the TGF-B2 coding sequences to ensure translation of the entire insertion sequence. These exogenous translation control signals and initiation codons can

  be of varied, natural or synthetic origin.

  The efficiency of the expression can be improved by inclusion of transcriptional attenuation sequences, enhancement elements, etc. Any of the methods previously described for insertion of DNA fragments into a vector can be used to construct expression vectors containing the TGF-B gene and appropriate transcription / translation control signals. These methods may include recombinant DNA techniques in vitro, synthetic techniques and

  in vivo recombination (genetic recombination).

  In cases where an adenovirus is used as an expression vector, the TGF-B2 coding sequence may be linked to an adenovirus transcription / translation control complex, for example the last promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the genome

  adenovirus by recombination in vitro or in vivo.

  Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) will result in the formation of a recombinant virus that is viable and capable of expressing TGF-B2 in affected hosts. Similarly, the vaccinia 7.5K promoter can

to be used.

  Another expression system that could be

  used to express TGF-B2 is an insect system.

  In such a system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The vire develops in the cells of Spodoptera frugiperda. The TGF-B2 coding sequence may be cloned into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g. polyhedrin promoter). Successful insertion of the TGF-B2 coding sequence will result in inactivation of the polyhedrin gene and production of recombinant viruses r occluded (i.e., virus lacking the protein coating for which the gene of polyhedrin). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the

inserted gene is expressed.

  In addition, one may select a host cell strain that modulates the expression of the inserted sequences, or that modifies and transforms the gene product c; the specific way desired. The expression of certain promoters can be increased in the presence of certain inducers, (for example zinc and cadmium ions for metallothionine promoters). Thus, the expression of TG - B2 obtained by genetic engineering can be controlled. This is important if the cloned foreign gene protein product is lethal to host cells. In addition, modifications (e.g., glycosylation) and transformations (e.g., cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for protein transformation and post-translational modification. Suitable cell lines or host systems can be chosen to ensure correct modification and transformation of the

foreign protein expressed.

  Host cells that contain the recombinant TGF-B2 coding sequence and express the biologically active mature product can be identified by at least four general approaches: (a)

  DNA-DNA hybridization; (b) presence or absence of -

  "marker" gene functions; (c) estimating the level of transcription as measured by the expression of TGF-B2 mRNA transcription sequences in the hte cell and (d) detecting the mature gene product as measured by immunoassay and ultimately by his

biological activity.

  In the first approach, the presence of the TGF-B2 coding sequence inserted into the expression vector can be detected by DNA-DNA hybridization using probes comprising the nucleotide sequences that are homologous to the coding sequence of TGF-B2 substantially as shown in Figure la, or parts thereof

or derivatives thereof.

  In the second approach, the recombinant expression vector system / hte can be identified and selected on the basis of the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, antibiotic resistance , resistance to methotrexate, phenotype of transformation, formation of an occlusion body in baculovirus, etc.). For example, if the TGF-B2 coding sequence is inserted into a vector marker gene sequence, the recombinants containing the TGF-B2 coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene may be placed in tandem with the TGF-B2 sequence under the control of the same or a different promoter used to control expression of the TGF-B2 coding sequence. Marker expression in response to induction or selection

  indicates the expression of the TGF-B2 coding sequence.

  In the third approach, transcriptional activity for the TGF-B2 coding region can be assayed by hybridization assays. For example, the polyadenylated RNA can be isolated and analyzed by Northern blot using a probe homologous to the sequence of

  encoding of TGF-B2 or particular parts thereof.

  Alternatively, the total nucleic acids of the host cell can be extracted and tested for

  relates to hybridization with such probes.

  In the fourth approach, expression of the mature protein produced can be immunologically assessed, for example by Western blots, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays and the like. However, the ultimate test for the success of the expression system includes the detection of the biologically active TGF-B2 gene product. When the cell secretes the gene product, the cell-free medium obtained from the cultured transfectant host cell can be tested for TGF-B2 activity. When the gene product is not secreted, cell lysates can be tested for such activity. In each case, bioassays such as the growth inhibition assay described herein or stimulation of growth independent of anchoring in target cells (Twardzik and Sherwin, 1985, J. Cell Biochem 28: 289-297, Delarco and Todaro, 1978, Proc Natl Acad Sci USA

  4001-4005) or the like may be used.

  Since a clone producing high levels of mature biologically active TGF-B2 is identified, the clone can be expanded and TGF-B2 can be purified using methods well known in the art. Such methods include immunoaffinity purification, chromatographic methods including high performance liquid chromatography, and

like.

  The following examples describe cDNA cloning of TGF-B2 precursor coding sequences from the human prostate adenocarcinoma cell line, PC-3, from which TGF-B2 has been

isolated previously.

  The following procedures, illustrating the materials and methods, were used to clone

  CDNAs encoding the human TGF-B2 precursor.

  The human prostate adenocarcinoma cell line, PC-3, was cultured in tamoxifen supplemented medium as described (Ikeda et al., 1987, Biochemistry 26: 2406-2410). MCF-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 6 units / ml insulin. All other cell lines were grown in the same medium without insulin. The polyadenylated RNA was isolated by oligo [dT] -cellulose chromatography as described (Purchio and Fareed,

1979, J. Virol. 29: 763-769).

  Double-stranded cDNA was synthesized from polyadenylated RNA isolated from tamoxifen-treated PC-3 cells for 24 hours as described (Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). Fractions of cDNA greater than 1000 base pairs were cloned into lambda gt10 as described (Webb et al., 1987, DNA 6: 71-79). The library was first screened in duplicate with a 132-P-labeled degenerate 24-fold probe complementary to amino acids WKWIHEP encoding DNA (probe 1) that are conserved between TGF-B1 and TGF-B2: [5] 'GGTTCGTGTATCCATTTCCA-3'i

C A G C

AT

  Positive clones were then screened with a second 128-fold degenerate probe complementary to CFRNVQD amino acids encoding DNA (probe 2); five of these seven amino acids are specific for TGFB2:

[5'TCTTGAACGTTTCTGAAGCA-3 ']

C C A C A A

  GT Hybridization was performed at 42C in 6X SSC, 5X Denhart's solution, 0.15mM pyrophosphate, in 100 micrograms / ml denatured calf thymus DNA, in 100 micrograms / ml d Yeast tRNA and EDTA lmM (Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). The filters were washed at 42 ° C in 2XSSC, 0.1% NaDodSO4, four times for minutes. Several cDNA clones that hybridized to both probes and were subcloned into pEMBL were isolated (Dante et al., 1983, Nucleic Acids Res., 11: 1645-1654). A clone (pPC-21) containing a 2.6 kb insertion sequence was sequenced on both strands by the dideoxy nucleotide termination reaction (Sanger et al., 1977, Proc Natl Acad Sci USA 74 5463-5467) using different restriction and deletion fragments of exonuclease III combined with specific oligonucleotide priming (Henikoff, 1984, Gene 28: 351-359). Another clone (pPC-14) containing a 2.2kb insertion sequence was partially sequenced. Dot matrix analysis was performed on an IBM PCAT using the Gene Pro software from

  Riverside Scientific Enterprises (Seattle, WA).

  The polyadenylated RNA was fractionated on a 1% agaroseformaldehyde gel (Lehrach et al., 1977, Biochemistry 16: 4743-4751), transferred to a nylon membrane (Hybond, Amrersham), and hybridized with a [2H-labeled probe]. 32P]. Hybridization was carried out at 42 ° C in 50% formamide containing 0.9 M NaCl, 50 mM sodium phosphate (pH 7), 5 mM EDTA, 0.1% NaDodSO4, 4X bromine solution. Denhardt, 0.4 mg / ml tRNA from

  yeast, and 0.25 mg / ml of denatured calf thymus DNA.

  The filters were washed at 65 ° C in 0.25X SSC, 0.1% NaDodSO4, dried and exposed to Cronex-4 X-ray film (DuPont) using intensifying screens.

Lightening Plus (DuPont).

  A cDNA library was constructed using polyadenylated RNA isolated from tamoxifen-treated PC-3 cells. Previous observations indicated that tamoxifen treatment increased the secretion of TGF-B2 by two to five times (Ikeda et al., 1987, Biochemistry 26-2406-2410). The library was screened with probes 1 and 2 as described above. Five clones hybridized to both probes were obtained. A clone, pPC-21, containing a 2.6kb insertion sequence, was chosen for sequencing. Another clone, pPC-14 containing a 2.2kb insertion sequence, was partially sequenced. The DNA and deduced amino acid sequences are shown in

Figure 1.

  pPC-21 contains a single open reading frame encoding a polypeptide derived from 442 amino acids; the 112 amino acids of the carboxy terminus comprise the mature TGF-B2 monomer (box in Figure la). The first methionine encoded by the open reading frame is immediately followed by an expanse of hydrophobic and uncharged amino acids (surmounted by a line in Figure la) characteristic of a signal peptide. Neither the nucleotide sequence encoding this methionine nor those encoding the following two methionines present in the open reading frame are homologous to the consensus sequence for the initiating methionine sequence (kozak, 1986, Cell 44: 283-292 ). Since translation usually begins with the first methionine in an open reading frame and because homologous regions of TGF-B1, as discussed below, occur upstream of the second methionine, attempts have been made to attribute the first methionine the site of the beginning of the translation. It appears then that TGF-B2, as well as TGF-B1, is expressed as part of a secreted precursor of much larger size. The clone of pPC-21 contains 467 bp upstream of the supposed priming methionine and a 3 'untranslated region of approximately 800 bp including a poly [A] terminus, fifteen bases upstream of which is located a signal sequence of polyadenylation (Proudfoot and Brownlee, 1976, Nature

263: 211-214).

  The homology of nucleotide sequences within the coding regions of the pPC-21 cDNA clone

  TGF-ElB1 and TGF-B2 were determined to be 53%.

  The mature protein coding regions have 57% homology while the upstream precursor regions have 48% homology. After optimal alignment of the two sequences, several nucleotide insertions were noted in the TGF-B2 precursor region, one of them extending over 75 nucleotides. It is not known if these insertions are due to the presence of additional exons in TGF-B2. No significant homology was detected between the DNA sequences in the non-cpdant regions of the two clones. In fact, while TGF-BEI had extensive non-coding regions rich in G-C, TGF-B2 had extensive non-coding regions rich in AT. Both cDNA clones contain repetitive structural units in the 3 'noncoding region, repeating units in TGF-B1 consisting of CCCC (purine) (Sharples et al., 1987, DNA 6: 239-244) and in TGF-B2 in ATG or

A (pyrimidine) (purine).

  The location of the restriction sites of many clones revealed that a clone, pPC-14, lacked a KpnI restriction site located in the amine portion of the TGF-22 coding sequence. The restriction sites of pPC-14 and pPC-21 are shown in Figure 1d. pPC-14 was sequenced over an extent of about 100 nucleotides corresponding to this region of the molecule by specific priming with a 20-mer oligonucleotide complementary to nucleotides 277-296 in Figure la. The results show that the clone pPC-14 contains a deletion of 87 nucleotides (nucleotide positions 346 to 432 in Figure la, see also Figure 1a) which explains the missing KpnI site and which is replaced by the AAT sequence, the codon for asparagine. The results suggest that clone pPC-14 encodes a shorter TGF-B2 precursor of 414 amino acids different from the sequence for which pPC-21 code only in that amino acid residues 116 to 144 are deleted and replaced by a single residue of asparagine. Although the entire coding region of pPC-14 has not been determined, it is probably in perfect agreement with the coding sequence of pPC-21 since, with the exception of the KpnI site, the restriction sites of both clones cover perfectly (Figure ld). In addition, a simian clone encoding a 414 amino acid TGF-B precursor containing the same 29 amino acid deletion and replacement was identified as described in Example Section 7 below. This simian clone has a coding sequence which is approximately identical to that of the human pPC-21 clone in the 'and 3' regions relative to the deletion. Figure 2A shows the deduced protein sequence of human TGF-B1 (Derynck et al., 1985, Nature 316: 701-705) compared to that of human TGF-B2-442. TGF-B2 was determined to be 71% homologous to human TGF-β1 throughout the mature portion of the molecule as

  previously described (Marquardt et al., 1987, J. Biol.

  Chem., In press). The amine portion of the precursor upstream of the mature molecule shows a 31% homology between TGF-B1 and TGF-B2-442. The dot matrix homology comparison shown in Figure 2B reveals that significant homology exists in several specific areas of the proteins. Comparison of the N-terminal amino acid sequences in the supposed signal peptide region does not reveal significant homology.

262 1,324

  In TGF-B2, the signal sequence cleavage site is predicted to be located after the amino acid (serine) and after amino acid 29 (glycine) in TGF-B1 (Von Heijne, 1983, Eur J Biochem. 133: 17-21). This cleavage site directly precedes the first block of homology between TGF-B1 and TGF-B2 which extends over 34 amino acids downstream. After elimination of the signal sequences, TGF-B1 and TGF-B2 precursors would share identical N terminations on the first four

  amino acids, including cysteine at position 4.

  Fourteen amino acids downstream of this supposed N-terminus, 19 amino acids out of the following 21 amino acids are conserved between TGF-B1 and TGF-B2, a block of greater homology than all those already seen even in the C-terminal region containing the mature TGF-B protein. Several other blocks of strong homology exist within the region upstream of the

  mature protein as shown in Figures 2A and 2B.

  These areas are separated by long stretches

non-homologous amino acids.

  The precursor of TGF-B2 has three potential sites of Nglycosylation (located at residues 72, 168 and 269 in Figure la). Only the first site is conserved in TGF-B1, and is located within a larger block of conserved residues, suggesting that this potential glycosylation site has important structural and / or functional characteristics. After removal of the signal sequence, the TGF-B2 precursor would contain 31 or 59 amino acids more than its TGF-B1 counterpart. An additional cysteine residue in TGF-B2 is located just upstream of a large non-homologous amino acid region that precedes the mature sequence. As with TGF-B1, the mature TGF-B2 protein cleavage site is just after a region of 4-5 basic amino acids as shown in Figure 2A. The mature region contains nine cysteines. Preservation of 7 of the 9 cysteines is characteristic of the different members of the TGF-B family. Hydropathy analyzes of TGF-B1 and TGF-B2 reveal similar patterns in the precursor and in the mature regions, the two proteins being generally hydrophilic in nature

(data not shown).

  Figure 3A shows Northern blot analysis using pPC-21 to test for polyadenylated RNA from BSC-40 (an African callikric kidney cell line) and tamoxifen-treated pPC-3 cells. PC-3 cells contain three major species of TGF-B2-specific mRNA with a size of 4.1, 5.1 and 6.5 kb (Fig. 3A, lane 2); BSC-40 cells predominantly contain 4.1 and 6.5 kb transcription sequences and smaller amounts of 5.1 kb RNA (Figure 3A, lane 1). It should be noted that the pPC21 probe does not detect the 2.5 kb TGF-B1 specific mRNA species present in this cell line under the hybridization conditions used herein. These results and previous observations (Sharples et al., 1987, DNA 6: 239-244) suggest that BSC-40 cells contain TGF-B1 and TGF-B2 specific mRNAs. In order to demonstrate this more clearly, Northern blots were hybridized to a mixture containing equal amounts of radio-labeled TGF-B1 and TGF-B2 probes with the same specific activity. Band 1 of Figure 3B shows that the BSC-40 cells contain the 2.5 kb TGF-B1 specific mRNA as well as the 4.1 and 6.5 kb TGF-B2 mRNA species. Band 2 of Figure 3B shows that tamoxifen-treated PC-3 cells also contain the 2.5 kb TGF-B1 specific mRNA. Figure 3B also demonstrates that tamoxifen-treated PC-3 cells contain more specific TGF-B1 message than message

specific to TGF-B2.

  Identification of TGF-B2 specific cDNA clones enabled us to screen for TGF-B2 mRNA in different cell lines. The Northern blot shown in Figure 4 shows that TGF-B2 specific transcription sequences could be detected in HBL100 (a normal human milk-derived epithelial cell line obtained from Dr. Greg Schultz), MCF-7 (a line human breast carcinoma cell), SK-MEL 28 (a melanoma cell line) and KB cells (a nasopharyngeal carcinoma cell line) containing very low

TGF-B2 mRNA levels.

  The following examples describe cDNA cloning of TGF-B2 coding sequences from the African callikric kidney cell line, BSC-40, which has been found to contain specific mRNAs.

  TGF-B2 (description above). The results

  indicate that simian TGF-B2, as well as human TGF-B2, is synthesized as one of at least two longer precursors from which the molecule is derived

  mature TGF-B2 by proteolytic cleavage.

  The following procedures were used to clone the cDNAs for the simian TGF-B2 precursor. BSC-40 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Polyadenylated RNA was isolated by oligo [dT] -cellulose chromatography as described.

  (Purchio and Fareed, 1979, J Virol 29: 763-769).

  Double-stranded cDNA was synthesized from polyadenylated BSC-40 RNA as described (Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 371-372). after treatment with EcoRI methylase, was ligated to oligonucleotide linkers containing an EcoRI restriction enzyme recognition site (EcoRI binders). The cDNA was digested with EcoRI and fractionated by Sephacryl S-1000 chromatography. The cDNA fractions greater than 750 base pairs were pooled and bound in lambda gt10 which had been cut with EcoRI (Davis et al., 1980, A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Sring Harbor, NY), packaged (Grosveld et al., 1981, Gene 13: 227-237) and plated on E. coli C600 rK mK + hfl. The library was screened by plaque hybridization (Bentonet et al., 1977, Science 196: 180-182) for [32p] labeled pPC-21 and pPC-14 probes. Clone pBSC-40-16, which hybridized the pPC-21 probe, and clone pBSC-40-1, which hybridized the pPC-14 probe, were isolated and subcloned into pEMBL. The TGF-B2 coding sequence of pBSC-40-1 was determined by sequencing both strands using the dideoxy nucleotide termination reaction (Sanger et al., 1977, Proc Natl Acad Sci. USA

  74: 5463-5467). pBSC-40-16 was partially sequenced.

  Two clones were obtained from a BSC-40 cDNA library which hybridized in turn with probes constructed from TGF-B2-442 and TGF-B2-414 precursor coding sequences. human. Clone pBSC-40-16, which hybridized to the TGF-B2-442 probe, was sequenced over a range of nucleotides (nucleotides 300 to 450 in Figure 1a) assumed to contain the coding sequence for the 29 amino acids from position 346 to position 432 in Figure la. The results show that, in this region, pBSC-40-16 encodes an amino acid sequence that is identical to the corresponding sequence of the human TGF-B2-442 cDNA clone, pPC-21, and suggests that pBSC -40-16 code for a

  TGF-B2 precursor of 442 amino acids.

  Clone pBSC-40-1, which hybridized to the TGF-B2-414 probe, was sequenced over the entire coding region. The results show that this clone encodes a 414 amino acid TGF-B2 precursor which is identical to the human TGF-B2-442 precursor except that human TGF-B2-442 amino acid residues 116 to 144

  deleted and replaced by a single residue of asphal-

  ragine. At the nucleotide level, pBSC-40-1 differs from human TGF-B2-442 in the deletion region: nucleotides 346 to 432 in Figure la are deleted and replaced by the codon for asparagine, AAT. With the exception of 13 silent base changes, the two structures are otherwise perfectly homologous

  on the rest of the coding sequence.

  The following examples describe the expression of mature biologically active TGF-B2 in Chinese hamster ovary cells (CHO cells) transfected with a recombinant plasmid containing the downstream-bound mature human TGF-B2 coding sequence and by frame with the coding sequence for the simian TGF-B1 precursor, under the regulatory control of the SV40 promoter sequences. The construction sequence directed the synthesis and secretion of mature biologically active TGF-B2 to a level of about

0.5 mg / L.

  Chinese hamster ovary cells (CH0) deficient in dihydrofolate reductase (dhfr) (Urlaub and Chasin, 1980, Proc Natl Acad Sci USA 77: 4216) were propagated in Ham's F-12 medium (Gibco Laboratories). , NY) supplemented with 10% fetal bovine serum (FBS) and 150 μg / ml L-proline. Penicillin and streptomycin were included at 100 U / ml and 100 μg / ml, respectively. CHO transfectants were cultured in Dulbecco's modified Eagle's medium containing the same supplements as those listed above. CHO cells and their derivatives were separated from their supports by trypsinization of

  routinely at a separation ratio of 1: 5.

  Methotrexate (Sigma, MO) was prepared at a stock concentration of 10 mg / ml in water. Dilute NaOH (0.2 M) was added to solubilize the drug (final pH of 6). The stock was sterilized by filtration and stored at -20 ° C. Stock solutions of methotrexate in medium (100 μM) were

  kept at 4 C for no more than one month.

  Restriction enzymes, T4 DNA ligase, calf intestinal phosphatase, Klenow fragment of DNA polymerase I and other DNA reagents were purchased from Bethesda Research Laboratories, MD. Standard DNA manipulations were performed as described in Maniatis, T., et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratory, New York.

  The plasmid pSV2 (B1-TGF-dhfr) which contains the TGF-B1 cDNA and the tandem mouse dhfr gene as well as intervening sequences of SV40, was

  constructed as described (Gentry et al., 1987, Mol Cell.

Biol. 7: 3418).

  Plasmid pSV2 / B1-B2 / dhfr was constructed as described further on pages 37, 38 and 39 first paragraph. Approximately 24 hours after seeding of 106 dhfr-deficient CHO cells into Omm trays, the cultures were transfected with 20 μg of plasmid pSV2- (BlTGF-dhfr) linearized with NdeI as a phosphate precipitate. of calcium (Wigler, M., and

  al., 1979, Proc. Natl. Acad. Sci. U.S.A. 76: 1373-1376).

  Briefly, 20 μg of linearized DNA was added to 1 ml of 250 mM sterile CaCl 2. A 1 ml portion of 2X HEPES solution (280 mM NaCl, 50 mM HEPES, 1.4 mM sodium phosphate, pH 7.1) was then added dropwise, and the mixture was allowed to stand. ice for 30 minutes. Then the precipitate was dispersed dropwise on cells containing 10 ml of F12 medium. After incubation at 37 ° C. for 4 hours, the medium was removed and replaced with 10 ml of F12 medium containing 25% glycerol for 90 seconds at room temperature. The cells were rinsed once with 20 ml of F12 medium and incubated in non-selective F12 medium (20 ml) for another 48 hours. Selection of dhfr-expressing transfectants was accomplished by replacing the medium with DMEM supplemented with 10% dialysed FBS (Gibco, N.Y.) and 150, ug / ml L-proline. Colonies were observed after culturing the cells for 10-14 days in the selection medium. Ten colonies were aspirated by means of a

Pasteur pipette and spread.

  Dihydrofolate reductase (dhfr) amplified cells were derived from primary transfectants essentially as described (Gasser, C.S. and Schimke, R.T., 1986, J. Biol Chem 261: 6938-6946). After plating, 105 cells were seeded in 100 mm tubs and adapted to increasing concentrations of methotrexate. The plate containing visible colonies at the highest concentration of methotrexate was trypsinized and adapted to this concentration of methotrexate for at least two further 1: 5 cell separations. Cells (105) were then sequenced in 100 mm dishes at 5 times the concentration of methotrexate. The tray containing visible colonies was again trypsinized and adapted to the medium containing methotrexate. The cells were fixed again at different amplification stages in media containing 40% FBS, 10% dimethyl sulfoxide and 50% DMEM. Methotrexate was not added to

freezing.

  Mink lung lung epithelial cells, Mv 1 Lu (CCL-64 serial number, American Type Culture Collection), which are extremely sensitive to TGF-B1,

  were used for the growth inhibition test.

  The assay was performed using the thymidine analog - 1253I! Iodo-2 desoxyuridine (125IdU) to estimate DNA synthesis. One unit of activity was defined as the amount required to inhibit 50% 125IdU incorporation compared to

* CCL-64 untreated.

  To test transfected cells for active TGF-B2 secretion, serum-free supernatants were collected from a 24-hour harvest on confluent cell cultures and extensively plated against acetic acid. , 2 M. The acetic acid was removed by lyophilization and the sample was redissolved in the medium of

  complete sterile culture for testing.

  A hybrid TGF-B precursor gene consisting of simian TGF-B1 precursor coding and 5 'flanked untranslated sequences with mature human TGF-B2 coding and with 3' untranslated sequences was constructed. as shown in the

Figure lc.

  pPC-21 was first digested with EcoRI, filled with the Klenow enzyme, the 2.3 kb fragment bound to pEMBL digested with HincII, and used to transform E. coli. Two clones, pPC-21 / HincII + and pPC-21 / HincII-, having opposite orientation insertional sequences, were used to produce overlapping ExoIII digests by digestion with SstI and BamHI followed by digestion with ExoIII, Klenow repair, DNA binding, and E. coli transformation. Two clones, Exo 5.9 and Exo c were found to contain different lengths of

  sequences 5 'and 3' respectively, and were sub-

  cloned into pEMBL to produce pEMBL 5.9 and pEMBL 25C.

  pEMBL 5.9 was digested with HindIII, cleaved with Klenow enzyme digests with KpnI, and the 0.6 kb fragment (fragment 1) was isolated. Exo 25C was digested with EcoRI and KpnI and the 1.1 kb fragment (fragment 2) was isolated. pGS62 was digested with BamHI, filled with Klenow enzyme, digested with EcoRI and ligated to fragments 1 and 2 (pGS62 was derived from pGS20 (Mackett et al, 1984, J. Virol 49: 857). ) by deletion of a single EcoRI site). The mixture was used to transform E. coli and

pGS62 / CIFB has been isolated.

  pGS62 / CIFB was digested with PstI and EcoRI and the 1600 bp fragment was isolated and further digested with XhoII. The XhoIIEcoRI fragment of 400 bp was isolated (fragment 3). pSV2-beta-TGF (Gentry et al., 1987, Mol Cell Biol 7: 3418) was digested with ApaI and EcoRI and the large 3000 bp fragment isolated.

(fragment 4).

  Two complementary strands of DNA with the sequences shown below were synthesized, phosphorylated, fused and linked to fragments "3" and "4"

described above.

  CAA CAT CTG CAA AGC TCC CGG CAC CGC CGA GCT TTG

  GAT GCG GCC TAT TGC TT AGA AAT GTG CAG GAT AAT

  TGC TGC CTA CGT CCA TTC TAC ATT GAT TTC AGG 3 '

  'GATC CCT CTT GAA ATC AAT GTA AAG TGG ACG TAG GCA

  GCA ATC ATT CTG CAC AT ATT AAA GCA ATA GGC CGC

  ATC CAA AGC TCG GCG GTG CGC GGA GCT TTG CAG ATG

TTG GGCC 3 '

  The binding mixture was used to transform E.

coli and pB1 / B2 was isolated.

  Plasmid pB1 / B2 was digested with EcoRI, filled in with the Klenow fragment of DNA polymerase I, cut with HindIII and the 1600bp fragment isolated: pSV2, B1 / B2 was constructed by inserting this fragment in pSV2, neo which had been previously put in

  digest with HindIII and HpaI to remove the neo gene.

  pSV2, B1 / B2 was digested with PvuI and EcoRI, filled with Klenow enzyme, digested with NdeI and the 2.6 kb NdeI-EcoRI fragment (approximately) isolated and bound to pSV2, dhfr that had been digested with NdeI and PvuII. The binding mixture was used to transform E. coli and

pSV2 / B1 / B2 / dhfr has been isolated.

  PSV / B1-B2 / dhfr was used to transfect CHO cells deficient in dhfr and dhfr-amplified cells were derived from transfectants

  described in materials and methods above.

  Positive clones were identified by bioassay (inhibition of mink lung epithelial cells (CCL-64) as described (Gentry et al., 1987, Mol Cell, Biol 7: 3418).) Recombinant proteins were also detected performing a Western blot using anti-peptide antisera prepared against the NH2-YNTINPEASASPC-COOH sequence (Gentry et al., 1987, Mol Cell, Biol 7: 3418) which is present in mature TGF-B2 One band, IB9, 12.5, was found to secrete 240 ng / ml of TGF-B2 (Figure 5).

  cloned by limiting the dilution in 96 cell plates.

  A clone, 1B9, 12.5, cl 36, produced approximately

500 ng / ml (Figure 5).

  The analysis of the protein secreted by this clone using a Western blot using anti-peptide anti-serum is presented in FIG. 6, showing the presence of the mature 24 kd TGF-B2 dimer of the same that the precursor form of larger size

(approximately 96 kd).

  The following microorganisms were deposited with the Agricultural Research Culture Collection, the Northern Regional Research Center (NRRL) and

  The following depots have been awarded to them.

  Microorganism Plasmid Deposit NO Escherichia coli HB101 pPC-21 B-18256 Escherichia coli HB101 pPC-14 B-18333 Escherichia coli HB101 pBSC-40-1 B18335 Escherichia coli HB101 pBSC-40-16 B-18334 Chinese hamster ovary pSV / B1-B2 / dhfr

Claims (30)

R E V E N D I C A T I 0 N S   A nucleotide sequence coding for the precursor of transforming growth factor 2, characterized in that it comprises the coding sequence of nucleotides substantially as shown in FIG. 1a for approximately the number of nucleotide number 1   up to about nucleotide residue number 1326.   2. A nucleotide sequence coding for the precursor of transforming growth factor B2, characterized in that it comprises the coding sequence of nucleotides substantially as shown in FIG. 1a from about nucleotide residue number 1 to about the residue of nucleotide number 1326 in which the nucleotide sequence from nucleotide residue number 346 to nucleotide residue number 432 is deleted and replaced by the nucleotide "AAT".   3. Nucleotide sequence coding for the mature transforming growth factor B2, characterized in that it comprises the nucleotide sequence substantially as shown in Figure la for approximately the number of nucleotide residue   991 to about nucleotide residue number 1326.   4. Precursor of the transforming growth factor B32 characterized in that it comprises the amino acid sequence substantially as shown in Figure la from about the amino acid residue number 1 to about the acid residue amine number 442.   5. Precursor of transforming growth factor B32, characterized in that it comprises the amino acid sequence substantially as shown in Figure la from about amino acid residue number 1 to about the acid residue amino acid number 442 in which the amino acid sequence from residue number 116 to residue number 144 is deleted and replaced by a single residue of asparagine. 6. Precursor of the transforming growth factor B2, characterized in that it comprises the amino acid sequence substantially as shown in Figure la from about the amino acid residue number 20 to about the acid residue amine number 442.   7. Precursor of the transforming growth factor B2, characterized in that it comprises the amino acid sequence substantially as shown in Figure la from about the amino acid residue number 20 to about the acid residue amino acid number 442 in which the amino acid sequence from residue number 116 to residue number 144 is deleted and replaced by a single residue of asparagine. 8. Transforming growth factor B2, characterized in that it comprises the amino acid sequence substantially as shown in Figure la from about the amino acid residue number 331 to   about amino acid residue number 442.   9. Nucleotide sequence coding for the precursor of transforming growth factor B1 / hybrid transforming growth factor B2, characterized in that it comprises the coding sequence of nucleotides substantially as shown in FIG. 1b from about the nucleotide residue number   -70 to about nucleotide residue number 1755.   10. Precursor of Transforming Growth Factor B1 / Transforming Growth Factor B2 Hybrid, characterized in that it comprises the amino acid sequence substantially as shown in Figure 1b from about the amino acid residue number 1   up to about amino acid residue number 390.   11. Precursor of the transforming growth factor B1 / transforming growth factor B2 hybrid characterized in that it comprises the amino acid sequence substantially as shown in Figure lb from about the number amino acid residue   up to about amino acid residue number 390.   12. Process for producing transforming growth factor B2, characterized in that it comprises: (a) culturing a eukaryotic cell containing a nucleotide sequence coding for the transforming growth factor B2 under the control of a second nucleotide sequence which regulates the expression of genes so that a peptide or protein having a transforming growth factor B2 activity is produced by the eukaryotic cell; and (b) obtaining the growth factor   transforming B2 from the culture.   13. The method of claim 12, characterized in that the nucleotide sequence encoding the transforming growth factor B2 comprises the nucleotide sequence substantially as shown in Figure la since the nucleotide number 1 to nucleotide number 1339.   14. Process according to claim 12, characterized in that the nucleotide sequence coding for the transforming growth factor B2 comprises the nucleotide sequence substantially as represented in FIG. 1b from the nucleotide number.   -70 to nucleotide number 1755.   15. Process according to claim 12, characterized in that the eukaryotic cell comprises a   Chinese hamster ovary cell.   16. The method according to claim 12, characterized in that the second nucleotide sequence which controls the expression of the gene comprises a promoter from SV40.   17. The method according to claim 12, characterized in that the second nucleotide sequence comprises a promoter and a coding sequence for a selectable marker for which the eukaryotic cell is deficient, so that the eukaryotic cell containing the coding sequence for the factor of   transforming growth B2 can be identified.   18. The method of claim 17, characterized in that the marker capable of being   selected comprises dihydrofolate reductase.   The method according to claim 18, characterized in that it further comprises exposing the eukaryotic cell to methotrexate, so that resistant colonies which contain amplified levels of the coding sequences for dihydrofolate reductase and the transforming growth B2, are selected.   20. The method of claim 19, characterized in that the eukaryotic cell comprises a Chinese hamster ovary cell deficient in dihydrofolate reductase.   21. Process for producing transforming growth factor B2, characterized in that it comprises (a) culturing the transfectant 1B9, 12.5, clone 36 as deposited with the ATCC; and (b) obtaining the growth factor   transforming B2 from the culture.   22. Method according to claim 21, characterized in that the transfectant is cultured. in the presence of methotrexate.   23. A eukaryotic cell, characterized in that it contains a nucleotide sequence coding for transforming growth factor 132 under the control of a second nucleotide sequence which regulates the expression of the gene so that the eukaryotic cell   produces the transforming growth factor B2 active.   24. A eukaryotic cell according to claim 23, characterized in that the nucleotide sequence coding for the transforming growth factor B2 comprises the nucleotide sequence substantially as shown in FIG. 1b from the nucleotide.   number -70 to nucleotide number 1755.   25. The eukaryotic cell according to claim 23, characterized in that it is a cell ovary of Chinese hamster.   26. A eukaryotic cell according to claim 23, characterized in that the second nucleotide sequence which controls the expression of the gene comprises a SV40 promoter.   27. A eukaryotic cell according to claim 23, characterized in that the second nucleotide sequence comprises a promoter and a sequence coding for a selectable marker for which the eukaryotic cell is deficient, so that the eukaryotic cell containing the coding sequence for the factor of   Transforming growth 132 simian can be identified.   28. A eukaryotic cell according to claim 27, characterized in that the marker can be   selected comprises dihydrofolate reductase.   29. The eukaryotic cell according to claim 28, characterized in that it is a Chinese hamster ovary cell deficient in dihydrofolate reductase. 30. Cell line, characterized in that it is the 139 line, clone 36 of Chinese hamster ovary cells carrying pSV / beta-beta 2 / dhfr as deposited with the ATCC, under the number d CRL 9800 access.