AU630075B2 - Cloning and expression of transforming growth factor 2b - Google Patents

Description

AUSTRALIA

630075 Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Complete Specification Lodged: Accepted; Published: Priority ':"ftelated Art:

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Int. Class 9 m o *Ntime(s) of Applicant(s): PPLICANT'S REFERENCE: CN-0029B T *Bri-stol- yex -s.-Copany S: dress(es) of Applicant(s): 4dldress(es) of Applicant(s): S

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345 Park Avenue, New York, New York, UNITED STATES OF AMERICA.

9 SAadress for Service is: PHILLIPS (RMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: CLCNING AND EXPRESSION OF TRANSFORMIN GROWTH FACTOR 2B Our Ref 107557 POF Code: 1490/1490 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6003q/1 I The preser expression of h -3- 1. INTRODUCTION it invention relates to the cloning and uman transforming growth factor-Beta2.

00 0 0 so 60000 06 0 0* 0 0000S 5 55 S 0 0*eS 2. BACKGROUND OF THE INVENTION Transforming growth factor-Beta (TGF-3) is a member of a recently described family of polypeptides that regulate cellular differentiation and proliferation. Other members of this family include Mullerian inhibitory substance (Cate et al., 1986, Cell 45:685-698), the inhibins (Mason et al., 1985, Nature 318:659-663) and a protein predicted from a transcript of the decapentaplegic gene complex of Drosophila (Padgett et al., 1987, Nature 325:81-84).

Transforming growth factor-Beta (TGF-p) consists of 15 two identical disulfide linked subunits having 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 2sources including placenta (Frolik et al., 1983, Nature 325:81-84), blood platelets (Childs et al., 1982, Proc.

Natl. Acad. Sci. USA 79-5312-5316; Assoian et al., 1983, J.

Biol. Chem. 258:7155-7160) kidney (Roberts et al., 1983, Biochemistry 22:5692-5698), and demineralized bone (Seyedin 2et al., 1985, Proc. Natl. Acad. Sci. USA 82:119-123). In the presence of 10% serum and epidermal growth factor, TGF-e promotes the anchorage independent growth of normal rat kidney fibroblasts (Roberts et al., 1981, Proc. Natl. Acad.

Sci. USA 78:5339-5343; Roberts et al., 1982, Nature 3295:417-419; Twardzik et al., 1985, J. Cell. Biochem.

3O 28:289-297); in the presence of 10% serum alone, it is able to induce colony formation of AKR-2B fibroblasts (Tucker et al., 1983, Cancer Res. 43:1518-1586). TGF-f has also been shown to cause fetal rat muscle mesenchymal cells to -4differentiate and produce cartilage specific macromolecules (Seyedin et al., 1986, J. Biol. Chem. 261:5693-5695).

In contrast to its effect on cell proliferation, TGF-P purified from human platelets as well as a functionally related protein isolated from African green monkey cells (BSC-1) has been shown to inhibit the growth of certain cells in culture (Tucker et al., 1984, Science 226:705-707). TGF-Beta 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/stimulatory effect of TGF-9 may depend on several factors including cell type and the physiological state of the cells (for review see Sporn et al., 1986, Science 233:532-534).

5 0 15 cDNA clones coding for human (Derynck et al., 1985, Nature 316:701-705), mouse (Derynck et al., 1986, J. Biol.

Chem. 261:4377-4379) and simian (Sharples et al., 1987, DNA 6:239-244) TGF-f have been isolated. DNA sequence analysis 0S of these clones indicates that TGF-8 is synthesized as a 20 large precursor polypeptide, the carboxy terminus of which is cleaved to yield the mature TGF-f monomer. Strong *Jog sequence homology has been found throughout the TGF-f8 S precursor protein from all of the above sources.

Very recently a protein isolated from bovine demineralized bone has been identified as being related to TGF-A (Seyedin et al., 1987, J. Biol. Chem. 262: 1946-1949).

The protein has also been isolated from porcine platelets (Cheifetz et al., 1987, Cell 48:409-415), a human prostatic adenocarcinoma cell line, PC-3 (Ikeda et al., 1987, Biochemistry 26:2406-2410), and a human glioblastoma cell line (Wrann et al., 1987, EMBO 6:1633-1636), Partial amino acid sequence of this protein indicated that it was homologous to TGF-9 and has been termed TGF-82. The human (Derynck et al., 1985, Nature 316:701-705), mouse (Derynck et al., 1986, J. Biol. Chem. 261:4377-4379) and simian I -C 7 0 0@ S

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5 5 S SO 0 (Sharples et al., 1987, DNA 6:239-244) TGF-f described previously has been termed TGF-31.

3. SUMMARY OF THE INVENTION The present invention relates to the production of large quantities of TGF-92 by eukaryotic host cells transfected with recombinant DNA vectors containing a TGF-32 coding sequence controlled by expression regulatory elements. In a specific embodiment, cDNA clones coding for human TGF-P2 precursor were obtained from a cDNA library made from a tamoxifen treated human prostatic adenocarcinoma cell line, PC-3. The cDNA sequence of one such clone predicts that TGF-82 is synthesized as a 442 amino acid polypeptide precursor from which the mature 112 amino acid TGF-92 subunit is derived by proteolytic cleavage. This TGF-32 precursor, termed TGF-92-442, shares a 41% homology with the precursor of TGF-l1. In another embodiment, cDNA clones coding for simian TGF-32 precursor were obtained from a cDNA library made from an African green monkey kidney cell 20 line, BCS-40. The cDNA sequence of one such clone predicts that TGF-32 is also synthesized as a 414 amino acid polypeptide precursor from which the mature 112 amino acid TGF-S2 subunit is derived by proteolytic cleavage. This TGF-32 precursor, termed TGF-f2-414, has an amino acid 2sequ.ence of 414 amino acid residues and is identical to the amino acid sequence of TGF-02-442, except that it contains a single Asparagine residue instead of the 29 amino acid sequence from residue numbers 116 to 135 of the human TGF-f2-442 sequence.

Clones from the BSC-40 cDNA library which encode a simian TGF-f2-442 precursor as well as clones from the human PC-3 cDNA library which encode a human TGF-32-414 precursor have also been identified. The human and simian TGF-32-442 precursors appear to be perfectly homologous at the amino 7

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-6acid level, as do the human and simian TGF-p2-414 precursors.

The mature 112 amino acid monomers of TGF-fl and TGF-2 show 71% homology.

Expression vectors containing the TGF-P2 mature coding sequence joined in-phase to the TGF-P1 signal and precursor sequences (Co-owned/pending United States Patent Application No. 189,984) were constructed and used to transfect Chinese Hamster Ovary cells (CHO cells). The resulting CHO transfectants produce and secrete mature, biologically active TGF-P2.

3.1. DEFINITIONS The following terms as used herein whether in the 15 singular or plural, shall have the meanings designated.

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TGF-,i2: TGF-P2 precursor: A transforming growth factor-Beta2 of human or simian origin comprising the amino acid sequence substantially as depicted in FIG. la from about amino acid residue number 331 to about amino acid residue number 442.

A family of transforming growth factor-Beta2 molecules of human or simian origin comprising an amino acid sequence substantially as depicted in FIG. la from about amino acid residue number 1 to about amino acid residue number 442, or from about amino acid residue number 1 to about amino acid residue number 442 where the amino acid sequence from amino acid residue number 116 to amino acid residue number 144 is deleted and replaced by a single Asparagine residue. The term shall mean a TGF-beta 2 precursor designated TGF-p2-442 or TGF-p2-414 whether of human or simian origin.

-7- Hybrid TGF-Il/ TGF-,92 precursor: A novel transforming growth factor-beta precursor molecule comprising the amino acid sequence substantially as depicted in FIG. lb from about amino acid residue number 1 to about amino acid number 390.

Simian-transforming growth factor-Betal precursor and signal sequences substantially as depicted in FIG. Ib from about amino acid residue number 1 to about amino acid residue number 278.

TGF-91 precursor: woee

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S S *5 S Sao O 0 0 r_ I i6YB~ -8- 4. BRIEF DESCRIPTION OF THE FIGURES FIG. la Nucleotide sequence of human TGF-82-442 cDNA and deduced amino acid sequence. The 2597 bp insert of PC- 21 was subcloned into pEMBL (Dante et al., 1983, Nucleic Acids Res. 11:1645-1654) and sequenced on both strands using the dideoxy chain-termination method (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-f2 sequence is boxed and the signal peptide is overlined. Potential glycosylation sites are indicated by asterisks. The arrow indicates the putative signal sequence cleavage site. The nucleotide sequence of simian TGF-02-414 cDNA is identical to the human TGF-p2-442 cDNA sequence except that 1 5 nucleotides 346 through 432 (bracketed) are deleted and replaced by the sequence AAT, and except that several silent O* nucleotide changes occur elsewhere in the structure (indicated by single letters directly below the changed nucleotide). The deduced amino acid sequence for simian TGF-p2-414 precursor is identical to the human TGF-p2-442 precursor amino acid sequence except that Asparagine replaces amino acid residues 116 through 144 in the human TGF-f2-442 structure. The nucleotide sequence of a human TGF-p2-414 cDNA has been sequenced through the region 25 indicated by broken underlining and was found to be perfectly homologous to the human TGF-92-442 cDNA sequence except that nucleotides 346 through 432 are deleted and replaced by the sequence AAT.

FIG. lb Nucleotide sequence of hybrid TGF-91/TGF-92 precursor DNA and deduced amino acid sequence. The coding sequence is shown and the deduced amino acid sequence is presented directly above. The mature TGF-p2 sequence is boxed and the precursor signal peptide is overlined.

Glycosylation sites are indicated by asterisks. The arrow Sindicates the putative signal sequence cleavage site. The TGF-P2 mature coding sequence depicted is of human origin.

The simian TGF-f2 mature coding sequence is nearly identical to the human sequence: only 3 silent base changes occur and are indicated by single letters directly below the changed nucleotide. Details of the cDNA cloning of TGF-92 and the construction of the hybrid TGF-fl/TGF-92 gene are given in the text.

FIG. Ic Schematic diagram of hybrid TGF-31/TGF-02 precursor gene.

FIG. Id Restriction endonuclease maps of pPC-14 (2.2kb) and pPC-21 (2.3 kb). The boxed regions indicate coding sequences for TGF-62 monomer. The ATG denotes the initiating methionine codon. The distance between the ATG .and KpnI site in pPC-21 (2.34 kb) is approximately 420 bp.

1" The darkened area indicates the position of the 84-bp insertion in pPC-21 (2.3 kb).

FIG. le Partial DNA sequence analysis of pPC-14 (2.2 kb). A synthetic oligonucleotide 5'-AGGAGCGACGAAGAGTACTA-3' which hybridized approximately 140 bp upstream from the KpnI site within the insert in pPC-21 (2.3 kb) was used to prime DNA sequencing reactions. In this region, the sequence of I pPC-14 (2.2 kb) (upper line) is identical to pPC-21 (2.3 kb) j up to nucleotides coding for Asn-116. The 84-bp insertion within the Asn-116 codon of pPC-14 (2.2 kb) which was found S 25 in pPC-21 (2.3 kb) is shown. The KpnI site within the 25 insert is denoted.

FIG. 2 Homologies of human TGF-31 and TGF-82-442 iprecursor sequences. a) Primary sequence homology: identical residues are boxed. Asterisks refer to potential glycosylation sites in TGF-A2. The potential signal sequence cleavage site and the cleavage site of the mature polypeptide are indicated, b) Dot matrix comparison using Gene Pro software. Each dot locates a point where 5 out of amino acids are identical. Diagonal lines indicate regions of homology.

r -23in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). cDNA fractions |i greater than 1000 base oairs wAer nlA r 4n- 1- FIG. 3 Northern blot analysis of BSC-40 and PC-3 polyadenylated RNA. Polyadenylated RNA was isolated from BSC-40 and PC-3 cells, fractionatcd on an agaroseformaldehye gel, transferred to Hybond-N filters and hybridized to [32P]-labelled TGF-/2 specific probe, pPC-21 (Panel A) or a mixture of [32P]-labelled TGF-2 and TGF-AL (Sharples et al., 1987) specific probes (Panel B) as described in Materials and Methods. Lane 1, BSC-40 polyadenylated RNA (5 micrograms); lane 2, PC-3 polyadenylated RNA (5 micrograms).

FIG. 4 Northern blot analysis of polyadenylated RNA from different sources. Polyadenylated RNA was isolated from MCF-7 (human mammary carcinoma), SK-MEL 28 (human melanoma), KB (nasopharangeal carcinoma) and HBL-100 (human mammary epithelial) cells and analyzed by Northern blot hybridization to a TGF-#2 specific probe (pPC-21) as described in Materials and Methods. Each lane contains 5 micrograms of polyadenylated RNA from SK-MEL 28 (lane MCF-7 (lane HBL-100 (lane 3) or KB (lane 4) cells.

FIG. 5 Bioactivity Assay of Recombinant TGF-A2. 1/9 12.5, clone 36 cells were grown to confluency in 100 mm tissue culture dishes. Cells were washed 3X with serum-free media and incubated for 24 hours in 5 ml of serum-free media. Media was collected, dialyzed against 0.2M acetic acid, and assayed for inhibition of DNA synthesis in CCL64 cells as described (Gentry et al., 1987, Mol. Cell. Biol. 7:3418). In this assay 3.3 pg of purified natural TGF-#l standard gave 50% inhibition; the specific activity of purified natural TGF-2 was calculated to be about half that of TGF-#l.

FIG. 6 Western Blot Analysis of Recombinant Proteins Secreted by S19, 12.5, clone 36. Acid dialyzed serum-free conditioned media from 1^9, 12.5 clone 36 cells was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with anti serum made against 0 0 00 0 0 0 0 00 00o 0 0 0 0 0S 0 0 0 oo0 0 00 00 0 0 0 0 0 0O00 0 00 0 7 0 0**0 0 00 *0 00 0 0 *0

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*0 0 0 0000 the synthetic peptide NH2-YNTINPEASASPC-COOH as described (Gentry et al., 1987, Mol. Cell. Biol. 7:3418).

DESCRIPTION OF THE INVENTION The present invention relates to the production of a biologically active, mature form of TGF-P2 from a TGF-p precursor gene coding sequence and its product. The mature biologically active TGF-p2 may be produced by the cloning and expression of the full-length nucleotide coding sequence of the TGF-92 precursor or its functional equivalent in a host cell which processes the precursor correctly so that a mature TGF-P2 is produced having a biological activity that is virtually indistinguishable from that of authentic natural TGF-P2. Functional equivalents of the full length 15 nucleotide coding sequence of the TGF-52 precursor include any DNA sequence which, when expressed inside an appropriate host cell, is capable of directing the synthesis, processing and export of mature TGF-92. In this regard, hybrid precursor coding sequences including, for example, the TGF- 20 fl precursor sequence joined in-frame to the TGF-p2 mature sequence, may be constructed and used to produce biologically active TGF-P2.

The method of the invention may be divided into the following stages solely for the purposes of description: (a) 25 isolation or generation of the coding sequence for a precursor form of TGF-92; construction of an expression vector which will direct the expression of a TGF-92 coding sequence; transfection of appropriate host cells which are capable of replicating and expressing the gene and processing the gene product to produce the mature, biologically active form of TGF-92; and identification and purification of the mature, biologically active TGF-P2.

Once a transfectant is identified that expresses high levels of bioactive, mature TGF-p2, the practice of the invention -12involves the expansion of that clone and isolation of the gene product expressed.

The method of the invention is demonstrated herein, by way of examples in which cDNAs of the TGF- 2 precursor coding region were prepared, cloned, se(plenced, and utilized to construct expression vectors which direct the high-level expression of TGF- 2 in CHO cells. In a specific embodiment, the complete amino acid sequence of the mature form of human TGF-P2 has been determined and shows an overall homology of 71% with TGF-fil. Using synthetic oligonucleotide probes we have identified clones from a PC-3 cDNA library coding for TGF-82. DNA sequence analysis of one of these clones revealed that TGF-fi2, like TGF-pl, is synthesized as a larger precursor protein, the carboxy seem.. 5 terminus of which is cleaved to yield the mature TGF- 2 monomer. While there is a 71% homology between TGF-01 and TGF-P2 throughout the mature portions of these molecules, only a maximum of 31% homology exists within the rest of the precursor, suggesting that the amino terminal regions of TGF-l and TGF-fi2 may be functionally distinct.

In a specific embodiment of the invention, expression of a novel TGF-fI/TGF-f2 hybrid gene in CHO cells is used to produce large amounts of biologically active TGF-82.

The various aspects of the method of the invention are described in more detail in the subsections below and in the 25 examples that follow.

see* 5.1. ISOLATION OR GENERATION OF THE TGF-BETA 2 CODING REGION The nucleotide coding sequence for TGF-02 is depicted in FIG. la. In the practice of the method of the invention, the nucleotide sequence depicted therein, or fragments or functional equivalents thereof, may be used to generate the recombinant molecules which will direct the expression of the TGF-P2 product in appropriate host cells. In a specific -13-

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embodiment, a TGF- l/TGF-2 hybrid gene (FIG. ib) was constructed and used to transfect CHO cells. Transfectants producing as much as 500 Mg mature, biologically active TGF-p2 per ml culture medium were isolated.

Due to the degeneracy of the nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequences as depicted in FIG. la and FIG. Ib may be used in the practice of the present invention for the cloning and expression of TGF-p2. Such alterations include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product may contain deletions, additions or substitutions of amino acid residues within the sequence, which result in a silent change thus producing a bioactive product. Such amino acid substitions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids 2include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, 2glutamine; serine, threonine; phenylalanine, tyrosine.

The nucleotide coding sequence for TGF-p2 may be obtained from cell sources that produce TGF-f2 like activity. The coding sequence may be obtained by cDNA cloning of RNA isolated and purified from such cellular 3sources or by genomic cloning. Either cDNA or genomic libraries of clones may be prepared from the DNA fragments generated using techniques well known in the art including but not limited to the use of restriction enzymes. The fragments which encode TGF-P2 may be identified by screening such libraries with a nucleotide probe that is substantially -14complementary to any portion of the sequence depicted in FIG. la. Full length clones, those containing the entire coding region for the TGF-P2 precursor may be selected for expression.

In an alternate embodiment of the invention, the coding sequence of FIG. la could be synthesized in whole or 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):2807-2817. Alternatively, the protein could be produced using chemical methods to synthesize the amino acid sequence depicted in FIG. la in whole or in part. For example, peptides can be synthesized by solid phase techniques on a Beckman 990 instrument, and cleaved from the resin as previously described (Gentry, et al., 1983, 0 J. Biol. Chem. 258:11219-11228; Gentry, L.E. nd Lawton, A., 1986, Virology 152:421-431). Purification can be accomplished by preparative high performance liquid S chromatography. The composition of the peptides was i confirmed by amino acid analysis.

In a specific embodiment, described in the examples herein, the TGF-P2 coding sequence was obtained by cDNA 0 25.cloning of human TGF-P2 precursor coding sequences derived 25 from polyadenylated RNA isolated from tamoxifen-treated human prostatic adenocarcinoma cell line, PC-3, previously shown to produce TGF-p2. The entire coding region of one 4i cDNA clone was sequenced and compared to the published sequence of human TGF-Il (see FIG. 2).

DNA sequence analysis of TGF-P2 cDNA clones indicates that TGF-P2, like TGF-P1, is synthesized as a large precursor protein, the carboxy terminus of which is cleaved to yield the mature 112 amino acid TGF-P2 monomer. TGF-P2 has been shown to have a nolecular weioght of 24,000 composed of two disulfide-linked 13,000 dalton subunits (Ikeda et al., 1987, Biochemistry 26:2406-2410; Cheifetz et al., 1987, Cell 48:409-415). Therefore, the production of mature TGFf2 requires proper proteolytic cleavage as well as the formation of intra- and inter-molecular disulfide bonds. An amino terminal hydrophobic leader sequence (residue 3-19) is present in the precursor and may be responsible for directing the protein out of the cell. The mature TGF-32 may still be associated with the remaining portion of the precursor during this process.

TGF-P2 shows 71% homology with TGF-#l throughout the mature portion of the precursor, implying a functional similarity which is supported by experimental evidence (Seyedin et al., 1987, J. Biol. Chem. 262:1946-1949; 15 Cheifetz et al., 1987, Cell 48:409-415). The amino portion 1 of the precursor region of TGF-#l from human, rodent and S* simian sources (Derynck et al., 1985, Nature 316:701-705; 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 may have an important biological function. In contrast, there is no more than 31% homology between the N-terminal precursor regions of TGF-1 and TGF-p2. After cleavage of the a" putative signal peptide, the TGF-P2 precursor would also contain more amino acids than TGF-pl precursor. The primary 25 structural differences within the amino terminal region of the TGF-pl and TGF- 2 precursor proteins may reflect functional differences. However, significant homologous regions within the precursors are found in isolated blocks 3suggesting the conservation of important functional domains 3O even within the N-terminal precursor region.

Northern blot analysis revealed two major size classes of TGF-p2-specific mRNA of 4.1 and 6.5kb in BSC-40 cells.

Tamoxifen-treated PC-3 cells contain three TGF-82 Stranscripts of 4.1kb, 5.1kb, and 6.5kb. These different- 2 -16sized messages 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. Nati. Acad. Sci. USA 84:2941-2945). Preliminary analysis of another TGF-P2 cDNA clone shows that it contains a 3'-untranslated region approximately 1kb larger than that of pPC-21 and pPC-14 and contains a different polyadenylation site suggesting that alternative polyadenylation is one factor responsible for the generation of multiple TGF-p2 mRNAs observed on Northern blots.

cells contain comparable levels of TGF-91 and TGF-f92-specific transcripts: tamoxifen-treated PC-3 cells 0 contain more TGF-f91 mRNA than TGF-p2 (FIG. 3B). The latter 15 result is unexpected since these cells produce more TGF-f2 fee: protein than TGF-l (Ikeda et al, 1987, Biochemistry 26:2406-2410) and suggests a post transcriptional level of regulation regarding the synthesis of this growth modulator.

i* Experiments aimed at understanding transcriptional and i 20 translational control mechanisms which lead to the i 20 ii *,.production of TGF-l and TGF-92 are in progress. Production of adequate amounts of TGF-92 by recombinant DNA techniques,

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as has been done for TGF-fI, should aid further in designing experiments to explore the different effects of this 25 protein.

25 In another embodiment, the TGF-92 coding sequence was o obtained by cDNA cloning of simian TGF-f92 precursor coding sequences derived from polyadenylated RNA isolated from an African green monkey cell line, BSC-40. The entire coding region of one cDNA clone was sequenced. The human and simian TGF-92 precursors appear to have identical amino acid sequences, and their nucleotide sequences are nearly identical.

-17- 5.2. CONSTRUCTION OF EXPRESSION VECTORS CONTAINING THE TGF-BETA 2 CODING SEQUENCE In order to express a biologically active, mature form of TGF-P2, an expression vector/host system should be chosen which provides not only for high levels of transcription and translation but for the correct processing of the gene product. This is especially important when employing the entire coding sequence of a TGF-92 precursor in the expression constructs because the mature form of TGF-f2 appears to be derived from the precursor product via cellular processing events. In addition, an expression/host cell system which provides for secretion of the product may be selected.

SIn particular, it appears that the mature TGF-f2, a disulfide-linked homodimer of 112 amino acids per subunit *fee •o 5may be formed by cellular processing involving proteolytic cleavage between the Arg-Ala amino acids of the precursor

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(residue numbers 330 and 331 in FIG. la). In addition, the STGF-32 precursor contains three potential N-glycosylation sites not found in the mature form; the proper glycosylation 20 20 of the precursor may be important to the cellular synthesis and release or secretion of the mature molecule. Moreover, the mature form of TGF-p2 comprises a disulfide-linked dimer involving nine cysteine residues per subunit. Some of these S are involved in interchain and others in intrachain 25 disulfide bonds which affect the tertiary structure and configuration of the mature molecule, and, as a result, its biological activity. Thus, the ability of a host cell used in the expression system to correctly express and process the TGF-P2 gene product is important to the production of a biologically active, mature TGF-92.

A variety of animal host/expression vector systems vectors which contain the necessary elements for directing the replication, transcription and translation of the TGF-92 coding sequence in an appropriate host cell) may 2 -18o0 io~e.

oC o4 be utilized equally well by the skilled artisan. These include, but are not limited to, virus expression vector/mammalian host cell systems cytomegalovirus, vaccinia virus, adenovirus, and the like); insect virus expression vector/insect cell systems baculovirus) or nonviral promoter expression systems derived from the genomes of mammalian cells the mouse metallothionine promoter).

The expression elements of these vectors vary in their strength and specificities. Depending on the host/vector system utilized, any one of a number of suitable transcription and translation elements may be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, mouse metallothionien promoter) or from viruses that grow in these cells, vaccinia virus 7.5K promoter) may be used.

Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted sequences.

20 Specific initiation signals are also required for sufficient translation of inserted protein coding sequences.

These signal:; include the ATG initiation codon and adjacent sequences. In cases where the entire TGF-f2 gene including its own initiation codon and adjacent sequences are inserted into the appropriate expression vectors, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be ctrid-d. Furthermore, the initiation codon must be in phase ith the reading frame of the TGF-P2 coding sequences to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of -19expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements, etc.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing the TGF-, gene and appropriate transcriptional/translational control signals.

These methods may include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombinations (genetic recombination).

In cases where an adenovirus is used as an expression vector, the TGF-32 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome 15 by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome region El or E3) will result in a recombinant virus that is viable and capable of expressing TGF-82 in infected hosts. Similarly, the vaccinia 7.5K promoter may be used.

An alternative expression system which could be used to express TGF-P2 is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The YGF-92 coding sequence 25 may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

Successful insertion of the TGF-P2 coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers, zinc and cadmium ions for metallothionein promoters). Therefore, expression of the genetically engineered TGF-fl2 may be controlled. This is important if the protein product of the cloned foreign gene is lethal to host cells. Furthermore, modifications glycosylation) and processing cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modificatin of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein 5.3. IDENTIFICATION OF TRANSFECTANTS OR TRANSFOR- MANTS EXPRESSING THE 2 TGF-BETA GENE PRODUCT 0 The host cells which contain the recombinant TG-f2 S coding sequence and which express the biologically active, mature product may be identified by at least four general approaches: DNA-DNA hybridization; the presence or see absence of "marker" gene functions; assessing the level *of transcription as measured by the expression of TGF-fl2 mRNA transcripts in the host cell; and detection of the 2mature gene product as measured by immnunoassay and, S ultimately, by its biological activity.

S.In the first approach, the presence of the TGF-fi2 coding sequence inserted in the expression vector can be detected by DNA-DNA hybridization using probes comprising nucleotide sequences that are homologous to the TGF-fl2 coding sequence substantially as shown in FIG. la, or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host syste~m can be identified and selected based upon .*6 *.o

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S

O* I 7 e -21the presence or absence of certain "marker" gene functions thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the TGF-P2 coding sequence is inserted within a marker gene sequence of the vector, recombinants containing the TGF-2 coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the TGF-P2 sequence under the control of the same or different promoter used to control the expression of the TGF-P2 coding sequence. Expression of the marker in response to induction or selection indicates expression of the TGF-p2 coding sequence.

In the third approach, transcriptional activity for the TGF-beta 2 coding region can be assessed by hybridization assays. For example, polyadenylated RNA can be isolated and analyzed by Northern blot using a probe homologous to the TGF-beta 2 coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the mature protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays and the like. The ultimate test of the success of the expression system, however, involves the detection of the biologically active TGF-P2 gene product. Where the host cell secretes the gene product the cell free media obtained from the cultured transfectant host cell may be assayed for TGF-P2 activity.

Where the gene product is not secreted, cell lysates may be assayed for such activity. In either case, biological assays such as the growth inhibition assay described herein or the stimulation of anchorage independent growth in target cells (Twardzik and Sherwin, 1985, J. Cell. Biochem.

CO0 _1 7

S

S

55 5 S S -22- 28:289-297; Delarco and Todaro, 1978, Proc. Natl. Acad. Sci.

U.S.A. 75:4001-4005) or the like may be used.

Once a clone that produces high levels of biologically active, mature TGF-f2 is identified, the clone may be expanded and the TGF-92 may be purified using techniques well known in the art. Such methods include immunoaffinity purification, chromatographic methods including high performance liquid chromatography, and the like.

6. EXAMPLE: cDNA CLONING OF TGF-BETA-2 PRECURSOR FROM PC-3 CELLS The following examples describe the cDNA cloning of TGF-92 precursor coding sequences from the human prostatic adenocarcinoma cell line, PC-3, from which TGF-beta-2 was previously isolated.

6.1. MATERIALS AND METHODS The following procedures were used to clone cDNAs encoding human TGF-beta 2 precursor.

6.1.1. GROWTH OF CELLS AND RNA EXTRACTION.

The human prostatic adenocarcinoma cell line, PC-3, was grown in tamoxifen-supplemented medium as described (Ikeda et al., 1987, Biochemistry 26:2406-2410). MCF-7 cells were grown in Dulbecco's modified Eagle's medium 25 containing 10% fetal calf serum and 6 units/ml insulin. All other cell lines were grown in the same medium without insulin. Polyadenylated RNA was isolated by oligo[dT]cellulose chromatography as described (Purchio and Fareed, 1979, J. Virol. 29:763-769).

6.1.2. cDNA LIBRARY CONSTRUCTION AND SCREENING Double-stranded cDNA was synthesized from polyadenylated RNA isolated from PC-3 cells treated with tamoxifen for 24 hours as described (Maniatis et al., 1982, s S.

sees 55

S

S

s 55 S 9 S 555 -23in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). cDNA fractions greater than 1000 base pairs were cloned into lambda gti0 as described (Webb et al., 1987, DNA 6:71-79). The library was 32 first screened in duplicate with a P]-labeled 24-fold degenerate probe complimentary to DNA encoding amino acids WKWIHEP (probe 1) which are conserved between TGF-il and TGF-92: [5'-GGTTCGTGTATCCATTTCCA-3'] C A G C

A

Positive clones were then screened with a second 128-fold degenerate probe complimentary to DNA encoding amino acids CFRNVQD (probe five out of these seven amino acids are specific for TGF-Beta 2: [5'TCTTGAACGTTTCTGAAGCA-3'] SC C A C A A

G

T

Hybridization was performed at 42°C in 6XSSC, 5X Denhart's solution, 0.15 mM pyrophosphate, 100 micrograms/ml denatured calf thymus DNA, 100 micrograms/ml yeast tRNA and 1 mM EDTA (Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). Filters were washed at 42*C in 2XSSC, 0.1% NaDodSO 4 ;o 25 4 four times for 30 min. Several cDNA clones were isolated which hybridized to both probes and were subcloned into .pEMBL (Dante et al., 1983, Nucleic Acids Res. 11:1645-1654).

One clone (pPC-21) containing a 2.6kb insert was sequenced on both strands by the dideoxy chain-termination method (Sanger et al., 1977, Proc. :5atl. Acad. Sci. USA 74:5463- 5467) using various restriction and exonuclease III deletion fragments combined with specific oligonucleotide priming (Henikoff, 1984, Gene 28:351-359). Another clone (pPC-14) containing a 2.2 kb insert was partially sequenced. Dot -24matrix analysis was performed on an IBM ATPC using Gene Pro software from Riverside Scientific Enterprises (Seattle,

WA).

6.1.3. NORTHERN BLOT ANALYSIS Polyadenylated RNA was fractionated on a 1% agaroseformaldehyde gel (Lehrach et al., 1977, Biochemistry 16:4743-4751), transferred to a nylon membrane (Hybond, 32 Amersham), and hybridized to [32 P-labeled probe.

Hybridization was carried out at 42*C in 50% formamide containing 0.9 M NaCI, 50 mM sodium phosphate (pH 5 mM EDTA, 0.1% NaDodSO 4 4X Denhardt's solution, 0.4 mg/ml of yeast tRNA, and 0.25 mg/ml of denatured calf thymus DNA.

Filters were washed at 65°C in 0.25X SSC, 0.1% NaDodS0 4 *4 15 dried, and exposed to Cronex-4 X-ray film (DuPont) with the aid of Lightening Plus intensifier screens (DuPont).

S* 6.2. RESULTS A cDNA library was constructed using polyadenylated 2RNA isolated from tamoxifen treated PC-3 cells. Earlier observations indicated that tamoxifen treatment resulted in a 2- to 5-fold increase in the secretion of TGF-92 (Ikeda et al., 1987, Biochemistry 26:2406-2410). The library was screened with probes 1 and 2 as described above. Five 25 clones were obtained which hybridized to both probes: one clone, pPC-21, containing a 2.6kb insert, was chosen for sequencing. Another clone, pPC-14, containing a 2.2 kb insert, was partially sequenced. The DNA and deduced amino acid sequences are shown in figure 1.

pPC-21 contains a single open reading frame coding for a deduced polypeptide of 442 amino acids; the 112 carboxy terminal amino acids comprise the mature TGF-82 monomer (boxed in FIG. la). The first methionine encoded by the open reading frame is immediately followed by a stretch of hydrophobic and uncharged amino acids (overlined in FIG. la) characteristic of a signal peptide. Neither the nucleotide sequence coding for this methionine nor those coding for the next 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). Because translation usually initiates with the first methionine in an open reading frame and because regions homologous to TGF-1, as discussed below, occur upstream of the second methionine, the first methionine has been tentatively assigned as the site of translation initiation. It appears then, that TGF-32, like TGF-B1, is expressed as part of a much larger secreted precursor. The pPC-21 clone contains 467 bp upstream of the putative initiating methionine and a 3' untranslated region of approximately 800 bp including a 15 poly track, fifteen bases upstream of which is located a polyadenylation signal sequence (Proudfoot and Brownlee, 1976, Nature 263:211-214).

'The nucleotide sequence homology within the coding regions of the TGF-P1 and TGF-p2 pPC-21 cDNA clone was Sdetermined to be 53%. The regions coding for the mature proteins have 57% homology while the upstream precursor regions have 48% homology. After optimal alignment of the S* two sequences, several nucleotide insertions were noted in the TGF-92 precursor region, one of which extended for 25 nucleotides. Whether these insertions are due to the presence of extra exons in TGF-P2 is unknown. No significant homology was detected between the DNA sequences in the non-coding regions of the two clones. In fact, while TGF-~1 had extended G-C rich non-coding regions, TGF-f2 had extensive A-T rich non-coding regions. Both cDNA clones contain repeating structural motifs in the 3' noncoding region with the repeats in TGF-91 consisting of (purine) CCCC (Sharples et al., 1987, DNA 6:239-244) and in TGF-f2 of ATG or A(pyrimidine)(purine).

AL I. :i~iI. ii -26- Restriction mapping of many clones revealed that one clone, pPC-14, lacked a KpnI restriction site located in the amino portion of TGF-p2 coding sequence. Restriction maps of pPC-14 and pPC-21 are shown in FIG. Id. pPC-14 was sequenced over a strech of about 100 nucleotides corresponding to this region of the molecule by specifically priming with a 20-mer oligonucleotide complementary to nucleotides 277 to 296 in FIG. la. The results show that the pPC-14 clone contains an 87 nucleotide deletion (nucleotide positions 346 to 432 in FIG. la; see also FIG.

le) that accounts for the missing KpnI site and which is replaced by the sequence AAT, the codon for Asparagine. The results suggest that the pPC-14 clone encodes a shorter TGF-p2 precursor of 414 amino acids differing from the 15 sequence encoded by pPC-21 only in that amino acid residues 116 through 144 are deleted and replaced by a single Asparagine residue.

Although the entire coding region of pPC-14 was not determined, it is probably in perfect agreement with the pPC-21 coding sequence since, except for the KpnI site, restriction maps of the two clones overlap perfectly (FIG.

Furthermore, a simian clone encoding a 414 amino acid TGF-p precursor containing the same 29 amino acid deletion and replacement has been identified, as described in example 25 Section 7, infra. This simian clone has a coding sequence 25 which is nearly identical to that of the human pPC-21 clone in the regions 5' and 3' to the deletion.

Figure 2A shows the deduced protein sequence of human TGF-pl (Derynck et al., 1985, Nature 316:701-705) compared 3to that of human TGF-32-442. It was determined that TGF-92 3O is 71% homologous with human TGF-pl throughout the mature portion of the molecule as reported previously (Marquardt et al., 1987, J. Biol. Chem., in press). The amino portion of the precursor upstream of the mature molecule shows a 31% homology between TGF-P1 and TGF-f2-442. The dot matrix -27- Shomology 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 I in the putative signal peptide region reveals no significant homology.

1| In TGF-32, the signal sequence cleavage site is i predicted to be after amino acid 20 (serine) and after amino acid 29 (glycine) in TGF-91 (Von Heijne, 1983, Eur. J.

j Biochem. 133:17-21). This cleavage site directly precedes the first block of homology between TGF-9 1 and TGF-32 which extends for 34 amino acids downstream. After removal of the signal sequences, the TGF-P1 and TGF-f2 precursors would share identical N-termini over the first four amino acids, including the cysteine at position 4. Fourteen amino acids f. 15 downstream of this putative N-terminus, 19 out of the next ,21 amino acids are conserved between TGF-5l and TGF-2, a homology block larger than any seen even in the C-terminal region containing the mature TGF-3 protein. Several more blocks of strong homology exist within the region upstream Sof the mature protein as seen in figure 2A and 2B. These i domains are separated by long stretches of non homologous I amino acids.

e 0 The TGF-92 precursor has three potential Nglycosylation sites (located at residues 72, 168, and 269 in 25 FIG. la). Only the first site is conserved in TGF-f1, and lies within a larger block of conserved residues, suggesting Sthat this potential glycosylation site has important structural and/or functional characteristics.

ii After removal of the signal sequence, the TGF-32 precursor would contain either 31 or 59 amino acids more than its TGF-p1 counterpart. An additional cysteine residue in TGF-f2 is located just upstream of a large region of non homologous amino acids that precedes the mature sequence.

As with TGF-1, the cleavage site of the mature TGF-92 protein occurs just after a region of 4-5 basic amino acids -28-

S

SS S S S S.i

S.

S~ *50556

S

*i S 0 *505 as shown in FIG. 2A. The mature region contains nine cysteines. Conservation of 7 of the 9 cysteines is characteristic for the different members of the TGF-p family. Hydropathy analyses of TGF-f1 and TGF-32 reveal similar patterns in both the precursor and mature regions with both proteins being generally hydrophilic in nature (data not shown).

Figure 3A shows a Northern blot analysis using pPC-21 to probe polyadenylated RNA from BSC-40 (an African green monkey kidney cell line) and tamoxifen-treated PC-3 cells.

PC-3 cells contain three major TGF-32-specific mRNA species of 4.1, 5.1 and 6.5 kb in size (FIG. 3A, lane cells contain predominantly the 4.1 and 6.5 kb transcripts and lesser amounts of the 5.1 kb RNA (FIG. 3A, lane 1).

15 Note that the pPC-21 probe does not detect the 2.5 kb TGFal-specific mRNA species present in this cell line under the hybridization conditions used here. These results and previous observations (Sharples et al., 1987, DNA 6:239-244) suggest that BSC-40 cells contain both TGF- 1- and TGF-f2specific mRNA's. In order to demonstrate this more clearly, Northern blots were hybridized to a mixture containing equal amounts of TGF-il and TGF-p2 probes radiolabelled to the same specific activity. Lane 1 of FIG. 3B shows that cells contain the 2.5kb TGF-91-specific mRNA as well as the 4.1 and 6.5kb TGF-Beta 2 mRNA species: lane 2 of figure 3B 25 shows that tamoxifen treated PC-3 cells also contain the 2.5kb TGF-pl-spacific mRNA. FIG. 3B also demonstrates that tamoxifen-treated PC-3 cells contain more TGF-61-specific than TGF-92-specific message.

30 The identification of TGF-p2-specific cDNA clones has 3O enabled us to screen for TGF- 2 mRNA ih various cell lines.

The northern blot shown in FIG. 4 shows that TGF-f2-specific transcripts could be detected in HBL100 (a normal epithelial cell line derived from human milk, obtained from Dr. Greg Schultz), MCF-7 (a human maniary carcinoma cell line), SK- -29- MEL 28 (a melanoma cell line), and KB cells (a nasopharyngeal carcinoma cell line) contain very low levels of TGF-P2 mRNA.

7. EXAMPLE: cDNA CLONING OF TGF-BETA-2 PRECURSOR FROM BSC-40 CELLS The following examples describe the cDNA cloning of TGF-P2 coding sequences from the African green monkey kidney cell line, BSC-40, shown to contain TGF-p2 specific mRNAs (Section 6, supra). The results indicate that simian TGF- 32, like human TGF-82, is synthesized as one of at least two Slonger precursors from which the mature TGF-P2 molecule is derived by proteolytic cleavage.

7.1. MATERIALS AND METHODS 15 The following procedures were used to clone cDNAs encoding simian TGF-P2 precursor.

7.1.1. GROWTH OF CELLS AND RNA EXTRACTION cells were grown in Dulbecco's modified Eagle 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).

7.1.2. cDNA LIBRARY CONSTRUCTION AND SCREENING 25 Double-stranded cDNA was synthesized from polyadenylated RNA as described (Maniatis et al., 1982, S: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor O Laboratory, Cold Spring Harbor, NY, 371-372) and after treatment with EcoRI methylase was ligated to oligonucleotide linkers containing an EcoRI restriction enzyme recogn 4 tion site (EcoRI linkers). The cDNA was digested with EcoRI and fractionated by chromatography on Sephacryl S-1000. cDNA fractions of greater than 750 base pairs were pooled and ligated into lambda gtl0 which had been cut with EcoRI (Davis et al., 1980, A Manual for Genetic Engineering: Advanced Bacterial Genetics; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), packaged (Grosveld et al., 1981, Gene 13:227-237) and plated on E.

coli C 600 rK-mK hfl. The library was screened by plaque hybridization (Bentonet et al., 1977, Science 196:180-182) to [32P]-labeled pPC-21 and pPC-14 probes. Clone 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-92 coding sequence of pBSC- 40-1 was determined by sequencing both strands using the dideoxy chain-termination method (Sanger et al., 1977, Proc.

Natl. Acad. Sci. USA 74:5463-5467). pBSC-40-16 was partially sequenced.

7.2. RESULTS Two clones were obtained from a BSC-40 cDNA library S* which hybridized alternatively to probes constructed from the human TGF-p2-442 and TGF-92-414 precursor coding sequences.

Clone pBSC-40-16, which hybridized to the TGF-P2-442 probe, was sequenced over a 150 nucleotide strech (nucleotides 300 to 450 in FIG. la) expected to contain the coding sequence for the 29 amino acid segment from positions 25 346 to 432 in FIG. la. The results show that, over this region, pBSC-40-16 encodes an amino acid sequence which is identical to the corresponding sequence in the human TGF- 2-442 cDNA clone, pPC-21, and suggest that pBSC-40-16 encodes a 442 amino acid TGF-p2 precursor.

Clone pBSC-40-1, which hybridized to the TGF-beta 2- 414 probe, was sequenced over the entire coding region. The results show that this clone encodes a 414 amino acid TGF-92 precursor which is identical to the human TGF-p2-442 precursor except that amino acid residues 116 through 144 human TGF-92-442 are deleted and replaced by a single 3 -31- Asparagine residue. At the nucleotide level, pBSC-40-1 differs from human TGF-92-442 in the deletion region: nucleotides 346 through 432 in FIG. la are deleted and replaced by the codon for Asparagine, AAT. Except for 13 silent base changes, the two structures are otherwise perfectly homologous over the rest of the coding sequence.

8. EXAMPLE: EXPRESSION OF TGF-§2 The following examples describe the expression of mature, biologically active TGF-92 in Chinese Hamster Ovary cells (CHO cells) transfected with a recombinant plasmid containing the coding sequence for mature human TGF-92 ligated down-stream and in-frame with the coding sequence for the simian TGF-pl precursor, under the regulatory 15 control of the SV40 promoter sequences. The construct directed the synthesis and secretion of mature, biologically active TGF-p2 at a level of about 0.5 mg/L.

8.1. MATERIALS AND METHODS 8.1.1. CELL CULTURE Dihydrofolate reductase (dhfr)-deficient Chinese Hamster Ovary (CHO) cells (Urlaub and Chasin, 1980 Proc.

Natl. Acad. Sci. U.S.A. 77:4216) were propagated in Ham's 25 F-12 medium (Gibco Laboratories, NY) supplemented with fatal bovine serum (FBS) and 150 ug/ml of L-proline.

Penicillin and streptomycin were included at 100 U/ml and 100 ug/ml, respectively. CHO transfectants were grown in Dulbecco's modified Eagle's medium containing the same supplements as those listed above. CHO cells and their derivatives were routinely passaged by trypsinization at a splitting ratio.

Methotrexate (Sigma, MO) was prepared at a stock concentration of 10 mg/ml in water. Dilute NaOH (0.2 M) was added in order to solubilize the drug (final pH of The I~ -32stock was filter-sterilized and stored at -20°C. Stock solutions of methotrexate in media (100 uM) were kept at 4°C for no longer than 1 month.

8.1.2. DNA MANIPULATIONS AND PLASMID CONSTRUCTIONS Restriction enzymes, T4 DNA ligase, calf intestinal phosphatase, the Klenow fragment of DNA polymerase I and other DNA reagents were purchased from Bethesda Research Laboratories, MD. Standard DNA manipulations were performed as outlined in Maniatis, et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

Plasmid pSV2 (81-TGF-dhfr), which contains the simian TGF-Il cDNA and the mouse dhfr gene in tandem as well as 15 intervening SV40 sequences, was constructed as described (Gentry et al., 1987, Mol. Cell. Biol. 7:3418).

Plasmid pSV2/l-p2/dhfr was constructed as outlined in SSection 8.2, infra.

8.1.3. DNA TRANSFECTIONS b. Approximately 24 hours after seeding 106 dhfrdeficient CHO cells onto 100 mm dishes, the cultures were transfected with 20 ug of NdeI linearized pSV2-(pl-TGF-dhfr) plasmid as a calcium phosphate precipitate (Wigler, et 25 al., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376).

Briefly, 20 ug of linearized DNA was added to 1 ml of 250 mM sterile CaCI 2 A 1 ml portion of 2X HEPES solution (280 mM NaC1, 50 mM HEPES, 1.5 mM sodium phosphate, pH 7.1) was then added dropwise, and the mixture was allowed to sit on ice for 30 minutes. The precipitate was then dispersed dropwise over the cells containing 10 ml of the F12 media. After incubation at 37°C for 4 hours, the media was removed and replaced with 10 ml of F12 media containing 25% glycerol for seconds at room temperature. Cells were rinsed once with Ir 20 ml of F12 media and incubated in the nonselective F12 -33media (20 ml) for an additional 48 hours. Selection for dhfr-expressing transfectants was accomplished by replacing the media with DMEM supplemented with 10% dialyzed FBS (Gibco, and 150 ug/ml L-proline. Colonies were observed after culturing the cells 10-14 days in the selection media. Ten colonies were aspirated by a pasteur pipet and expanded.

8.1.4. SELECTION OF METHOTREXATE RESISTANT CELLS Dihydrofolate reductase (dhfr)-amplified cells were derived from the primary transfectants essentially as described (Gasser, C.S. and Schimke, 1986, J. Biol.

Chem. 261:6938-6946). After expansion, 10 cells were S seeded onto 100 mm dishes and adapted to increasing 5 concentrations of methotrexate. The plate containing 15 visible colonies at the highest methotrexate concentration Swas trypsinized and adapted to that concentration of methotrexate for at least two additional 1:5 cell passages.

5 Cells (10 were then seeded onto 100 mm dishes in 5 times the concentration of methotrexate. The dish containing i visible colonies was again trypsinized and adapted in the i methotrexats containing medium. Cells were frozen back at various stages of amplification in media containing 40% FBS, .10% dimethyl sulfoxide and 50% DMEM. Methotrexate was not included in the freezing media.

8.1.5. GROWTH INHIBITION ASSAY Mink lung epithelial cells, Mv 1 Lu (Accession Number JI CCL-64, American Type Culture Collection), which are extremely sensitive to TGF-P1, were utilized for the growth inhibition assay. The assay was performed using the thymidine analog 125 I]-iodo-2'deoxyuridine (125IdU) to assess DNA synthesis. One unit of activity was defined as the amount required to inhibit 50% incorporation of 125IdU compared to untreated CCL-64 cells.

-34- To assay transfected cells for secretion of active TGF-P2, serum free supernatants were collected from one 24hour collection on confluent cultures of cells and dialyzed extensively against 0.2 M acetic acid. The acetic acid was removed by lyophilization and the sample was re-dissolved in sterile complete culture medium for assays.

8.2. CONSTRUCTION OF TGF-BETA 1/TGF-BETA 2 HYBRID PRECURSOR GENE FOR TGF-BETA 2 EXPRESSION A hybrid TGF-beta precursor gene consisting of simian TGF-Beta 1 precursor coding and 5' untranslated sequences joined in-frame with human TGF-Beta 2 mature coding and 3' untranslated sequences was constructed as illustrated in I FIG. Ic.

pPC-21 was first digested with EcoRI, filled-in with 15 I Klenow enzyme, the 2.3 Kb fragment ligated into HincII digested pEMBL, and used to transform E. coli. Two clones, pPC-21/HincII and pPC-21/HincII-, having inserts in opposite orientations, were used to generate overlapping ExoIII digest fragments by digesting both with SstI and 20 0 BamHI followed by ExoIII digestion, Klenow repair, religation of the DNA, and transformation of E. coli. Two clones, Exo 5.9 and Exo 25C were found to contain different s* lengths of 5' and 3' sequences, respectively, and were G es: 2 subcloned into pEMBL to generate pEMBL 5.9 and pEMBL S* 25 pEMBL 5.9 was digested with HindIII, blunt ended with S. Klenow enzyme, digested 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.

SpGS62 was digested with BamHI, filled in 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 was isolated.

r~ pGS62/CIFB was digested with PstI and EcoRI and the 1600 bp fragment was isolated and further digested with XhoII. The resulting 400 bp XhoII-EcoRI fragment was isolated (fragment pSV2-beta-TGF (Gentry et al., 1987, Mol. Cell. Biol. 7:3418) was digested with Apal and EcoRI and the large 3000 bp fragment was isolated (fragment 4).

Two complimentary strands of DNA with the sequences shown below were synthesized, phosphorylated, annealed and ligated to fragments and described above.

CAA CAT CTG CAA AGC TCC CGG CAC CGC CGA GCT TTG GAT GCG GCC TAT TGC TTT AGA AAT GTG CAG GAT AAT TGC TGC CTA CGT CCA CTT TAC ATT GAT TTC AAG AGG 3' ee*** S 5' GATC CCT CTT GAA ATC AAT GTA AAG TGG ACG TAG GCA GCA ATT ATC CTG CAC ATT TCT AAA GCA ATA GGC CGC 15 ATC CAA AGC TCG GCG GTG CCG GGA GCT TTG CAG ATG TTG GGCC 3' The ligation mixture was used to transform E. coli and ppl/p2 was isolated.

Plasmid ppl/p2 was digested with EcoRI, filled in with 20 20 the Klenow fragment of DNA polymerase I, cut with HindIII and the 1600 bp fragment was isolated: pSV2,P1/P2 was constructed by inserting this fragment into pSV2, neo which had been previously digested with HindIII and HpaI to eliminate the neo gene.

25 pSV2,P1/P2 was digested with PvuI and EcoRI, filled in with Klenow enzyme, digested with Ndel and the 2.6 kb (approx.) NdeI-EcoRI fragment was isolated and ligated to pSV2,dhfr which had been digested with NdeI and PvuII. The ligation mixture was used to transform E. coli and pSV2/Blp2/dhfr was isolated.

-36- 8.3. EXPRESSION OF TGF-BETA 2 IN CHO CELLS pSV//l-92/dhfr was used to transfect dhfr-deficient CHO cells and dhfr-amplified cells were derived from the primary transfectants as described in Materials and Methods, supra.

Positive clones were identified by 1l .assay (inhibition of mink lung epithelial cells (CCL-64) as described (Gentry et al., 1987, Mol. Cell, Biol. 7:3418). Recombinant proteins were also detected by Western blotting using an anti-peptide antisera made against the sequence NH2- YNTINPEASASPC-COOH (Gentry et al., 1987, Mol. Cell, Biol.

7:3418) which is present in mature TGF-beta 2.

One line, 1p9, 12.5, was found to secrete 240 ng/ml TGF-92 (FIG. This line was then cloned by limiting dilution in 96 well plates. One clone, 1i9, 12.5, cl 36, produced approximately 500 ng/ml (FIG. Analysis of the protein secreted by this clone by Western blotting using anti-peptide antiserum is shown in FIG. 6, revealing the presence of the mature 24kd TGF-82 dimer as well as the larger (approx. 90kd) precursor form.

9. DEPOSIT OF MICROORGANISMS The following microorganisms have been deposited with '*the Agricultural Research Culture Collection, Northern Regional Research Center (NRRL) and have been assigned the following accession numbers: Microorganism Plasmid Accession No.

Escherichia coli HB101 pPC-21 B-18256 Escherichia coli HB101 pPC-14 B-18333 Escherichia coli HB101 pBSC-40-1 B-18335 Escherichia coli HB101 pBSC-40-16 B-18334 Chinese Hamster Ovary pSv/Bl-B2/dhfr CRL 9800, deposited 23 August 1988

Claims (21)

1. A nucleotide sequence encoding biologically active transforming growth factor-132 precursor comprising the nucleotide coding sequence substantially as depicted in FIG. la from nucleotide residue number 1 to nucleotide residue number 1326.

2. A nucleotide sequence encoding biologically active transforming growth factor-B2 precursor comprising the nucleotide coding sequence substantially as depicted in FIG. la from nucleotide residue number 1 to nucleotide residue number 1326 in which the nucleotide sequence from nucleotide residue numbers 346 to 432 is deleted and replaced by the nucleotide sequence "AAT".

3. A nucleotide sequence encoding biologically active mature transforming growth factor-32 comprising the nucleotide sequence substantially as depicted in FIG. la from nucleotide residue number 991 to nucleotide residue number 1326.

4. A biologically active transforming growth factor-32 precursor comprising the anino acid sequence substantially as depicted in FIG. la from amino acid residue number 1 to amino acid residue number 442.

5. A biologically active transforming growth factor-B2 precursor comprising the amino acid sequence 2tO substantially as depicted in FIG. la from amino acid residue number i to amino acid residue number 442 in which the amino acid sequence from residue numbers 116 to 144 is deleted and replaced by a single Asparagine residue.

6. Biologically active transforming growth factor-82 precursor comprisini the amino acid sequence substantially as depicted in FIG. la from amino acid residue number 20 to amino acid residue number 442. factor substa residu amino delete factor 10 as de to ami active 15 growth coding nucleo

1755. I I I i i i growth compri depict amino growth compr depict amino transf 37

7. Biologically active transforming growth factor-B2 precursor comprising the amino acid sequence substantially as depicted in FIG. la from amino acid residue number 20 to amino acid number 442 in which the amino acid sequence from residue numbers 116 to 144 is deleted and replaced by a single Asparagine residue.

8. Biologically active transforming growth factor-B2 comprising the amino acid sequence substantially as depicted in FIG. la from amino acid residue number 331 to amino acid residue number 442.

9. A nucleotide sequence encoding biologically active hybrid transforming growth factor-El transforming growth factor-S2 precursor comprising the nucleotide coding sequence substantially as depicted in FIG. lb from nucleotide residue number -70 to nucleotide residue number 1755.

10. A biologically active hybrid transforming growth factor-El tranforming growth factor-E2 precursor comprising the amino acid sequence substantially as depicted in FIG lb from amino acid r esidue number 1 to amino acid residue number 390.

11. A biologically active hybrid transforming growth factor-El transforming growth factor-82 precursor comprising the amino acid sequence substantially as depicted in FIG. lb from amino acid residue number 30 to amino acid residue number 390.

12. A method for producing biologically active transforming growth factor-132 comprising: culturing a eucaryotic cell containing a nucleotide sequence encoding transforming 3 9 38 A oo 6 *6 o o S soo oooooo oSI oooo -39- growth factor-82 under the control of a second nucleotide sequence that regulates gene expression so that a peptide or protein having transforming growth factor-fi2 activity is produced by the eucaryotic cell; and recovering the transforming growth factor- 2 from the culture.

13. The method according to claim 12 in which the nucleotide sequence encoding transforming growth factor-i2 comprises the nucleotide sequence substantially as depicted in FIG. la from nucleotide number 1 to 1339.

14. The method according to claim 12 in which the nucleotide sequence encoding transforming growth factor-S2 comprises the nucleotide sequence substantially as depicted in FIG. lb from nucleotide number -70 to 1755.

15. The method according to claim 12 in which the 20 eucaryotic cell comprises a Chinese Hamster Ovary cell.

16. The method according to claim 12 in which the second nucleotide sequence which controls gene expression comprises an SV40 promoter.

17. The method according to claim 12 in which the second nucleotide sequence comprises a promoter and a sequence encoding a selectable marker for which the eucaryotic cell is deficient, so that the eucaryotic cell containing the transforming growth factor-i2 coding sequence can be identified.

18. The method according to claim 17 in which the selectable marker comprises dihydrofolate reductase.

19. The method according to claim 18 further comprising exposing the eucaryotic cell to methotrexate, so that resistant colonies are selected which contain amplified levels of the coding sequences for dihydrofolate reductase and transforming growth factor- 62. The method according to claim 19 in which the eucaryotic cell comprises a dihydrofolate reductase-deficient Chinese Hamster Ovary cell. 21. A method for producing transforming growth factor- 2, comprising culturing transfectant 149, 12.5, clone 36 as deposited with the ATCC; and recovering the transforming growth factor-Beta2 from the culture. 22. The method according to claim 21 in which the transfectant is cultured in the presence of methotrexate. 23. A eucaryotic cell containing a nucleotide sequence encoding transforming growth factor-yf2 under the control of a second nucleotide sequence that regulates gene expression so that the eucaryotic cell produces active transforming growth factor-,92. 24. The eucaryotic cell according the claim 23 in which the nucleotide sequence encoding the transforming growth factor- 2 comprises the nucleotide sequence substantially as depicted in FIG. lb from nucleotide number -70 to 1755. The eucaryotic cell according to claim 23 which comprises a Chinese Hamster Ovary cell. WD a .0 a 26. The eucaryotic cell according to claim 23 in which the second nucleotide sequence which controls gene expression comprises an SV40 promoter. 27. The eucaryotic cell according to claim 23 in which the second nucleotide sequence comprises a promotor and a sequence encoding a selectable marker for which the eucaryotic cell is deficient, so that eucaryotic cell containing the simian transforming growth factor-f2 coding sequence can be identified. 28. The eucaryotic cell according to claim 27 in which .the selectable marker comprises dihydrofolate redu'tase. 29. The eucaryotic cell according to claim 28 which comprises a dihydrofolate reductase-deficient Chinese Hamster Ovary cell. 30. A cell line comprising i, 9, 12.5, clone 36 as deposited in the ATCC. 31. A nucleotide sequence encoding transforming growth factor-f,2 precursor substantially as hereinbefore, e:cirbod with reference to any one of the figures or examples. 0 32. A transforming growth factor-,8 2 precursor substantially as hereinbefore described with reference to any one of the figures or examples. 33. A method for producing transforming growth factor- 2 substantially as hereinbefore described with reference to any one of the examples. 34. A eucaryotic cell containing a nucleotide sequence encoding transforming growth factor-A62 substantially as hereinbefore described with reference to any one of the examples. DATED: 5 October 1988 PHILLIPS ORMONDE FITZPATRICK Attorneys for: f f. .BR-I-STOL-MYERS-COMPANY- r- i j Mo^ r ^i i ^wj -41 21'2''(CTC2'2 VAi (01"II'1L'2~f'0l 0'AC cO2Ao'oIr(' lct llllcr0'.,10l(;2100 -391 ClAC IIl 'A0iA1 MXVC2Ar 'C(CCC;CtI.CAA:I(AA*CCCIICicrcrAh--ArrrAIIATG(ACT-r111'*r -29 B C0C0hTI 'CAIlCI cCc AA1-r71 02-r11':'"Icl-ro*C2 (,tlcC'~'Aoomc,; 'rCCC'roGCCCGToCC't A'rrOA'Iol-r1CCAo01-1,1'-r oo'2 21~*[C -199 OccO2I'rc-Il-2-irAAArO2IAAri-cAAlArAcG2'2'ir'rrici,1l'I0JLCllGC'~rr7CAAiOi2A7VlrcA0O1.rrcTCAA,\ 0 Cp,A 0 0 AC 0 -100 AAO('CAAACAACTOI'CI0TrI'OI ArACTT'rl'llO210T'liri112'1il"--l-I-l'r.rAcnj1'0AA 0CAAL l..n..CCA,~rln.IAAAAA I 229Iti Tyr C y, Vol Siet 020 Pike IAM~ 1ie10 lIS le Val Th r Val Ala Leou SerI Sep Tlkr CyOr AlC VAC TAC 101' 1122 010 AIIO ocr 111' CIO AlO 012G CAT Cr0 GTC 000 OTC 000 crc GCo C"c Tcr ACC TUC ACC T1, 45 'r cAlp Hut As'p G2 1 H,,t Org1 Ii' Ar q 11. G 10 A I lie Arg Oly Gii, Ii a2.00 Sur Lys I~ yn

20, At:A ClO' 210r AIG CAC CAG 'T21 ATG 022 AAG 0210 020 GAGOC00 ATC COC 000 Car1 ATC CTG ACO MG0 el2 C AG crC ISO I ir 02,,Ii Ii Al, Ty r Pi, it Gil Gill r2, I Ii. V.I2rP. ,l Val 11. S, IeI Tyr Ann S. r 12l,, h-, AC C A T C0 C 0A GAO G00 TAT' CUIT GAG 000 (10 GAA arc cCo CCG GAG OCr Oir' Toc or e AC 0 000 CC AGO 225 0 S 95 Aspt- 2o,, 20 21 n1, GiLs Ain Ser Aril Atg 020 1 Ala, Ala Cys Clu Arg GIG 0,9 Ser A.11C, G il 0 T, yr Tyr Al. GAC 120 U1 CT A UG. 0021 (102 0210 020 02 GCU 000 000 TOO GAG 000 GA0 0210 AGO 00 00 00 rACAGTc TOO COCC 300 2210 1.20 IV C 02, 092 V '1yr Lyn 2Ile Asp Hitt Iro N (1.0 ro, SrOr G2, I u 21r Vol Cy. P'ro VolI V.o l r Ill r Pro Ser 00G0 00 02 TAC AAA 010 GAC AOrO 000 000 '2C 'prO ccc TO 000 L~cr 010 TOO CC0 011' 01 000 0C0 000 TIcr 1 0 00 2IS 1245 G ly Sur Vol ay IVSet' 2.0, Oyo Sur Art; .1.i Ser- ln Vol LAU cys Oly Tyr 1,a0 h0.V02Al 1, plrs TlIle *G *C C1 0000 20 A0 0C 21 100 icc 000 000 TOO 000 010 crc TOT 000 TOO Cli' CA] CCC ArC 0CCC 1 10 202 170 ***TrArtl Iro Tyr 1:11c Org 1 I1c Vol 0, ,9 Ili2e Asp V.1 I Sur Alo Hot Gb AlYa 00ti Alo Sep 00(2i 2.00 Val LYe AlIe 10 AC AA ccc 100 I1 02C10, APIr OTT 0210 112' (100 Gcc rco 2100 AT U0 00 O A AT ocT oCQ OLAT 210G 011 AAA 600 525 r1 G *6 00 Il2li, 222 Ie 2(1 IVc, 02 09,, A ~k nirPe -lyeR Ala Org Vol Pro 010 Oln Org Ile t2il(, Leo Tyr Ole Ile LE- e A a0 Vrc ,0A G I C 11T Cr TI1 0U 2 CA(; 000 000 600 000 020 COT 000 CAO 000 OIl' 000 Cr& TAT CooG Ai' crc 600 S2220 220 lysa SOP Alip(.D 2,0 ~r Iro 1l, r 02,, li A Tyr li. Aep Lys Vol' V.ol l.Ye Ir O Al. 020 ly 02,, a. aA C A AA t00 V0 102 22 oCA 'r Co oC. co A C CooC TAC 010 000 A C A00 OT'G11 000 000 00A, GCA 00),UC 000 25 0, 22 245 *Trp 1i.0l Sop l'2e Aep 0,2 I Ir hall, Ole Vo I2We,. Ilit lI( II I-lila 11. LYS Asp Org Ac l. IlUIy ly ie. 1021e 01 100 1-rO 002' 010 ACT COT 001' OTT 001UA 100 TOO CI' CA0 OAT A00 60,. 0AV0 000 OraG 060 TIT 000 AT 150 Scr 2I0, 1"sa Cye Ir Cys Oye i PI,le v l I P Ser A0.0 ?,an Tyr Ile lie Pro Acas Ly. Oar Gl,62 leo 020Ul 22(5 295 so 02a Org I'lie 020 Oly lie Aep Cly Tihr Sear Ilr Tyr Ti, SoP Oly Aop Gln Ly a Thr Ile Lys aSr TI,, Org Ly. a G0 A00 00 iTT 000 GOT 01'I~ COT 000 000 ,0 0C00e TAT00 ACAT GOT OAT 006 AAA OCT ATO 000 TOO ACT 000 0A0 900 3 1 320 YS Aynn 09, Oc ly l.ys 1Iher 1,0 .il 1 7" Hot 1-u 1eu P'ro Sar Tyr Org Ie., G2 I cSr 0G, I l,,i Tir Anti a ta 00 AA 0 0211' ACT 2 001'A, AC0 000 CATer cr0 (22 cr0 oO TT0 WC0 000 TOO TOO 000 0'21' 000 TOO 000 C00 000 000 915 *e 00 02 00 006 001hGCG GOT T121 OAT (100 000 TOT TOO '111 0G0 OAT OTO 000 OAT OAT TU OO oCT C1 UT 00001rT12050 eat a 361 330 TV IeI Aslp PIhe Ly. Org Asp leo dly T i'.lYe Trp Ie III 2, Me a Gill NOLY y Tyr A i Ala Ann l'le Oya Ala' a TAO OITT OAT 17C 000 060 GOT 010 000 100 AAA TOO ATA 000 600 000 A00 006 TAO OAT 000 000c rr0 TOT GOT 1125 Oly l Cys Pro 'ryr 1.01, Trp 05', Ser Ault TI, (11 nIlls Oar Arg Valkm Ler Im leo Tyr Aan TI,, lie Ann Pro a(1 00 1A 1 U 1 j OCtiTT 110h I GU. AlIT TINh 1100 002 000 C00 0C0 000 GTC 06 000 110 TOT OAT 000 OTO OAT 000 1200 O 00 420 40 a o At u1 0& OCar AlT Oa .r Oey Vol 0 Ole 1 oOP 1.00 010 Pro 1.0 TIhr II.La Tyr Tyr lie GIYly L Thr 00 000 Ter COT 1o'i cer Icc ioc GIG TOO ('00 OAT 'rro 000 C Cro ACC on' crc TOO 011 000 AAA 000 023'. P .ir. LYS lie Gila Oln les Stir Ann(e Val lye Otir Cy. S, Oar* 000 006 Oil' 000 COO 011' Ter OAT OTU 011' OThAA00 Ter' TOO 00 TOO AGO TOO 0I11'T OOOOAAhGTOOOA0000000 1356 AI'CTACA1rTAih(ATA I002A1 CGCAC011000000210000ff11T111AAOOAOAOOATAA\ACrOOOOOOAlhl. O1'Irwcr22iTOO irOiOAAOOOOOO 2050 000211 0101 AAoAoCA OOTAAMC1i'ArAOO~IIyWI OO GCTo21V1'000600cl1000 01 OAAAO Ac A1ooOoioOTOTAGC00i' 2950 T tT1'T11T000000T00000000101"IAAI AAAOAOOhCIT1'002T000 1l'oly A) 22112 0 S. S S S 0 161 GGiAchiincGG-.-nnarln rotliririircnocir rcculIc IlroI I ArrlccaranlwlFcwGlrcrCCCrcccnnrCCTCCCCrCCnCCnCCOCCCiiccinrn-Ir-.it (nriIrin--icr nl L..S V L Il- A L L"r L- nI. I. V.1 Lf A I P It- il.1 l .10 C Al To anCI t to. Ti.. A T :in C l l. TO C11, I UTf AIn r. I I u G uIln I. 1',i I q,. PPA l. t- T ly Lij .L S I V.1- ri-. q V. II 11.1 t V-1. L. SI.. II. L A n U .Pi y c.Cr CDc rci cii pcAl A O r c n r c cn rc GTTia G r cur a r-IG nri O_ tc ai cr GIG n1l' crIli f. O 1)?a Gin. A r l* GL- Ly L- L. Ar i.S.,IU 9 P- P- a S 6 1 GI I,r v.1 IV- Or G r L T. Tcc cn aCf Ow cIar ci c IX~ irc flA IT Ac Ar A (IO GA cinn i G irl GA CiG aT. C crl A n ir 'isr AL- in- A. Tn. lr g u V.1 Al ar, j S. flr.5.. a.,a an Lf l. fl 1 rC C 0 o CC a cc G C I, inc I crc hc vrc cT Cia Cra Oca ccc, aGC uic tc cO r IG CUG1,. riG.. Ci ill l.3 "a 1 1 30 Vl T1. V.1 i. A. 01. Vr TO i. a2 V* ar- a 1 GO, 1 i. Ar S. Si 1P An i r c r c n ll aT cl nA a a a arir n nCC ci il cn C r o cr c-i it a IIL ric 13 19 1 31 I~ nU M AI, i. ,U h. .1 Th A ti 01 nl, 1 Ir ,at V A.T I Al T 1 I i S XA OA G r AC cn7 L.inL n' ro L A Ic an V.c AcI 1 Gil-It, dGn I III nc l Ay Air CC aAa IC car cc nc cm Ci ccG ncc AI nccan cn C11. ni1 r.C P CA C IA Cnll Miin wi 5 5* *5 S* S S. S S S 55 5 S. 'W" A.-~Ai Alm. A. T. l. ~I611N,11.yr Cyn MrI.T3r Ar .Ia Gin A.1) hall cyir Cy.Ir t LirrI lr. IrA 'l C 13ca COA 1 11cr 2; TGA 0131 0cc 1c TAT 1-cc TITT AG0A MT 01G TO 11131-T 1313 1d 1TM CT3 COT 1(13 cTSI _31o U10 Tyr Ilaml O I. 3 1.V~ Ay Nirl Aftp lal DIV TtIt 1.y T Trp. Illi Gilt p~ro yn TOOy 1-yr 61111 Ann nlII. CV. Aim _T1C All' GTM 10 h33 G31 G131- CTA 0COG -GO 1313A Too AT1A CAC GMA ccc MAA GO 1-Ac AAT OG C 330rC TOT OcT j,- Gly AIR Cyn II,. Tyr loll Tip siir 0Si Anmp 1-hr Gil, IlI. Olr Ag i-q 11111 I"11 t0. i T.11yr man, Thr I I" A3."1 PIT, GO13 OCA IOaC ccG 1-AT1 i'A TOO1 13T 1-cA GAc ACT Cho 0130 PGc AGO O1-C CTIG A3CC '111 1-AT1 13131 Act: 11 13131 CCA l)I Gi him Ser Nl, Ser P'ro Cyr cys COTTi q1at.I Oil r I 13'I.41 MI Pro- 1.11U 1lrI it lll 1-yr 1-yr Iiia Gly lyn 1-hr 0131 lCN 1-CT OCT 1-CT COT 'Ic GC I al 1-c O NN 0131 1-13hCI CATM h e TA A3CC Ni CTC 1-NC TP. C Ari- GOC 1313 AGA i't a yr F, d ill Gin ,LoG Sor 131.n Htit Ili Val yn a get Cyri lyrit Cyri sl' ccc 13131 1311 013 CAD0 Cli' 1CT 13131- O Al-0 ITA 1N AG TCT 1-GO MAN C t 1-11TAA111 lVc'lT033GGcAGc1N 2C. ACAcrTAOCNccICC1311c IACAcIi'ACiA~c~nrrl~nlih~tN1-1-GATTG.1CAC1T131-N~lIo13Nc'l-GCI~rl-Occcl,TGIIAAt(A1333I~ A CA CAmC A CAt, A C mA A I;GA AA Ar C VAII At .1 itI A 111;CI'll CGA CC (ll CGC C CAC~lU MT 11 M0TA I I l 1 I- 1C ATG110 C lrCCITC I T TCT AG A3cGI'TAA A I I-r-rcN0ITcN I al TO Tr6)TTT AlUAAC T171)C7 l GAAAT FIG. Ic a 0w a a a a. S 0O S SS 4 TGF-b.*a I Precursor Mature TGF-bota I 3' S. a 5* 4 5* 6 5 iITGF-beta I -I TGF-beta 2 as.. 5.59 *a9505 0 S S S. 5* 5 0* 05 A,S1Plld A6- 1.2 APrillursor 66174 9 9 9 9 S 9 9 9 99 0 9 9 9 9 999 99 99 49 9 S 9 9 9 9 9 9* 9 *C FiT&.1J *d49 9949 9 S ~.9 pPC-14 (2-2 kb) EcoRI PstI Sst I I i ATG HindlE SphI 4 Hinc RL HindilI 1 1 pPC-21 (2.3 kb) ElcoRIL PstI Ssf I ATG KpnI HndI SphI HinclI HindII[ I FIG.1 J 101 Li-5 L'i s Clu Val Tyr Lys Ile Asp M'et Pro Pro Phe Phe Pro 5cr Giu Asn Ala Ile P1. P ro Thr Phe Tyr Arg Pro -ryr pthe Arg MAG GAG G 7 TAC AAA ATA CAC AIG CCC CCC iTTC CCC TCC GMA MT CCC ATC CCG CCC ACT ri-C TAC AGA CCC TAC TIC AGA Thr Val Cys Pro Val Val Thr Thr Pro Ser Gly 5cr Val Gly 5cr Leu Cys 5cr Arg Gin 5cr Gin Val Leu Cys Gly tylr Leu CT C TGC CCA CiT CiTT ACA ACA CCC iTT CCC 'iCA GTG CCC AGC =1C M- TCC AGA CAG -FCC CAC G-iG CI-C IGT GCCC TAC C1T iKpnl 130 ile Val A-Pr CIT Asp CAT pFC-14 (2.2kS5) pPC-21 (2.3kb) n, t. I B0t6 2 D-t. I 13ot a I De t 2 n.t. 2 b~9 FY

2 1 KIYCV SA-IP LIILTL VJ I, L T S I, D I 47 G ,S R AM Q C P (;PL E M AL "ST; )1 A G E: A E3 K I V Q P V VC I (4 N S T LK 0 C K A iYKrITCL E Vt ECV-Ui 11 SLCS 0S0V IP~ n. C 12 l V0 A Ippr VA V K VTRK r b V S A ~x K o SI~~ 2C2 L-tS -E QII [TA- KYVY~ O P r~ V CVITM IIM.AI 72 T VAlVJJQI PAVM~JK~22.2 h.S~T~TQjjIS V K 2 (ltll T 1 LqVDIIIG 1 rITTMRIoK G(ATIIIOKIV-;-KIPrLLK 267 1IP HRS EC E JA R FA y T V S 0 KT I KS T K K S K T 1 2621 A TI'L MAQ Q11 LQ 5 IIKKt 2 T 11~f M S S T E .K V V Q I' V ID r 1KID)L 209 L 0 T NIC K Rh KlFE K~ AF Fpyj R 1S KV Q L PTQYiI D r L 229 1 KWIEKyJI JAiJJIA2 JII MQAMQ 1jjJI0cj 225 A CViPEA t I'bIF1VU7IMR K[ K V s TL I V R 405 Sm T I LJ L~I[ a K T I [CQ1 tSI I V F 6 6 6 6 e 6 *666 0 0660 6e 6 60 *6 6 300 F 66 6 6 6 6 *6 L 0C I- *6 6 6666 66*006 6 66 0 6@ 6 6 6* 100 200 300 '100 TGF-f32 FIG. 3 A B JS444@ 4 4 4 4 4 4 4.4. 28S- a 4. 44 4 4 44 -18S 18S~ I ~r -2.5kb (p1) 44. 12 2 .4 4 94 44 4 4 44 a 4 4 4.44 4.444. 4 4 .4 4 4 4 44 44 4 4 44 4 44 FIG. 4 S S S S 0SOS S S* 55 S S SO S S S SS S S S. S. S S SS*S S 55.. 55.5.. S S 55 SS S S S S S 55 1 23 4 6 S S S S S S S S S S S S U S S S SS S S S. **SS S 5555 S S S S*S S S 5555 5 S S S S S S ,go 310 12 -j /Stae Q~oI0.1