WO1993007173A1 - Transforming growth factor-e - Google Patents

Transforming growth factor-e Download PDF

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WO1993007173A1
WO1993007173A1 PCT/US1992/008417 US9208417W WO9307173A1 WO 1993007173 A1 WO1993007173 A1 WO 1993007173A1 US 9208417 W US9208417 W US 9208417W WO 9307173 A1 WO9307173 A1 WO 9307173A1
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tgfe
pro
cells
seq
glu
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PCT/US1992/008417
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French (fr)
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Jaroslava Halper
Pamela G. Parnell
Royal A. Mcgraw
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University Of Georgia Research Foundation, Inc.
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Publication of WO1993007173A1 publication Critical patent/WO1993007173A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • This invention relates to the isolation in substantial purified form of a protein found in both normal and neoplast epithelial tissue and body fluids, which protein acts as a mitog and progression factor for epithelial and fibroblastic cells.
  • this material is useful in cell culture and i wound healing and the treatment of burns in humans and animals as well as for treatment of cell proliferation defects i epithelial tissue.
  • TGFs Transforming growth factors
  • TGFs are a family of acid- an heat-stable transforming polypeptides which reversibly caus nontransformed, anchorage-dependent cells to assume a transforme morphology and to form progressively growing colonies in soft aga (De Larco and Todaro (1978) Proc. Natl. Acad. Sci. USA 75:4001 4005; Ozanne et al. (1981) J. Cell Physiol. 105:163-180; Robert et al. (1980) Proc. Natl. Acad. Sci. USA 77:3494-3498; Moses e al. (1981) Cancer Res. 41:2842-2848).
  • SGF sarcoma growth factor
  • M Moloney murine sarcoma virus
  • TGF ⁇ competes with EGF for membr receptors and induces the formation of small colonies of nor rat kidney (NRK) cells in soft agar; its colony-forming respo is not potentiated by EGF (De Larco and Todaro (1978) sup Todaro et al (1980) supra; Massague (1983) J.
  • TGF ⁇ is a M. 5,600 single chain polypeptide with intrachain disulfide bonds. Amino acid sequence analysis of r TGF ⁇ shows that it shares limited homology (30-35%) with mouse E (Marquardt and Todaro (1982) J. Biol. Chem. 257:5220-522 Marquardt et al. (1984) Science 221:1079-1082); Frolik (198 Proc. Natl. Acad. Sci. USA jJO:3676-3680) .
  • TGF ⁇ a potent mitogen for mesenchymal and epithelial cells in monolay culture, but only a weak stimulator of anchorage-independe growth of mesenchymal cells in soft agar (Anzano et al. (198 supra) .
  • TGF ⁇ acts synergistically with TGFB promoting soft agar growth of nontransformed NRK cells (Anzano al. (1983) supra: Anzano et al. (1982) supra) .
  • TGF ⁇ has be identified in embryonic tissue (Twardzik et al. (1982) supr . tumor tissue and transformed cells (Marquardt et al (1984) supr Anzano et al. (1983) supra; Anzano et al. (1982) supra: Marquar et al. (1983) Proc. Natl. Acad. Sci. USA £0:4684-4688) , as we as in normal adult cells, including skin keratinocytes (Coffey al. (1987) Nature 328:817-820) , human eosinophils (Wong et a (1990) J. Exp. Med.
  • TGFB does not compete for EGF recepto (Roberts et al. (1981) supra) and requires the presence of EGF TGF ⁇ to induce formation of large colonies of NRK cells in so agar.
  • TGFB is a M-.25,000 polypeptide consisting of two identic 112-amino acid chains held together with disulfide bonds (Assoi et al. (1983) J. Biol. Chem. 258:7155-7160; Frolik et al. (198 Proc. Natl. Acad. Sci. USA 80.:3676-3680; Roberts et al. (19 Biochemistry 22.:5692-5698; Derynck et al.
  • TGFe epithelial tissue-derived transforming grow factor
  • TGFe Parti purification and characterization of TGFe from bovine kidn revealed it to be a single , 23,000-25,000 polypeptide chai requiring disulfide bonds for maximal activity (Halper and Mos (1987) Cancer Res. .47:4552-4559).
  • TGFe is distinct from othe characterized growth factors and hormones in its ability t stimulate soft-agar growth of SW-13 cells.
  • the only other growt factors known to possess this activity are basic and acidi ibroblast growth factor (bFGF) , which are distinguished from TGF by molecular weight, the inability of bFGF to stimulate the sof agar growth of A431 cells and the inability of TGFe to bind to th bFGF receptor.
  • bFGF basic and acidi ibroblast growth factor
  • TGF ⁇ , TGFB, EGF, and bombesine do not possess SW 13-stimulating activity.
  • SW-13 cell soft agar growth is inhibite by TGFB, and SW-13 cells do not bind EGF.
  • TGFe is not related t the family of insulin-like growth factors. Neither insulin Insulin-like Growth Factor 1, nor Insuin-like Growth Factor stimulate the multiplication of SW-13 cells, thus indicating th TGFE is not related to the family of insulin-like growth factor
  • TGFe acts as a competence or a progressi factor, and to examine its effect on several fibroblast a epithelial cell lines of nonneoplastic origin (Brown and Hal (1990) Exp. Cell Res. 190.:233-242) .
  • the results establish t TGFe is involved in both monolayer growth and anchora independent growth.
  • the mitogenic activity of T for monolayer growth is slightly less than that of bFGF or PD equipotent to that of EGF, and greater than that of IGF-1.
  • time course of stimulation of DNA synthesis induced by TGFe AKR-2B cells is similar to that induced by EGF, IGF-1, FGF, PDGF.
  • TGFe either alone or in combination with IGF-1, stimula [ 3 H]-thymidine incorporation after a G remedy/Gj lag of 10-14 h and pe at 19-22 h after growth factor addition.
  • T stimulates DNA synthesis in AKR-2B cells after a GQ/G X lag of h (Shipley et al. (1985) Proc. Natl. Acad. Sci. USA 82_:4147-4151 which may be attributable to an indirect mitogenic effect of TG through the autocrine production of PDGF-related peptides (Le et al. (1986) Proc. Natl. Acad. Sci.
  • TGFe appea to act as a progression factor in both Balb/c-3T3 and AKR- cells, as suggested by the requirement for continuous (rather th transient) TGFe exposure to achieve maximal stimulation of D synthesis (Brown and Halper (1990) supra) .
  • the granulins are candidate growth factors discovered human and rat inflammatory leukocytes and bone marrow.
  • cD analysis revealed that prepropeptide for the human granulins a 593 amino acid glycoprotein, with seven tandem repeats of t 12-cysteine granulin domain.
  • the message corresponding to th cDNA is found in myelogenous leukemic cell lines of promonocyti promyelocytic and proerythroid lineage, in fibroblasts, epithelial cell lines and in kidney.
  • Granulin A is believ identical to epithelin 1, and inhibits A431 cell proliferation culture (Bhandari et al. (1992) Proc. Natl. Acad. Sci. US 19.1715-1719) .
  • Epithelins 1 an 2 are cysteine-rich growth modulatin proteins, and have been purified from rat kidney. Both epithelin 1 and 2 inhibit the growth of the A431 cells, which are derive from a human epidermal carcinoma (Shoyab et al. (1990) Proc. Nat Acad. Sci. USA 87:7912-7916) . By contrast, bovine TGFe suppor anchorage-independent growth of A431 cells.
  • TGFe differs from keratinocyte growth factor (KGF) , a memb of the FGF family produced solely by mesenchymal tissue whi stimulates paracrine growth of adjacent epithelial tissue (Fin et al. (1989) Science 245:752-755) . Limited studies indicate K to be a specific mitogen for epithelial cells (Rubin et al. (198 Proc. Natl. Acad. Sci. USA 16:802-806). In contrast, TGFe present in higher concentrations in epithelial organs than mesenchymal tissues (Halper and Moses (1983) supra) .
  • KGF keratinocyte growth factor
  • TGFe w first described as a growth factor possibly involved in autocri stimulation of anchorage-independent neoplastic cell grow (Halper and Moses (1983) supra)
  • its presence in normal tissue plasma, and platelets demonstrates a role for TGFe in normal ce growth.
  • Bovine TGFe has cross-species activity on human cell for example, it supports anchorage-independent growth of hum A431 cells, D562 squamous cell carcinoma cells and T24 bladd carcinoma cells.
  • bovine TGFe stimulates growth monolayer culture of normal human epidermal keratinocytes.
  • Bovine kidney was selected as a source of TGFe because of t high SW-13-stimulating activity content present in nonneoplast kidney and because of its activity on other human cell lines.
  • T acid-ethanol procedure of Roberts et al. (1980) supra f was us as a first purification step. Protein recovery was 15-20 protein/g of wet tissue. EDso, defined as the concentration protein required to give 50% maximal response measured as SW- colony-stimulating activity, was 40 ⁇ g (Halper and Moses (198 supra) .
  • a second purification step was accomplished with a Bi Gel P-60 molecular sieve column. Protein recovery was 5-10% material applied to the column and the ED 50 was 10 ⁇ g, representi a 4-fold purification.
  • Active P-60 fractions were pooled a applied to a molecular sieve Spherogel TSK SW3000 high performa liquid chromatography (HPLC) column. Protein recovery was 5- of that applied to the HPLC column and the ED J Q was 50 representing an approximately 800-fold purification over the ac ethanol extracted material. The next purification step, utilizi reverse phase high performance liquid chromatography (RP-HPLC yielded a protein recovery of 3.5% and an ED 50 of 3.5 ng.
  • HPLC liquid chromatography
  • Protein recovery from the P-60 column wa approximately 10-15% (of the material subjected to molecular siev chromatography) with an ED 50 of 200 ng.
  • the most active fraction from molecular sieve chromatography were pooled and furthe purified on a heparin-Sepharose column (Shing et al. (1984 Science 223:1296-1299) .
  • TGFe eluted as a major peak from th heparin-Sepharose column, with a protein recovery of 4.7% and a ED 5 o of 5.0 ng.
  • ED S0 could not be determined on this and subseque steps due to the small amounts of protein recovered.
  • Pool samples were rerun on the same column and the most acti fractions from 6-8 HPLC purification runs pooled for fin purification. Proteins were separated in a 10% to 20% gradie SDS-PAGE gel. Five percent of the total protein was run in separate lane and silver stained for assessment of purity a identification of active bands eluted from the gel containing 9 of the protein. Protein from the lane containing 95% of t protein was electroeluted (Jacobs and Clad (1986) Anal. Bioche 154.:583-589) , and repurified by HPLC using a microbore RP-3 column.
  • FIG. 2 HPEC Purification of TGFe 60 ⁇ l fractions w collected and 5 ⁇ l aliquots were assayed for SW-13 soft a stimulatory activity.
  • FIG. 3 SDS-PAGE of TGFe after HPEC purification left-h column purified TGFe, right-hand column molecular wei standards: A single band with an apparent molecular mass
  • TGFe The transforming growth factor, TGFe, has been isolated f the irst time with sufficient purity and in sufficient quantiti to allow determination of its amino acid composition and sequenc TGFe is a glycoprotein with an M, of about 25,000.
  • TGFe Three partial amino acid sequences have been determined f deglycosylated bovine TGFe: an amino-terminal amino acid sequen of purified TGFe, N-Asp-Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pr Asp-Asp-Tyr-Thr-C (SEQ ID NO: 1) , and internal amino ac sequences N-Pro-Glu-Pro-Lys-Lys-Pro-Glu-C (SEQ ID NO: 2) and Xaa Gly-Leu-Ala-Ala-Ala-Gly-Pro-Ala-Pro-Ser-Glu-Ser-Gln-Glu-Lys-Lys Pro-Leu-Lys-Pro-Glu-Gly-Ala, where Xaa is Ala or Pro (SEQ I NO:48).
  • the invention provides full-length cDNA encoding animal o human TGFe, which coupled with a known expression system, can b used to synthesize active TGFe in a cell culture system. Hos cell synthesis of TGFe allows production of commerciall significant quantities of TGFe protein, either glycosylated o unglycosylated depending on choice of host cell. The inventi further provides a method of purifying TGFe in high yield, wi an ED 50 of about 0.5 - 1.5 ng for stimulating soft agar growth SW-13 cells.
  • the cDNA encoding bovine TGFe can be used as a pro to isolate cDNA encoding human TGFe. Primers derived from t cDNA encoding bovine TGFe can be used to amplify DNA sequenc encoding human TGFe.
  • SDS-PAGE Sodium Dodecyl Sulfate polyacryla ide gel electrophores
  • Samples were heated in boiling water for 3 mi and run on a 10% - 20% gradient acrylamide gel, or on a 12 acrylamide resolving gel in a Tris-glycine-SDS buffer. The g were then fixed in 50% methanol with or without 10% acetic ac washed with water and 10% glutaraldehyde, and stained with sil nitrate.
  • cDNA methods involve the in vi synthesis of a double-stranded DNA sequence by enzymatic rever transcription of messenger RNA (mRNA) isolated from donor cell cDNA methodology is the method of choice for isolating the D sequence of a gene when the entire sequence of amino acid residu of the desired polypeptide is not known. Standard procedures a known in the art for the preparation of plasmid-borne cD libraries derived from reverse transcription of mRNA.
  • Expression refers to the transcription and translation of structural gene so that a protein is synthesized.
  • bovine TG is exemplary of, and homologous to, TGFe from other mammali sources including humans. Many growth factors share extensi homology with one another.
  • Bovine TGFe is sufficiently homologo to human TGFe to bind to the appropriate receptors on human cell
  • the purified DNA encoding bovine TGFe is suitable to provi primers for amplifying DNA encoding human TGFe, or for probing nucleic acid library to isolate or identify nucleic acids encodin human TGFe.
  • the techniques of purification disclosed herein wil be understood to be applicable to purifying human TGFe to comparable level of purity.
  • TGFe exis various forms of TGFe exis and are included within the present invention. All have th biological activity defined for TGFe and include high molecula weight TGFe, low molecular weight TGFe, glycosylated forms o TGFe, including forms having various intermediate stages o glycosylation. Also included herein are forms of TGFe of human bovine and other animal origins, provided these have the define biological activity on human cells.
  • DNA encoding TGFe include various sequences which encode the foregoing forms of TGFe itse including sequence variants, coding sequences bearing one or m deletions and DNA isolated from human, bovine or other ani cells encoding TGFe. Such TGFe-encoding DNA sequences obtainable by those of ordinary skill in the art by the use known techniques and information disclosed herein.
  • epithelin 1 stimulat keratinocytes but inhibits the growth of mink lung CC164 cells a other epithelial cells.
  • Granulin A also inhibits the growth epithelial cells in culture.
  • Both granulin A and epithelin 1 a of relatively low molecular weight (about 6 kDa) have numero disulfide bonds, and relatively low proline content.
  • Intact TG has a molecular weight of about 22-28 kDa and a relatively hi proline content.
  • the 6kDa degradation product of TG substantially lacks biological activity and differs in N-termin amino acid sequence from granulin A and epithelin 1.
  • Example 1 Tissue Culture
  • SW-13 cell line derived from a human small cell carcin of the adrenal cortex (Leibovitz et al. (1973) supra), obtained ATCC CCL 105 from the American Type Culture Collect (Rockville, MD) .
  • Cells were maintained in McCoy's 5a (Gr Island Biological Co., Grand Island, NY) medium supplemented w 5% (v/v) calf serum (Hazelton Biologies, Inc., Lenexa, KS) .
  • Ce were grown at 37°C in a humidified atmosphere of 5% C0 2 and 9 air. Cells were replaced with frozen stock within 10 to passages and regularly examined after Hoechst 33258 staining ensure they remained free of mycoplasmas (Chen (1977) Exp. Ce Res. 104:255-262).
  • Soft agar growth assay using SW-13 cells as indicator cel was used to monitor the biological activity of TGFe duri purification.
  • Solidified base layers of 1 ml of 0.8% agarose McCoy's Medium 5a with 10% fetal bovine serum (FBS) were overla with 1 ml of upper layer of 0.4% agarose (SeaPlaque, F BioProducts, Rockland, ME) in McCoy's Medium 5a with 10% FBS, 7 x 10 3 SW-13 cells, and appropriate quantities of TGFe in 35- tissue culture dishes. The dishes were incubated at 37°C in humidified atmosphere of 5% C0 2 for 5-7 days.
  • the pH of the combined supernatants was k in the acidic range by adjustment to pH 5.2 with concentra ammonium hydroxide followed by the addition of 1 ml of 2 ammonium acetate buffer, pH 5.3, per 85 ml of extract. volumes of 95% ethanol and four volumes of anhydrous ether we immediately added, after which the mixture was precipitat overnight at -20°C. The precipitate was collected under vacu by filtration through No. 1 Whatman paper.
  • the filter was wash three times with 1 M acetic acid (final volume of 3-4 ml per gr of tissue) to recover the precipitate, which was then lyophiliz to dryness and stored at -20°C.
  • Initial homogenization and aci ethanol extraction yielded 11 mg of protein per gram of wet tiss weight, with ED 50 of 40 ⁇ g (Table 1) .
  • the extract was subjected to Bio-Rex 70 batch cation-exchan chromatography and concentration and diafiltration with an Amic concentrator equipped with an S1Y10 spiral membrane cartrid (Parnell et al. (1990) J. Cell. Biochem. 42:111-116).
  • Prote from acid-ethanol extraction resuspended in 1 M acetic acid, w adsorbed to Bio-Rex 70 ion-exchange resin (Bio-Rad, Richmond, C pre-equilibrated with 1 M acetic acid at a volume of 100 ml res per 1-2 gm of protein (Savage and Harper (1981) Anal. Bioche 111:195-202).
  • Bio-Gel P-60 Molecular Sieve Chromatography. Two-hundred protein aliquots suspended in 1 M acetic acid were loaded on x 90 cm molecular sieve Bio-Gel P-60 (Bio-Rad) col (polyacrylamide beads) equilibrated with 1 M acetic acid. protein was eluted at 22°C with 1 M acetic acid, at a flow ra of 30 ml/h. Ten ml fractions were collected and 10 ⁇ l aliquo tested for SW-13 colony-stimulating activity. Protein content w determined by the colorimetric method of Bradford (1976) Ana Biochem. 22:248-254.
  • TGFe elutes as either one broad peak several peaks of activity on the Bio-Gel P-60 molecular sie column after the majority of protein (Parnell et al. (1990) supr Dunnington et al. (1988) Anal. Biochem. 174:257-264) .
  • acti fractions were collected in two pools: higher molecular weight ( 25,000) fractions were labelled pool A, and fractions from t second active peak were labeled pool B (M,.15,000-20,000) .
  • Pool P-60 material had an ED 50 of 0.2 ⁇ g (Table 1).
  • LMW TGFe The low molecular weight form of TGFe is biological active. The origin of the LMW TGFe is probably either at leas partial deglycosylation, partial degradation of the primary amin acid sequence, or both. LMW TGFe can be further purified a described below for the high molecular weight form.
  • the Protein Plus HPLC colum is a silica gel support to which is bonded a C 3 -like entity, w pore size of 300 angstroms, 6 ⁇ m particles, and action essentia a reverse phase column.
  • the protein was eluted with a lin gradient of 8-38% acetonitrile in 0.085% TFA/water over 40 min a flow rate of 1.5 ml/ in.
  • One minute fractions were collec and 50 ⁇ l aliquots assayed for TGFe biological activity.
  • an SDS-polyacrylamide gel a showed 4-5 protein bands in active fractions.
  • One min fracti with a peak biological activity eluting at 29-30 min from f runs were pooled and evaporated to dryness.
  • the ED 50 was 5 (Table 1) .
  • HPE High Performance Electrophoresis Chromatography
  • the tube gel was mounted between two electrodes and upp (25mM Tris, 0.19 M glycine, 0.1% SDS, pH 8.3) and lower buff (25mM Tris-HCl, pH 8.3). Eletrophoresis buffers were filter through 0.22 ⁇ m filters prior to use. Elution buffer was 25 Tris-HCl (pH 8.3). Protein was eluted into the lower buffer a flow of 12 ⁇ l/ in, and collected in 5 minute fractions, and ⁇ l aliquots were assayed for SW-13 soft agar stimulatory activi (Fig. 2).
  • the Model 230A HPEC instrument w initialized by installation of a 25 mM X 50 mm 10% polyacrylami gel between the upper and lower elution blocks.
  • the gel w prepared according to the manufacturer's instruction incorporated by reference herein, and allowed to polymerize room temperature.
  • Upper buffer 0.025 M Tris, 0.192 M glycin 0.1% SDS, pH 8.8
  • lower electrode buffer and elution buf 0.025 M Tris-HCl, pH 8.3 were prepared with double distil water, and filtered as above.
  • a prerun (constant current of 0.8 mA for 90 min) performed to remove polymerization byproducts from the gel. Up and lower buffer flow rates were each 2 ml/minute, with elution buffer flow rate at 20 ⁇ l/min. Baseline stabilization achieved after about 60 min.
  • Peak activity fractions from 5 HPLC runs were poole evaporated to dryness, resuspended in lO ⁇ l 0.004 N HCl, a diluted with an equal volume of 2X sample buffer, to a fin concentration of 0.0625 M Tris-HCl, 3% SDS, 10% glycerol, 0.2% mercaptoethanol, pH 6.8.
  • the 20 ⁇ l sample was applied to the ge and electrophoresed at constant current (0.5 mA) for 30 min a then at constant current (1.5 mA) for 400 min at 21°C.
  • Bovine kidney 500 mg was used as a source. b Initial low yields of activity may be due to coprecipitation of an inhibitory 5 factor which is removed in subsequent steps.
  • a M j of 25,000 was used for TGFe to calculate the empirica formula.
  • the content of tryptophan was not determined.
  • Th reported glycine content may be an overestimate due to the glycin content of the HPEC sample buffer.
  • N- glycanase B-glucosaminyl asparagine amidase which is available as N- glycanase (Genzyme, Boston, MA) .
  • the sample was resuspended 0.5% SDS in 0.4 M sodium phosphate buffer pH 7.4, deglycosylated at 37 ⁇ C for 18 h with 0.875 units of N-glycan enzyme.
  • the reaction was terminated by heat treatment of sample at 100°C for 5 min.
  • the protein was recovered by desalt on a 2.1 mm x 30 mm reverse phase (RP-300 C 8 ) HPLC column wit 15 min linear gradient of 0-80% acetonitrile in 0.085% TFA/wa at a flow rate of 200 ⁇ l/min.
  • GenBank library search revealed no sequence homology previously cloned genes or peptide sequences.
  • SEQ N0:1 exhibits considerable amino acid sequence identity to t deduced N-terminal amino acid sequence of human granulin A: As Val-Lys-Cys-Asp-Met-Glu-Val-Ser-Cys-Pro-Asp-Gly-Tyr-Thr- (SEQ NO:49) (Bhandari et al (1992) supra).
  • SEQ ID N0:1 also exhibi considerable amino acid sequence identity with the N-terminus epithelin 1: Val-Lys-Cys-Asp-Leu-Glu-Val-Ser-Cys-Pro-Asp-Gly-Ty Thr (SEQ ID NO:50) (Shoyab et al. (1990) supra).
  • the N-terminal sequence of a spontaneous TGFe degradati product of about 6 kDa was also determined by automated Edm degradation, as described above.
  • This internal amino acid sequence appears to be unique; there no homology to the published sequence of granulin A (See, e.g Bhandari et al. (1992) supra) . Thus, it was concluded that TG is not identical to granulin A.
  • Two mixed-sequence PCR primers were constructed based on t available partial amino acid sequence for TGFe (see Table 3)
  • the Primer 1 sequence corresponds to SEQ ID NO:3 through SEQ I NO:10.
  • a BamHI restriction site (underlined) was incorporate into the primer near its 5'-end to facilitate subsequent clonin of the PCR product into an M13 vector. Two additional base precede the BamHI site to stabilize it and improve cutting b BamHI. Best guesses were made with regard to codon usage for th first three amino acids (Asp Val Lys) , according to establishe guidelines (Lathe, R. (1985) J. Mol. Biol. .181:1-12). Al possible nucleotides were incorporated into the third position o the codon for Pro, and both of the two third position choices were included for the following Asp. The remaining five nucleotide at the 3' end of this primer were unambiguous, as Met has only on possible codon, and both of the two codons for Glu begin with GA. As an alternative the following primer was constructed ba on the same amino acid sequence.
  • Primer 2 collectively represents SEQ ID NO:12 through SEQ NO:43.
  • a restriction site was not incorporated as the PCR prod is cloned using a T-vector.
  • T-vector advantage taken of the fact that Taq polymerase adds a single adenosine the 3' end of the amplified fragment.
  • a T-vector was construc by incubation of Smal-cut M13 vector with Taq polymerase wit mM dTTP under standard conditions. This resulted in the addit of a single thymidine at the 3' end of each fragment (Marchuk al. (1991) Nucl. Acids Res. .19:1154).
  • PCR products after purification, are ligated to the vector as both the vector and PCR products have complementary single base 3' overhangs.
  • Prim 2 is less degenerate, but more specific, though it could hav maximum of 20% mismatch.
  • an authentic DNA sequence can be synthesized by PCR e with this high degree of base pair mismatches between the pri and the cDNA. Use of Primer-2 and this approach, however, was successful.
  • T-tailed primer at the 3' end used to produce first strand cDNA.
  • the T-tailed primer pri cDNA synthesis at the poly(A) tail of the mRNA. This resemb the procedure reported by Frohman et al. (1988) as the RACE (Ra Amplification of cDNA Ends) method.
  • the sequence of the T-tai primer is:
  • primer D 5'- GCGAATTCTGCAGGATCCAAAC(T) 18 -3' (SEQ ID NO:44)
  • First-strand cDNA was used as the starting template. Fir strand cDNA was reverse transcribed from total RNA extracted fr bovine kidney using primer-D and murine leukemia virus rever transcriptase. The TGFe-specific cDNA was amplified using eith Primer-l or -2 in conjunction with Primer-E which is essential primer D lacking the T-tail:
  • primer E 5'- GCGAATTCTGCAGGATCCAAAC -3' (SEQ ID NO:45)
  • the amplification was performed using standard PCR buff with 1.5 mM MgCl 2 in 30 PCR cycles consisting of denaturation 94°C for 45 sec, annealing at 60°C for 45 sec, and elongation 72°C for 90 sec.
  • PCR products are eluted from a 2% agarose gel and reamplifi with the same primers using 65°C temperature for annealing.
  • T reamplified product is electroeluted from 2% agarose gels, and D is precipitated.
  • Each reamplified PCR product is inserted in an M13 vector and cloned. Where the product is derived from primer lacking a restriction site, the DNA is ligated into vector, a modified version of an M13 vector, obviating the ne for a restriction site, and cloned.
  • PRIMER1 1 28-mer 5'-GCGGATCC-GAT-GTG-AAG-CCA- GAC-ATG-GA- 3' 5'-GCGGATCC-GAT-GTG-AAG-CCC--GAC-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCG--GAC-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCT--GAC-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCA--GAT-ATG-GA- 3' 5'-GCGGATCC-GAT-GTG-AAG-CCC--GAT-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCC--GAT-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCC--GAT-ATG-GA- 3 5'-GCGG
  • a BamHI restriction site shown underlined, is incorporated into 5'-end of each primer.
  • the nucleotide sequences of the PCR products are determi by known techniques, for example, by the dideoxy method (Sambr et al. (1989) supra) . From the sequence thus obtained, it possible to design less degenerate, or nondegenerate primers amplify the remainder of the cDNA, as demonstrated by Su et
  • a different strategy is used to amplify the 5' end of TG cDNA.
  • a specific antisense primer based on available TGFe ami acid sequence is used to prime cDNA synthesis initially, then t first strand cDNA is treated with terminal transferase to add polyA sequence at the 3'-end.
  • a T-tailed nonspecific prim e.g., primer D
  • primer D is used in conjunction with the specif antisense primer to amplify the 5'-end of the TGFe cDN essentially as described by Frohman et al. (1988) Proc. Nat. Aca Sci. 85:8998-9002.
  • oligonucleotide probe for screening bovine or human kidney cDN or genomic libraries.
  • a single (unique sequence) relatively lon oligonucleotide probe is synthesized using best-guess nucleotide at every position where the code is ambiguous.
  • Probe 1 is base on the known amino acid sequence of TGFe as follows: N-Asp Val Lys Pro Asp Met Glu Val Ser Pro Pro Asp Asp 5'-GAT-GTG-AAG-CCT-GAC-ATG-GAG-GTG-TCC-CCC-CCC-GAT-GAC-
  • Choice of which nucleotide to use at each position is based several factors including codon usage frequency tables and known under-representation of 5'-CG-3' dinucleotides in mammal DNA. (See, e.g., Sambrook et al. (1989) supra) .
  • oligonucleot probing just described can be used in combination.
  • a unique, or less degenerate oligonucleotide, obtained by amplification can be used as a probe to screen an oligo(dT)- random-primed bovine or human cDNA library, as described by Mor et al. (1990) Cell 61:203-211 and by Li et al (1990) Scie 250:1690-1694.
  • Primer 1 (collective SEQ ID NO:3 through SEQ ID NO:10) is used in conjunction wit specific primer (Primer F) in place of the nonspecific antise primer.
  • the sequence of the specific antisense primer is ba on internal amino acid sequence data.
  • This specific antise primer was used, together with Primer 1 to amplify a partial T cDNA, i.e., about the first 420-480 bp of the TGFe mRNA.
  • Pri F was made with 32-fold degeneracy and a 5' BamHI restricti site:
  • R is A or G; Y is C or T.
  • R is A or G
  • S is C or G
  • H is A,C or T.
  • Primer G is used in conjunction with Primer E (SEQ ID NO:45), nonspecific T-tailed primer for the 3' end of mRNA.
  • PC polymerase chain reaction
  • t 3SR Trademark, Baxter Laboratories
  • TG cDNA is introduced into an appropriate expression syste
  • Nonglycosylated TGFe can be synthesized in a prokaryot expression system.
  • Full-length TGFe cDNA is amplified wit suitable primers (the first primer is based on the cDNA sequence and the second primer is again primer E) using PCR.
  • the PC product is isolated from an agarose gel, digested with EcoRI an Bglll and directionally cloned into the polylinker region of th pFLAG-1 vector (International Biotechnology, Inc.
  • This vector allows for IPTG-inducible expression of cloned gen products as fusions with an amino-terminal vector-derived peptide
  • Presence of the FLAG peptide facilitates purification of th product by anti-FLAG monoclonal antibody immunoaffinit chromatography.
  • the FLAG fusion protein is purified by affinit chromatography employing anti-FLAG monoclonal antibody coupled agarose. Following purification, the protein is recovered removal of the FLAG peptide with enterokinase.
  • TGFe is solubilized in urea and further purified as necessary. Because TGFe conta post-translational modifications, including disulfide b formation and glycosylation, a mammalian expression system, s as Chinese hamster ovary (CHO) cells or COS cells is preferred production of glycosylated TGFe.
  • TGFe cDNA is inserted into the plasmid V19.8 transfected into COS cells. This approach was recently used express human mast cell growth factor by Mortin et al. (19 supra. For long term production Chinese hamster ovary (CHO) ce are employed. Wong et al. (1985) Science 228:810-815 u cotransformation of dihydrofolate reductase (DHFR)-deficient cells with CSF cDNA cloned into p3A, a plasmid expressing DH The initial transfectants are selected for growth in increas concentrations of methotrexate.
  • DHFR dihydrofolate reductase
  • T is purified by a sequencial molecular sieve chromatography, HP and high performance electrophoresis chromatography, as necessar
  • identity of this recombinant protein with TGFe can be verifi by SDS-PAGE and glycosylation analysis, by amino acid sequenc and, most importantly, by the ability of neutralizing antibody block its biological activity, i.e., stimulation of colo formation of SW-13 cells in soft agar.
  • Natural or recombinant TGFe is used as an antigen intradermal or intraperitoneal immunization to develop monoclo antibodies to TGFe. At least 5 ⁇ g to 10 ⁇ g of natural TGFe immunization is administered. The relatively greater abunda of recombinant TGFe (TGFe synthesized by transformed ce expressing cDNA or genomic DNA encoding TGFe) makes it possi to use larger immunizing doses of rTGFe.
  • Alternativel intrasplenic immunization is carried out. The advantage intrasplenic immunization is that even smaller amounts of antig are required.
  • mice Female Balb/c mice are immunized with 3 doses TGFe 4-6 weeks apart with several intraperitoneal and intraderm injections administered each time. First dose of TGFe emulsified in complete Freund's adjuvant, the subsequent doses a in incomplete Freund's adjuvant. Alternatively, (using natur or rTGFe, or synthetic peptides) intrasplenic immunization done. Serial dilutions of serum from the mouse to be used f fusion are tested for anti-TGFe activity. The immunized mouse ( mice) are killed, splenocytes are isolated and fused with SP2 myeloma cell line using polyethylene glycol. After screening hybridomas in the antibody capture assays active cell lines a cloned, maintained and frozen so they can be used for futur antibody production.
  • selected peptides of 15-20 amino acids are prepared with flan cysteine or tyrosine residues and coupled to BSA or soy trypsin inhibitor with m-maleimidobenzoyl sulfosuccinimide es as a coupling agent.
  • Alternative carriers such as keyhole lim hemocyanin, and alternative coupling agents such as glutaraldeh or bisimido esters can be determined experimentally, as descri by van Regenmortel et al. (1988) , Laboratory Techniques Biochemistry and Molecular Biology. Vol 19, (R.H. Burdan and P vanKnoppeberg, eds.) , Elsevier, New York.
  • a number of methods anchoring the synthetic peptide to the carrier are kno
  • both amino acid carboxy ends are linked to the carr to form a loop of the synthetic peptide that provides maxi exposure of the peptide to the antibody.
  • Polyclonal chicken and rabbit antibodies have been rai against TGFe after immunization with a peptide of the seque Asp-Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pro-Asp-Asp-Tyr (SEQ NO:51) conjugated to bovine serum albumin.
  • IgG is precipitated from immune rabbit serum with ammon sulfate.
  • IgY was precipitated from chicken egg yolk w polyethylene glycol. The chicken IgY recognizes the pept (unconjugated) in a Western blot assay and in an ELISA, altho relatively weakly. Both rabbit and chicken antibodies have w TGFe-neutralizing activity, as measured in the SW-13 colony as in soft agar. SW13 colony formation was reduced by 50%.
  • SEQ ID NO:l N-terminal sequence of TGFe with epithelins 1 and 2 and w granulin
  • a pept corresponding in sequence to amino acids 2-20 of SQ ID NO:48 synthesized and aggregated by heat treatment before injecti
  • a shorter peptide, corresponding in sequence to amino acids 2 of SEQ ID NO:48 is synthesized and conjugated to bovine se albumin for use in immunizing for polyclonal antibody producti
  • Polyclonal antibodies can also be produced. Polyclo antibodies can be raised in the rabbit or in the sheep, or ot species known to elicit an antibody response. It is preferred raise antibodies against conserved mammalian antigens in avi species, such as turkey or chicken. Chickens and rabbits a preferred as animals requiring small amounts of antigen f immunizations. Repeated injections of low doses (2-5 ⁇ g) antigen will also allow selection for high affinity antisera. to five adult leghorn chickens are immunized with at least 5 of native bovine TGFe or rTGFe each. Before the fir administration of TGFe 5 ml of control serum is obtained from wing vein.
  • the animals are bled 10 to 14 da after each boost from one of the wing veins.
  • Production chicken egg yolk antibodies is a particularly efficient and che source of antibodies against a conserved mammalian protei (Gassmann et al (1990) FASEB J. 4.:2528-2532) .
  • IgY is purified from egg yolks and chicken serum b precipitation with Polyethylene glycol as described by Poison e al. (1980) Immunol. Commun. 9.:495-514. This method achieves 90 pure IgY.
  • rabbit antibodies up to five rabbits ar immunized with native TGFe according to the protocol described fo immunizing chickens.
  • Preimmune serum (5 ml) is obtained befor immunization.
  • Antisera are further purified using either combination of ammonium sulfate precipitation and anion exchang chromatography or a protein A bead column (Harlow and Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  • An antibody capture assay is preferred for rapid screen of antibodies in production.
  • Ten to twenty ng of pure natural recombinant TGFe are bound to the bottom of the wells o microtiter plate in a phosphate or carbonate buffer overnight 4°C.

Abstract

A substantially pure epithelial growth factor (TGFe) and a method for purification are provided. Bovine TGFe is a monomeric glycoprotein of Mr 22,000 - 25,000, as determined by SDS-PAGE under nonreducing conditions, and is characterized by the N-terminal amino acid sequence N-Asp-Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pro-Asp-Tyr-Thr (SEQ ID NO:1) and internal peptide sequences of Pro-Glu-Pro-Lys-Lys-Pro-Glu (SEQ ID NO:2) and Xaa-Gly-Leu-Ala-Ala-Ala-Gly-Pro-Ala-Pro-Ser-Glu-Ser-Gln-Glu-Lys-Lys-Pro-Leu-Lys-Pro-Glu-Gly-Ala, where Xaa is Ala or Pro (SEQ ID NO:48). The characteristic biological activity of TGFe includes stimulation of soft agar colony formation of SW-13 cells, and stimulation of A431 cells, epithelial cells, fibroblasts, epidermal keratinocytes and certain transformed cell lines.

Description

TRANSFORMING GROWTH FACTOR-E
This invention was made, in part, with funding from t National Institutes of Health (Grant No. NIH RO1-CA44039) . T United States government may have certain rights in th invention.
This invention relates to the isolation in substantial purified form of a protein found in both normal and neoplast epithelial tissue and body fluids, which protein acts as a mitog and progression factor for epithelial and fibroblastic cells.
Accordingly, this material is useful in cell culture and i wound healing and the treatment of burns in humans and animals as well as for treatment of cell proliferation defects i epithelial tissue.
BACKGROUND OF THE INVENTION
Transforming growth factors (TGFs) are a family of acid- an heat-stable transforming polypeptides which reversibly caus nontransformed, anchorage-dependent cells to assume a transforme morphology and to form progressively growing colonies in soft aga (De Larco and Todaro (1978) Proc. Natl. Acad. Sci. USA 75:4001 4005; Ozanne et al. (1981) J. Cell Physiol. 105:163-180; Robert et al. (1980) Proc. Natl. Acad. Sci. USA 77:3494-3498; Moses e al. (1981) Cancer Res. 41:2842-2848).
The original description of TGF activity was that of De Larc and Todaro (1978) (supra) who described the properties of polypeptide, termed sarcoma growth factor (SGF) , isolated from th conditioned medium of Moloney murine sarcoma virus (MuSV)-infecte mouse 3T3 cells. SGFs are low molecular weight (M, 6,000-25,0 acid- and heat-stable polypeptides which compete with epider growth factor (EGF) for binding to membrane receptors, yet do cross-react with antibodies to EGF, and reversibly confer transformed phenotype upon nonneoplastic cells in tissue cult (De Larco and Todaro (1978) supra; Todaro and De Larco (19 Cancer Res. 8.:4147-4154) . The transforming activity of SGF subsequently shown to result from the presence of TG specifically TGFα and TGFB (Anzano et al. (1983) Proc. Natl. Aca Sci. USA 80.:6264-6268) .
Polypeptides similar to SGF have been reported in the cultu of other rodent sarcoma virus-transformed cells (Ozanne (198 supra: Kryceve-Martinerie et al. (1982) J. Cell. Physiol. 113:36 372), leukemia virus-transformed cells (Twardzi et al. (198 Science 216:894-896) , certain chemically transformed cells (Mos et al. (1981) supra; Zwiebel et al. (1982) Cancer Res. 42:511 5125) , and human tumor cells lines (Todaro et al. (1980) Pro Natl. Acad. Sci. USA 77:5258-5262) ; intracellularly in virally a chemically transformed cells (Roberts et al. (1980) supra embryonic mice (Twardzik et al. (1982) Cancer Res. 42:590-59 Proper et al. (1982) J. Cell. Physiol. 110.:169-174) and ra (Matrisian et al. (1982) Biochem. Biophys. Res. Commun. 107:76 769); and in fetal calf serum (Childs et al. (1982) Proc. Nat Acad. Sci. USA 79.:5312-5316) and urine of normal pregnant tumor-bearing humans (Twardzik et al. (1982) J. Natl. Cancer Ins »59_:793-798) . Other polypeptides from chemically transformed cell (Moses et al. (1981) supra) and from nonneoplastic human, murine and bovine tissues (Roberts et al. (1981) Proc. Natl. Acad. Sci USA 78:5339-5343; Roberts et al. (1982) Cold Spring Harbor Conf Cell Proliferation 9.:319-332) have been described which do no compete for binding to the EGF receptor but do induce anchorage independent growth of indicator cells.
Subsets of the TGF family have been classified as type α o β based on interaction with the EGF receptor and the requirement for EGF for growth in soft agar (Anzano et al. (1982) Cancer Res 4_2.:.4776-4778; Roberts et al. (1983) Fed. Proc. Fed. Am. Soc. E Biol. 42.:2621-2626) . TGFα competes with EGF for membr receptors and induces the formation of small colonies of nor rat kidney (NRK) cells in soft agar; its colony-forming respo is not potentiated by EGF (De Larco and Todaro (1978) sup Todaro et al (1980) supra; Massague (1983) J. Biol. Ch 258:13614-13620: Pike et al. (1982) J. Biol. Chem. 217:1462 14631). TGFα is a M. 5,600 single chain polypeptide with intrachain disulfide bonds. Amino acid sequence analysis of r TGFα shows that it shares limited homology (30-35%) with mouse E (Marquardt and Todaro (1982) J. Biol. Chem. 257:5220-522 Marquardt et al. (1984) Science 221:1079-1082); Frolik (198 Proc. Natl. Acad. Sci. USA jJO:3676-3680) . Antibodies to EGF not crossreact with TGFα (De Larco and Todaro (1978) supra; Larco and Todaro (1980) J. Cell. Physiol. 102:267-277) . TGFα a potent mitogen for mesenchymal and epithelial cells in monolay culture, but only a weak stimulator of anchorage-independe growth of mesenchymal cells in soft agar (Anzano et al. (198 supra) . TGFα, like EGF, acts synergistically with TGFB promoting soft agar growth of nontransformed NRK cells (Anzano al. (1983) supra: Anzano et al. (1982) supra) . TGFα has be identified in embryonic tissue (Twardzik et al. (1982) supr . tumor tissue and transformed cells (Marquardt et al (1984) supr Anzano et al. (1983) supra; Anzano et al. (1982) supra: Marquar et al. (1983) Proc. Natl. Acad. Sci. USA £0:4684-4688) , as we as in normal adult cells, including skin keratinocytes (Coffey al. (1987) Nature 328:817-820) , human eosinophils (Wong et a (1990) J. Exp. Med. 172:673-681) f gastric mucosa (Beaucha p et a (1989) J. Clin. Invest. 84.:1017-1023) and pituitary (Kobrin et a (1987) Endocrinology 121:1412-1416) .
In contrast to TGFα, TGFB does not compete for EGF recepto (Roberts et al. (1981) supra) and requires the presence of EGF TGFα to induce formation of large colonies of NRK cells in so agar. TGFB is a M-.25,000 polypeptide consisting of two identic 112-amino acid chains held together with disulfide bonds (Assoi et al. (1983) J. Biol. Chem. 258:7155-7160; Frolik et al. (198 Proc. Natl. Acad. Sci. USA 80.:3676-3680; Roberts et al. (19 Biochemistry 22.:5692-5698; Derynck et al. (1985) Nature 316:7 705) . While platelets and bone are the major source of T (Assoian et al. (1983) supra Childs et al. (1982) supra) , it also produced by transformed and nontransformed cell lines (Mo et al. (1981) Cancer Res. 41:2842-2848; Roberts et al. (19 Nature 295.:417-419) , solid human neoplasms (Tucker et al. (198 Cancer Res. 41:1581-1586; Nickell et al. (1983) Cancer. R 43:1966-1971) , and by virtually all normal tissues; Roberts et a (1988) Brit. J. Cancer 57:594-600; Sporn et al. (1986) Scien 231:532-534; Halper and Moses (1983) Cancer Res. 41:1972-1979) a mouse embryos (Proper et al. (1982) supra) .
A distinctive epithelial tissue-derived transforming grow factor, TGFe, was described by Halper and Moses (1983) supr TGFe induces anchorage-independent growth of certain epitheli cell lines, such as the SW-13 cell line derived from a human sma cell carcinoma of the adrenal cortex (Leibovitz et al. (1973) Natl. Cancer Inst.152.:691-697) , human squamous cell carcinoma ce lines (A431 and D562) , and mouse embryo AKR-2B cells. TGFe found in both neoplastic and nonneoplastic tissue (Halper a Moses (1983) supra) of humans and other mammals. Parti purification and characterization of TGFe from bovine kidn revealed it to be a single , 23,000-25,000 polypeptide chai requiring disulfide bonds for maximal activity (Halper and Mos (1987) Cancer Res. .47:4552-4559). TGFe is distinct from othe characterized growth factors and hormones in its ability t stimulate soft-agar growth of SW-13 cells. The only other growt factors known to possess this activity are basic and acidi ibroblast growth factor (bFGF) , which are distinguished from TGF by molecular weight, the inability of bFGF to stimulate the sof agar growth of A431 cells and the inability of TGFe to bind to th bFGF receptor. TGFα, TGFB, EGF, and bombesine do not possess SW 13-stimulating activity. SW-13 cell soft agar growth is inhibite by TGFB, and SW-13 cells do not bind EGF. TGFe is not related t the family of insulin-like growth factors. Neither insulin Insulin-like Growth Factor 1, nor Insuin-like Growth Factor stimulate the multiplication of SW-13 cells, thus indicating th TGFE is not related to the family of insulin-like growth factor
Normal cell growth is dependent on a complex, incomplete understood series of interactions between the cell and i environment. Growth factors, as modulators of cell growth, a polypeptides that stimulate or inhibit cell proliferation throu binding with specific cell membrane receptors. Growth facto primarily act on cells locally, in an autocrine or paracri manner (Sporn and Roberts (1985) Nature 111:745-747). The action is rather nonspecific, as growth factors usually stimula several different cell types. The requirement for multiple grow factors for optimal growth of a particular cell is reflected the presence of specific receptors for many growth factors in t cell membrane and by the production of several growth factors most individual cells (Leof et al. (1983) Exp. Cell Res. 147:20 208) .
A model of growth factor controlled cell cycle traverse h been developed with mouse embryo Balb/c-3T3 cells in which grow factors exert their effects in a coordinated, ordered fashio resulting in cell proliferation (Pledger et al. (1978) Proc. Nat Acad. Sci. USA 75:2839-2843; Leof et al. (1982) Exp. Cell. Re 141:107-115; Okeefe and Pledger (1983) Mol. Cell. Endocri H:167-186). Competence factors, such as platelet-derived grow factor (PDGF) or FGF, are necessary to first remove quiesce Balb/c-3T3 cells from the G0 phase of the cell cycle preparation for Gj traverse (Stiles et al. (1979) J. Cel Physiol. £9:395). The subsequent action of progression factor such as EGF and IFG-1, is necessary for Balb/c-3T3 cells to ent the S phase of the cell cycle (Leof et al. (1982) supra) . AKR- cells have been used to detect the competence activity of TG (Goustin et al. (1987) Exp. Cell. Res. 172.:293-303) .
Studies were conducted using Balb/c-3T3 and AKR-2B cells determine whether TGFe acts as a competence or a progressi factor, and to examine its effect on several fibroblast a epithelial cell lines of nonneoplastic origin (Brown and Hal (1990) Exp. Cell Res. 190.:233-242) . The results establish t TGFe is involved in both monolayer growth and anchora independent growth. In general, the mitogenic activity of T for monolayer growth is slightly less than that of bFGF or PD equipotent to that of EGF, and greater than that of IGF-1. time course of stimulation of DNA synthesis induced by TGFe AKR-2B cells is similar to that induced by EGF, IGF-1, FGF, PDGF. TGFe, either alone or in combination with IGF-1, stimula [3H]-thymidine incorporation after a G„/Gj lag of 10-14 h and pe at 19-22 h after growth factor addition. In contrast, T stimulates DNA synthesis in AKR-2B cells after a GQ/GX lag of h (Shipley et al. (1985) Proc. Natl. Acad. Sci. USA 82_:4147-4151 which may be attributable to an indirect mitogenic effect of TG through the autocrine production of PDGF-related peptides (Le et al. (1986) Proc. Natl. Acad. Sci. USA 11:2453-2457; Soma a Grotendorst (1989) J. Cell. Physiol. 140:246-253) . TGFe appea to act as a progression factor in both Balb/c-3T3 and AKR- cells, as suggested by the requirement for continuous (rather th transient) TGFe exposure to achieve maximal stimulation of D synthesis (Brown and Halper (1990) supra) .
The granulins are candidate growth factors discovered human and rat inflammatory leukocytes and bone marrow. cD analysis revealed that prepropeptide for the human granulins a 593 amino acid glycoprotein, with seven tandem repeats of t 12-cysteine granulin domain. The message corresponding to th cDNA is found in myelogenous leukemic cell lines of promonocyti promyelocytic and proerythroid lineage, in fibroblasts, epithelial cell lines and in kidney. Granulin A is believ identical to epithelin 1, and inhibits A431 cell proliferation culture (Bhandari et al. (1992) Proc. Natl. Acad. Sci. US 19.1715-1719) .
Epithelins 1 an 2 are cysteine-rich growth modulatin proteins, and have been purified from rat kidney. Both epithelin 1 and 2 inhibit the growth of the A431 cells, which are derive from a human epidermal carcinoma (Shoyab et al. (1990) Proc. Nat Acad. Sci. USA 87:7912-7916) . By contrast, bovine TGFe suppor anchorage-independent growth of A431 cells.
TGFe differs from keratinocyte growth factor (KGF) , a memb of the FGF family produced solely by mesenchymal tissue whi stimulates paracrine growth of adjacent epithelial tissue (Fin et al. (1989) Science 245:752-755) . Limited studies indicate K to be a specific mitogen for epithelial cells (Rubin et al. (198 Proc. Natl. Acad. Sci. USA 16:802-806). In contrast, TGFe present in higher concentrations in epithelial organs than mesenchymal tissues (Halper and Moses (1983) supra) . stimulat the proliferation of epithelial cells and is an effective mitog for fibroblasts (Brown and Halper (1990) supra) . While TGFe w first described as a growth factor possibly involved in autocri stimulation of anchorage-independent neoplastic cell grow (Halper and Moses (1983) supra) , its presence in normal tissue plasma, and platelets demonstrates a role for TGFe in normal ce growth. Bovine TGFe has cross-species activity on human cell for example, it supports anchorage-independent growth of hum A431 cells, D562 squamous cell carcinoma cells and T24 bladd carcinoma cells. In addition, bovine TGFe stimulates growth monolayer culture of normal human epidermal keratinocytes.
Prior Attempts to Purify TGFe
Bovine kidney was selected as a source of TGFe because of t high SW-13-stimulating activity content present in nonneoplast kidney and because of its activity on other human cell lines. T acid-ethanol procedure of Roberts et al. (1980) supraf was us as a first purification step. Protein recovery was 15-20 protein/g of wet tissue. EDso, defined as the concentration protein required to give 50% maximal response measured as SW- colony-stimulating activity, was 40 μg (Halper and Moses (198 supra) . A second purification step was accomplished with a Bi Gel P-60 molecular sieve column. Protein recovery was 5-10% material applied to the column and the ED50 was 10 μg, representi a 4-fold purification. Active P-60 fractions were pooled a applied to a molecular sieve Spherogel TSK SW3000 high performa liquid chromatography (HPLC) column. Protein recovery was 5- of that applied to the HPLC column and the EDJQ was 50 representing an approximately 800-fold purification over the ac ethanol extracted material. The next purification step, utilizi reverse phase high performance liquid chromatography (RP-HPLC yielded a protein recovery of 3.5% and an ED50 of 3.5 ng. Wh analyzed by sodium dodecyl sulfate-polyacrylamide g electrophoresis (SDS-PAGE) (Laemmli (1970) Nature 227:680-68 (12.5% (w/v) gel) under nonreducing conditions, a number of ban were observed (see. Fig 3 or Fig 5 from Halper and Moses (198 Cancer Res. 47:4552-4559) . The M,.23,000 band eluted from the g slice, resuspended in medium, and tested for activity account for all the SW-13 colony-stimulating activity in soft ag cultures. Although this protocol yielded an 11,000-fold degr of purification and 40% recovery of initial biological activit the purity and amounts recovered was insufficient to all structural analysis of TGFe or biological studies (Halper a Moses (1987) supra) .
An altered purification protocol was used in an attempt improve final protein recovery and purity (Parnell et al. (1990 J. Cell. Biochem. 42:111-116) . Protein from the initial aci ethanol extraction of bovine kidney was adsorbed to Bio-Rex 7 ion-exchange resin (Savage and Halper (1981) Anal. Biochem 111:195-202) and the eluant concentrated with an Amico concentrator. The ED^ of the concentrated product was 0.5 μg with 10-15% protein recovery. Active protein from the Bio Rex 7 ion-exchange chromatographywas further purified by P-60 molecula sieve chromatography. Protein recovery from the P-60 column wa approximately 10-15% (of the material subjected to molecular siev chromatography) with an ED50 of 200 ng. The most active fraction from molecular sieve chromatography were pooled and furthe purified on a heparin-Sepharose column (Shing et al. (1984 Science 223:1296-1299) . TGFe eluted as a major peak from th heparin-Sepharose column, with a protein recovery of 4.7% and a ED5o of 5.0 ng. The purification steps involving Bio-Rex 70, Bio Gel P-60, and heparin affinity chromatography resulted in mar increase in recovery of activity, which may be attributable removal of growth inhibitors (e.g., TGF-B) from TGFe present the acid-ethanol extraction. Peak active fractions from 4 heparin-Sepharose runs were pooled and further purified microbore C8 RP-HPLC. Peak active fractions from 10-12 HPLC ru were pooled for further purification. Protein recovery w estimated to be 5%, based on protein absorbance at 214 nm from t chromatograph and silver stained SDS-PAGE gels of each fracti collected. EDS0 could not be determined on this and subseque steps due to the small amounts of protein recovered. Pool samples were rerun on the same column and the most acti fractions from 6-8 HPLC purification runs pooled for fin purification. Proteins were separated in a 10% to 20% gradie SDS-PAGE gel. Five percent of the total protein was run in separate lane and silver stained for assessment of purity a identification of active bands eluted from the gel containing 9 of the protein. Protein from the lane containing 95% of t protein was electroeluted (Jacobs and Clad (1986) Anal. Bioche 154.:583-589) , and repurified by HPLC using a microbore RP-3 column. Individual peaks were collected and aliquots tested f colony-stimulating activity. The major biologically active ba migrated at 22,000-25,000, with a minor biologically inactive ba at 40,000 (See Fig. 5 of Parnell et al. (1990) supra. showing SD PAGE of final purification product) . Based solely on intensi of staining, protein recovery was estimated to be between 10- ng, with an estimated purity of greater than 90%. The foregoi procedure for TGFe purification proved to be inadequate becau it did not provide sufficient quantities of TGFe for measuremen of specific activity or for structural analyses, including ami acid composition and sequence determinations. Furthermore, t estimated degree of purity obtained by the prior process w subsequently found to be inaccurate, the actual purity being mu lower than estimated. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: HPLC Purification of TGFe
Figure 2: HPEC Purification of TGFe 60 μl fractions w collected and 5 μl aliquots were assayed for SW-13 soft a stimulatory activity.
Figure 3: SDS-PAGE of TGFe after HPEC purification left-h column purified TGFe, right-hand column molecular wei standards: A single band with an apparent molecular mass
28,000 was observed with silver staining after migration of 75 of the sample in a 10-20% gradient nonreducing SDS-PAGE.
SUMMARY OF THE INVENTION
The transforming growth factor, TGFe, has been isolated f the irst time with sufficient purity and in sufficient quantiti to allow determination of its amino acid composition and sequenc TGFe is a glycoprotein with an M, of about 25,000. The parti amino acid sequence of purified bovine TGFe indicated no homolo to other known growth factors.
Three partial amino acid sequences have been determined f deglycosylated bovine TGFe: an amino-terminal amino acid sequen of purified TGFe, N-Asp-Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pr Asp-Asp-Tyr-Thr-C (SEQ ID NO: 1) , and internal amino ac sequences N-Pro-Glu-Pro-Lys-Lys-Pro-Glu-C (SEQ ID NO: 2) and Xaa Gly-Leu-Ala-Ala-Ala-Gly-Pro-Ala-Pro-Ser-Glu-Ser-Gln-Glu-Lys-Lys Pro-Leu-Lys-Pro-Glu-Gly-Ala, where Xaa is Ala or Pro (SEQ I NO:48).
The invention provides full-length cDNA encoding animal o human TGFe, which coupled with a known expression system, can b used to synthesize active TGFe in a cell culture system. Hos cell synthesis of TGFe allows production of commerciall significant quantities of TGFe protein, either glycosylated o unglycosylated depending on choice of host cell. The inventi further provides a method of purifying TGFe in high yield, wi an ED50 of about 0.5 - 1.5 ng for stimulating soft agar growth SW-13 cells. The cDNA encoding bovine TGFe can be used as a pro to isolate cDNA encoding human TGFe. Primers derived from t cDNA encoding bovine TGFe can be used to amplify DNA sequenc encoding human TGFe.
DETAILED DESCRIPTION OF THE INVENTION
In general the terminology used herein is standard, understood by those ordinarily skilled in the fields of molecul biology, protein chemistry, cell biology, and biochemistry. F added clarity, certain terms are further defined. Standa abbreviations are used, such as approved by scientific journa in the field (Nature, Science, Proceedings of the National Acade of Science, etc.). All references cited herein are incorporat by reference.
Methods used herein are either specifically referenced sufficiently well known as to be available in one of sever published collections of methods, for example, Sambrook et a (1989) Molecular Cloninσ. A Laboratory Manual (2d ed.). Co Spring Harbor Laboratory, Cold Spring Harbor, NY, and Innis et a (1990) PCR Protocols: A Guide to Methods and Application Academic Press, Inc., San Diego, CA.
Sodium Dodecyl Sulfate polyacryla ide gel electrophores (SDS-PAGE) is a technique well known in the art for the separati of proteins on a basis of size. A modification of the origin procedure of Laemmli (1970) supraf with a discontinuous buff system was used. Molecular weight standards were obtained fr Bethesda Research Laboratories (Gaithersburg, MD) or Bio-Rad. S was used as a dissociating agent and bismercaptoethanol as reducing agent. Lyophilized aliquots of fractions fr chromatography columns were dissolved in 10 μl 0.004 M HCl and 4 μl Laemmli buffer. Samples were heated in boiling water for 3 mi and run on a 10% - 20% gradient acrylamide gel, or on a 12 acrylamide resolving gel in a Tris-glycine-SDS buffer. The g were then fixed in 50% methanol with or without 10% acetic ac washed with water and 10% glutaraldehyde, and stained with sil nitrate.
Complementary DNA (cDNA) methods involve the in vi synthesis of a double-stranded DNA sequence by enzymatic rever transcription of messenger RNA (mRNA) isolated from donor cell cDNA methodology is the method of choice for isolating the D sequence of a gene when the entire sequence of amino acid residu of the desired polypeptide is not known. Standard procedures a known in the art for the preparation of plasmid-borne cD libraries derived from reverse transcription of mRNA.
Expression refers to the transcription and translation of structural gene so that a protein is synthesized.
It is understood by those skilled in the art that bovine TG is exemplary of, and homologous to, TGFe from other mammali sources including humans. Many growth factors share extensi homology with one another. Bovine TGFe is sufficiently homologo to human TGFe to bind to the appropriate receptors on human cell The purified DNA encoding bovine TGFe is suitable to provi primers for amplifying DNA encoding human TGFe, or for probing nucleic acid library to isolate or identify nucleic acids encodin human TGFe. The techniques of purification disclosed herein wil be understood to be applicable to purifying human TGFe to comparable level of purity.
It is therefore understood that various forms of TGFe exis and are included within the present invention. All have th biological activity defined for TGFe and include high molecula weight TGFe, low molecular weight TGFe, glycosylated forms o TGFe, including forms having various intermediate stages o glycosylation. Also included herein are forms of TGFe of human bovine and other animal origins, provided these have the define biological activity on human cells. DNA encoding TGFe inclu various sequences which encode the foregoing forms of TGFe itse including sequence variants, coding sequences bearing one or m deletions and DNA isolated from human, bovine or other ani cells encoding TGFe. Such TGFe-encoding DNA sequences obtainable by those of ordinary skill in the art by the use known techniques and information disclosed herein.
It has been determined that there is partial amino a sequence identity of the N-terminus of bovine TGFe with the ami acid sequence of human granulin A, as given in Bhandari et (1992) supra. and with the amino acid sequence of epithelin 1, given in Shoyab et al. (1990) supra. The kidney express granulin A and epithelin 1 , as well as TGFe. However, t biological activity of TGFe differs from that of granulin A a epithelin 1. TGFe stimulates the .in vitro growth of keratinocyt and mink lung CC164 cells. By contrast, epithelin 1 stimulat keratinocytes but inhibits the growth of mink lung CC164 cells a other epithelial cells. Granulin A also inhibits the growth epithelial cells in culture. Both granulin A and epithelin 1 a of relatively low molecular weight (about 6 kDa) , have numero disulfide bonds, and relatively low proline content. Intact TG has a molecular weight of about 22-28 kDa and a relatively hi proline content. The 6kDa degradation product of TG substantially lacks biological activity and differs in N-termin amino acid sequence from granulin A and epithelin 1.
The following examples are provided for illustrative purpos only and are not intended to limit the scope of the inventio
The examples utilize many techniques well known and accessible those skilled in the arts of molecular biology and prote chemistry. Example 1: Tissue Culture
SW-13 cell line, derived from a human small cell carcin of the adrenal cortex (Leibovitz et al. (1973) supra), obtained ATCC CCL 105 from the American Type Culture Collect (Rockville, MD) . Cells were maintained in McCoy's 5a (Gr Island Biological Co., Grand Island, NY) medium supplemented w 5% (v/v) calf serum (Hazelton Biologies, Inc., Lenexa, KS) . Ce were grown at 37°C in a humidified atmosphere of 5% C02 and 9 air. Cells were replaced with frozen stock within 10 to passages and regularly examined after Hoechst 33258 staining ensure they remained free of mycoplasmas (Chen (1977) Exp. Ce Res. 104:255-262).
Example 2: Soft Agar Colony Stimulation Assay
Soft agar growth assay using SW-13 cells as indicator cel was used to monitor the biological activity of TGFe duri purification. Solidified base layers of 1 ml of 0.8% agarose McCoy's Medium 5a with 10% fetal bovine serum (FBS) were overla with 1 ml of upper layer of 0.4% agarose (SeaPlaque, F BioProducts, Rockland, ME) in McCoy's Medium 5a with 10% FBS, 7 x 103 SW-13 cells, and appropriate quantities of TGFe in 35- tissue culture dishes. The dishes were incubated at 37°C in humidified atmosphere of 5% C02 for 5-7 days. Colonies of diameter greater than 50-60 μm in 20 medium-power fields (invert microscope) were counted. The background (unstimulated) coun obtained with SW-13 cells generally ranged from 10 to 30 colonie (Halper and Moses (1983) supra) . The ED50 was defined as th quantity of protein necessary to elicit 50% maximal stimulatio of soft agar growth of SW-13 cells.
Example 3: Purification of TGFe
Initial Purification. Bovine kidneys from freshl slaughtered cows were trimmed of fat and were extracted with aci ethanol according to Roberts et al. (1980) supraf as modified b Proper et al. (1982) supra. Tissue was homogenized with polytron (lOg of tissue in 60 ml) of a solution consisting of ml of 95% (v/v) ethanol and 7.5 ml of concentrated HCl, plus mg of phenylmethylsulfonyl fluoride and 1.9 mg of pepstatin protease inhibitors. The mixture was stirred for 20 h at 4 then centrifuged at 20,000 x g for 30 min. The pellet reextracted for 4 h with 40 ml of a solution consisting of 375 of 95% ethanol, 105 ml of distilled water, and 7.5 mol concentrated HCl. The pH of the combined supernatants was k in the acidic range by adjustment to pH 5.2 with concentra ammonium hydroxide followed by the addition of 1 ml of 2 ammonium acetate buffer, pH 5.3, per 85 ml of extract. volumes of 95% ethanol and four volumes of anhydrous ether we immediately added, after which the mixture was precipitat overnight at -20°C. The precipitate was collected under vacu by filtration through No. 1 Whatman paper. The filter was wash three times with 1 M acetic acid (final volume of 3-4 ml per gr of tissue) to recover the precipitate, which was then lyophiliz to dryness and stored at -20°C. Initial homogenization and aci ethanol extraction yielded 11 mg of protein per gram of wet tiss weight, with ED50 of 40 μg (Table 1) .
The extract was subjected to Bio-Rex 70 batch cation-exchan chromatography and concentration and diafiltration with an Amic concentrator equipped with an S1Y10 spiral membrane cartrid (Parnell et al. (1990) J. Cell. Biochem. 42:111-116). Prote from acid-ethanol extraction, resuspended in 1 M acetic acid, w adsorbed to Bio-Rex 70 ion-exchange resin (Bio-Rad, Richmond, C pre-equilibrated with 1 M acetic acid at a volume of 100 ml res per 1-2 gm of protein (Savage and Harper (1981) Anal. Bioche 111:195-202). The mixture was stirred overnight at 4°C, th poured into a 2.6 x 40 cm column. The column was washed with 5 ml of 1 mM HCl, followed by 100-150 ml of 0.5 M ammonium acetat pH 8.0. Both the HCl and the ammonium acetate eluants we discarded. The protein was then eluted with 500 ml of IM ammoni acetate. The eluant was concentrated and diafiltered against 4 volumes of 1 M acetic acid to a final volume of 100 ml. The E of the extract after Amicon concentration using an SIYIO spi membrane and diafiltration was 0.5 μg, with 10-15% prot recovery (Table 1) .
Bio-Gel P-60 Molecular Sieve Chromatography. Two-hundred protein aliquots suspended in 1 M acetic acid were loaded on x 90 cm molecular sieve Bio-Gel P-60 (Bio-Rad) col (polyacrylamide beads) equilibrated with 1 M acetic acid. protein was eluted at 22°C with 1 M acetic acid, at a flow ra of 30 ml/h. Ten ml fractions were collected and 10 μl aliquo tested for SW-13 colony-stimulating activity. Protein content w determined by the colorimetric method of Bradford (1976) Ana Biochem. 22:248-254. TGFe elutes as either one broad peak several peaks of activity on the Bio-Gel P-60 molecular sie column after the majority of protein (Parnell et al. (1990) supr Dunnington et al. (1988) Anal. Biochem. 174:257-264) . To achie more complete separation of several forms of TGFe, acti fractions were collected in two pools: higher molecular weight ( 25,000) fractions were labelled pool A, and fractions from t second active peak were labeled pool B (M,.15,000-20,000) . Pool P-60 material had an ED50 of 0.2 μg (Table 1).
The material in Pool B was designated low molecular weig
(LMW) TGFe. The low molecular weight form of TGFe is biological active. The origin of the LMW TGFe is probably either at leas partial deglycosylation, partial degradation of the primary amin acid sequence, or both. LMW TGFe can be further purified a described below for the high molecular weight form.
High Performance Liquid Chromatography. Pool A wa lyophilized to dryness, resuspended in 1 M acetic acid to a fina protein concentration of 20 mg/ml, and further purified by HPLC Lyophilized 5 mg protein aliquots were resuspended in 250 μl o 1 M acetic acid and loaded on a semi-preparative 4.6 mm x 25 c Protein Plus HPLC column (Zorbax, DuPont, Wilmington, DE) , whic had been pre-equilibrated with 8% acetonitrile in 0.085 trifluoroacetic acid (TFA)/water. The Protein Plus HPLC colum is a silica gel support to which is bonded a C3-like entity, w pore size of 300 angstroms, 6μm particles, and action essentia a reverse phase column. The protein was eluted with a lin gradient of 8-38% acetonitrile in 0.085% TFA/water over 40 min a flow rate of 1.5 ml/ in. One minute fractions were collec and 50 μl aliquots assayed for TGFe biological activity. T eluted as a broad peak between 22 and 35 min (Fig. 1) . Simila to the HPLC chromatogram (Fig. 1) , an SDS-polyacrylamide gel a showed 4-5 protein bands in active fractions. One min fracti with a peak biological activity eluting at 29-30 min from f runs were pooled and evaporated to dryness. The ED50 was 5 (Table 1) .
High Performance Electrophoresis Chromatography (HPE Final puri ication to homogeneity was achieved with HPEC using Applied Biosystems Model 230A HPEC instrument (Foster City, C This separation technique allows separation of proteins throu combination of gel electrophoresis and sample elution. Pool HPLC fractions resuspended in modified Laemmli buffer (1 glycerol 2% bismercaptoethanol and 3% SDS in 0.625 M Tris-HCl, 6.8) as recommended by the manufacturer, and was separated in 2.0 x 50 mm, 5% polyacrylamide tube gel at a constant current 0.7 mA. The tube gel was mounted between two electrodes and upp (25mM Tris, 0.19 M glycine, 0.1% SDS, pH 8.3) and lower buff (25mM Tris-HCl, pH 8.3). Eletrophoresis buffers were filter through 0.22 μm filters prior to use. Elution buffer was 25 Tris-HCl (pH 8.3). Protein was eluted into the lower buffer a flow of 12 μl/ in, and collected in 5 minute fractions, and μl aliquots were assayed for SW-13 soft agar stimulatory activi (Fig. 2).
For micropreparative HPEC, the Model 230A HPEC instrument w initialized by installation of a 25 mM X 50 mm 10% polyacrylami gel between the upper and lower elution blocks. The gel w prepared according to the manufacturer's instruction incorporated by reference herein, and allowed to polymerize room temperature. Upper buffer (0.025 M Tris, 0.192 M glycin 0.1% SDS, pH 8.8) and lower electrode buffer and elution buf (0.025 M Tris-HCl, pH 8.3) were prepared with double distil water, and filtered as above.
A prerun (constant current of 0.8 mA for 90 min) performed to remove polymerization byproducts from the gel. Up and lower buffer flow rates were each 2 ml/minute, with elution buffer flow rate at 20 μl/min. Baseline stabilization achieved after about 60 min.
Peak activity fractions from 5 HPLC runs were poole evaporated to dryness, resuspended in lOμl 0.004 N HCl, a diluted with an equal volume of 2X sample buffer, to a fin concentration of 0.0625 M Tris-HCl, 3% SDS, 10% glycerol, 0.2% mercaptoethanol, pH 6.8. The 20 μl sample was applied to the ge and electrophoresed at constant current (0.5 mA) for 30 min a then at constant current (1.5 mA) for 400 min at 21°C. Buff flow rates were the same as in the prerun; eluant absorbance 220 nm was monitored 5 min fractions (100 μl each) were collect beginning at 100 min into the run (30 min at 0.5 mA, then at 1 mA) . Three μl aliquots of each fraction were assayed for TG activity using the SW-13 soft agar assay. Fractions were al analyzed by SDS-PAGE using 10 - 20% gradient gels and silv staining as described by Eschenbuch and Burke (1982) Ana Biochem. 125:96-99. It was estimated that the TGFe eluted w about 95% homogeneous.
Table 1. Purification of TGFe*
to
Figure imgf000021_0002
"Bovine kidney (500 mg) was used as a source. bInitial low yields of activity may be due to coprecipitation of an inhibitory 5 factor which is removed in subsequent steps.
Figure imgf000021_0001
Example 4: Amino Acid Analysis and Sequence
Amino Acid Analysis. An aliquot of pure bovine TGFe (75 μ (260 pmol) was subjected to acid hydrolysis in 6M HCl for 1 h 65°C, and amino acid analysis performed using an Appli Biosystems Model 420A Amino Acid Analyzer. Results of amino ac analysis are summarized in Table 2. Calculations of the empiric formula do not take glycosylation into account.
Figure imgf000022_0001
"A Mj of 25,000 was used for TGFe to calculate the empirica formula. The content of tryptophan was not determined. Th reported glycine content may be an overestimate due to the glycin content of the HPEC sample buffer.
Determination of Partial Amino Acid Seguence. Due t unsuccessful attempts at amino acid sequencing of native TGFe an low content of methionine (Table 2) , TGFe was subjected t cleavage with cyanogen bromide with the expectation of obtainin
2-3 peptide fragments. This attempt was also unsuccessful. A a result, the native protein was deglycosylated prior to amin acid sequence analysis. A portion of the pure bovine TGFe sampl used for amino acid analysis was evaporated to dryness an subjectedto enzymatic deglycosylation usingpeptide-N4-(N-acetyl-
B-glucosaminyl) asparagine amidase which is available as N- glycanase (Genzyme, Boston, MA) . The sample was resuspended 0.5% SDS in 0.4 M sodium phosphate buffer pH 7.4, deglycosylated at 37βC for 18 h with 0.875 units of N-glycan enzyme. The reaction was terminated by heat treatment of sample at 100°C for 5 min. The protein was recovered by desalt on a 2.1 mm x 30 mm reverse phase (RP-300 C8) HPLC column wit 15 min linear gradient of 0-80% acetonitrile in 0.085% TFA/wa at a flow rate of 200 μl/min. SDS-PAGE revealed t deglycosylated TGFe migrated faster than glycosylated TGFe, wi M-. = 18,000-20,000. This band was slightly sharper t nontreated TGFe when silver stained. Deglycosylated TGFe al retained its biological activity.
Approximately 150 pmol of the deglycosylated sample w subjected to N-terminal amino acid sequencing by automated Edm degradation using an Applied Biosystems Model 470 Prote Sequencer with on-line Applied Biosystems Model 120A HPLC (Appli Biosystems, Foster City, CA) . The initial yield was estimated be 100 pmol, with a repetitive yield of 95%. Two sequences we obtained: the amino-ter inal amino acid sequence N-Asp-Val-Ly Pro-Asp-Met-Glu-Val-Ser-Pro-Pro-Asp-Asp-Tyr-Thr-C (SEQ ID NO: 1 and a downstream sequence from the same polypeptide, N-Pro-Gl Pro-Lys-Lys-Pro-Glu-C (SEQ ID NO: 2) .
A GenBank library search revealed no sequence homology previously cloned genes or peptide sequences. However, SEQ N0:1 exhibits considerable amino acid sequence identity to t deduced N-terminal amino acid sequence of human granulin A: As Val-Lys-Cys-Asp-Met-Glu-Val-Ser-Cys-Pro-Asp-Gly-Tyr-Thr- (SEQ NO:49) (Bhandari et al (1992) supra). SEQ ID N0:1 also exhibi considerable amino acid sequence identity with the N-terminus epithelin 1: Val-Lys-Cys-Asp-Leu-Glu-Val-Ser-Cys-Pro-Asp-Gly-Ty Thr (SEQ ID NO:50) (Shoyab et al. (1990) supra).
The N-terminal sequence of a spontaneous TGFe degradati product of about 6 kDa was also determined by automated Edm degradation, as described above. The resulting amino ac sequenceXaa-Gly-Leu-Ala-Ala-Ala-Gly-Pro-Ala-Pro-Ser-Glu-Ser-Gl Glu-Lys-Lys-Pro-Leu-Lys-Pro-Glu-Gly-Ala, where Xaa is Ala or Pr (SEQ ID NO:48) is internal to the intact TGFe of about 25-28 kD This internal amino acid sequence appears to be unique; there no homology to the published sequence of granulin A (See, e.g Bhandari et al. (1992) supra) . Thus, it was concluded that TG is not identical to granulin A.
Example 5: Isolation of the TGFe cDNA
Methods have been developed in the art to use D amplification methods such as the polymerase chain reaction (PC for cDNA cloning. From a partial amino acid sequence of t protein of interest, oligonucleotides coding for a particul tract of amino acids in the protein have been synthesized.
Two mixed-sequence PCR primers were constructed based on t available partial amino acid sequence for TGFe (see Table 3)
PRIMER-1 (28-mer) :
5'-GCGGATCCGAT GTG AAG CC(ACGT) GA(CT)ATG GA -3' N- Asp Val Lys Pro Asp Met Glu -C
The Primer 1 sequence corresponds to SEQ ID NO:3 through SEQ I NO:10. A BamHI restriction site (underlined) was incorporate into the primer near its 5'-end to facilitate subsequent clonin of the PCR product into an M13 vector. Two additional base precede the BamHI site to stabilize it and improve cutting b BamHI. Best guesses were made with regard to codon usage for th first three amino acids (Asp Val Lys) , according to establishe guidelines (Lathe, R. (1985) J. Mol. Biol. .181:1-12). Al possible nucleotides were incorporated into the third position o the codon for Pro, and both of the two third position choices wer included for the following Asp. The remaining five nucleotide at the 3' end of this primer were unambiguous, as Met has only on possible codon, and both of the two codons for Glu begin with GA. As an alternative the following primer was constructed ba on the same amino acid sequence.
PRIMER-2 (23-mer) :
5'- GAT(C) GTG(C) AAA(G) CCC GAT(C) ATG GAA(G) GT -3' N- Asp Val Lys Pro Asp Met Glu Val-C
Primer 2 collectively represents SEQ ID NO:12 through SEQ NO:43. A restriction site was not incorporated as the PCR prod is cloned using a T-vector. In using a T-vector advantage taken of the fact that Taq polymerase adds a single adenosine the 3' end of the amplified fragment. A T-vector was construc by incubation of Smal-cut M13 vector with Taq polymerase wit mM dTTP under standard conditions. This resulted in the addit of a single thymidine at the 3' end of each fragment (Marchuk al. (1991) Nucl. Acids Res. .19:1154). PCR products, after purification, are ligated to the vector as both the vector and PCR products have complementary single base 3' overhangs. Prim 2 is less degenerate, but more specific, though it could hav maximum of 20% mismatch. However, as shown by Su et al. (19 Mol. Biochem. Parasitol. 4j>:331-336; and Lee et al. (1988) Scie 239:1288. an authentic DNA sequence can be synthesized by PCR e with this high degree of base pair mismatches between the pri and the cDNA. Use of Primer-2 and this approach, however, was successful.
A nonspecific T-tailed antisense primer at the 3' end used to produce first strand cDNA. The T-tailed primer pri cDNA synthesis at the poly(A) tail of the mRNA. This resemb the procedure reported by Frohman et al. (1988) as the RACE (Ra Amplification of cDNA Ends) method. The sequence of the T-tai primer is:
primer D: 5'- GCGAATTCTGCAGGATCCAAAC(T)18-3' (SEQ ID NO:44)
First-strand cDNA was used as the starting template. Fir strand cDNA was reverse transcribed from total RNA extracted fr bovine kidney using primer-D and murine leukemia virus rever transcriptase. The TGFe-specific cDNA was amplified using eith Primer-l or -2 in conjunction with Primer-E which is essential primer D lacking the T-tail:
primer E: 5'- GCGAATTCTGCAGGATCCAAAC -3' (SEQ ID NO:45)
The amplification was performed using standard PCR buff with 1.5 mM MgCl2 in 30 PCR cycles consisting of denaturation 94°C for 45 sec, annealing at 60°C for 45 sec, and elongation 72°C for 90 sec.
PCR products are eluted from a 2% agarose gel and reamplifi with the same primers using 65°C temperature for annealing. T reamplified product is electroeluted from 2% agarose gels, and D is precipitated. Each reamplified PCR product is inserted in an M13 vector and cloned. Where the product is derived from primer lacking a restriction site, the DNA is ligated into vector, a modified version of an M13 vector, obviating the ne for a restriction site, and cloned.
Table 3. Oligonucleotide Primers
S
Name Size Sequence ID
PRIMER11 28-mer 5'-GCGGATCC-GAT-GTG-AAG-CCA- GAC-ATG-GA- 3' 5'-GCGGATCC-GAT-GTG-AAG-CCC--GAC-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCG--GAC-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCT--GAC-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCA--GAT-ATG-GA- 3' 5'-GCGGATCC-GAT-GTG-AAG-CCC--GAT-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCG--GAT-ATG-GA- 3 5'-GCGGATCC-GAT-GTG-AAG-CCT--GAT-ATG-GA- 3 N- Asp Val Lys Pro Asp Met Glu-C
PRIMER2 23-mer 5 -GAT -GTG -AAA -CCC -GAT -ATG -GAA -GT-3
5 -GAC -GTG -AAA -CCC -GAT ATG -GAA -GT-3
5 -GAT -GTC -AAA -CCC -GAT -ATG -GAA -GT-3
5 -GAC -GTC -AAA -CCC -GAT -ATG -GAA -GT-3
5 -GAT -GTG -AAG -CCC -GAT -ATG -GAA' -GT-3
5 -GAC -GTG -AAG -CCC -GAT -ATG -GAA- -GT-3
5 -GAT -GTC -AAG -CCC -GAT -ATG -GAA -GT-3
5 -GAC' -GTC' -AAG- -CCC -GAT -ATG- -GAA- -GT-3
5 -GAT' -GTG' -AAA- -CCC -GAC -ATG- -GAA- -GT-3
5 -GAC- -GTG- -AAA- -CCC- -GAC -ATG- -GAA- ■GT-3
5 -GAT- -GTC- -AAA- •CCC- -GAC -ATG- -GAA- ■GT-3
5 -GAC- -GTC- -AAA- -CCC- -GAC -ATG- -GAA- ■GT-3
5 -GAT- -GTG- -AAG- •CCC- -GAC -ATG- -GAA- ■GT-3
5 -GAC- ■GTG- -AAG- •CCC- -GAC -ATG- -GAA- ■GT-3
5 -GAT- ■GTC- -AAG- ■CCC- ■GAC' -ATG- ■GAA- ■GT-3
5 -GAC- ■GTC- -AAG- ■ccc- ■GAC- -ATG- ■GAA- ■GT-3
5 -GAT- ■GTG- -AAA- •ccc- ■GAT- -ATG- ■GAG- •GT-3
5 -GAC- ■GTG- -AAA- •CCC- ■GAT- -ATG- ■GAG- •GT-3
5 -GAT- ■GTC- -AAA- ■CCC- ■GAT- -ATG- •GAG- •GT-3
5 -GAC- •GTC- ■AAA- •ccc- •GAT- -ATG- ■GAG- •GT-3
5 -GAT- •GTG- •AAG- ■ccc- •GAT- ■ATG- ■GAG- •GT-3
5 -GAC- ■GTG- ■AAG- •ccc- •GAT- -ATG- ■GAG- •GT-3
5 -GAT- ■GTC- ■AAG- ■ccc- GAT- -ATG- •GAG- ■GT-3
5 -GAC- •GTC- •AAG- ccc- •GAT- ■ATG- ■GAG- GT-3
5 -GAT- •GTG- ■AAA- ccc- ■GAC- ■ATG- •GAG- GT-3
5 -GAC- GTG- •AAA- ccc- GAC- •ATG- •GAG- GT-3
5 -GAT- GTC- •AAA- ccc- GAC- •ATG- GAG- GT-3
5 -GAC- GTC- ■AAA- ccc- GAC- •ATG- GAG- GT-3
5 -GAT- GTG- •AAG- ccc- GAC- •ATG- GAG- GT-3
5 -GAC- GTG- AAG- ccc- GAC- •ATG- GAG- GT-3
5 -GAT- GTC- AAG- ccc- GAC- ATG- GAG- GT-3
5 -GAC- GTC-. AAG- ccc- GAC- ATG- GAG- GT-3
PRIMER D 5 -GCGAATTCTGCAGGATCCAAAC (T) ι8-3 ' PRIMER E 5 -GCGAATTCTGCAGGATCCAAAC-3 '
A BamHI restriction site, shown underlined, is incorporated into 5'-end of each primer. The nucleotide sequences of the PCR products are determi by known techniques, for example, by the dideoxy method (Sambr et al. (1989) supra) . From the sequence thus obtained, it possible to design less degenerate, or nondegenerate primers amplify the remainder of the cDNA, as demonstrated by Su et
(1991) Mol. Biochem. Parasitol. 45.:331-336. Another approach reduce degeneracy is to employ primers in which inosine substituted at one or more positions of codon ambiguity (Sambro et al. (1989) supra; Koth et al. (1988) Nucl. Acids Re 16:10932).
A different strategy is used to amplify the 5' end of TG cDNA. A specific antisense primer based on available TGFe ami acid sequence is used to prime cDNA synthesis initially, then t first strand cDNA is treated with terminal transferase to add polyA sequence at the 3'-end. A T-tailed nonspecific prim (e.g., primer D) is used in conjunction with the specif antisense primer to amplify the 5'-end of the TGFe cDN essentially as described by Frohman et al. (1988) Proc. Nat. Aca Sci. 85:8998-9002.
The foregoing techniques are applicable to cDNA amplificati based on longer primers, less degenerate primers and prime complementary to other cDNA segments encoding other regions of t TGFe amino acid sequence as such data are obtained. For exampl amino acid sequence data of various regions of TGFe can obtained by sequencing tryptic digest peptides of TGFe in whol or in part. The resulting amino acid sequences make it a matte of ordinary skill to generate new primers for cDNA amplification as previously described.
Another cloning strategy well-known in the art is to use a oligonucleotide probe for screening bovine or human kidney cDN or genomic libraries. A single (unique sequence) relatively lon oligonucleotide probe is synthesized using best-guess nucleotide at every position where the code is ambiguous. Probe 1 is base on the known amino acid sequence of TGFe as follows: N-Asp Val Lys Pro Asp Met Glu Val Ser Pro Pro Asp Asp 5'-GAT-GTG-AAG-CCT-GAC-ATG-GAG-GTG-TCC-CCC-CCC-GAT-GAC-
Tyr Thr-C
TAC-ACC-3' (SEQ ID NO:l)
Choice of which nucleotide to use at each position is based several factors including codon usage frequency tables and known under-representation of 5'-CG-3' dinucleotides in mammal DNA. (See, e.g., Sambrook et al. (1989) supra) .
The techniques of cDNA amplification and oligonucleot probing just described can be used in combination. For examp a unique, or less degenerate oligonucleotide, obtained by amplification, can be used as a probe to screen an oligo(dT)- random-primed bovine or human cDNA library, as described by Mor et al. (1990) Cell 61:203-211 and by Li et al (1990) Scie 250:1690-1694.
In further cDNA cloning experiments. Primer 1 (collective SEQ ID NO:3 through SEQ ID NO:10) is used in conjunction wit specific primer (Primer F) in place of the nonspecific antise primer. The sequence of the specific antisense primer is ba on internal amino acid sequence data. This specific antise primer was used, together with Primer 1 to amplify a partial T cDNA, i.e., about the first 420-480 bp of the TGFe mRNA. Pri F was made with 32-fold degeneracy and a 5' BamHI restricti site:
5'-GCG-GAT-CCG-GCT-TCA-GRG-GYT-TYT-TYT-CYT-G-3' (SEQ ID NO:47
R is A or G; Y is C or T.
To amplify the remainder of the cDNA (about 120-180 b Primer G, a sense primer with a 5' BamHI site, was made, wi nucleotide sequences derived from the internal amino ac sequence:
5'-GCG-GAT-CCA-GGA-GAA-GAA-RCC-HCT-SAA-RCC-3' (SEQ ID NO:46) R is A or G, S is C or G, and H is A,C or T. Primer G is used in conjunction with Primer E (SEQ ID NO:45), nonspecific T-tailed primer for the 3' end of mRNA.
Satisfactory cDNA libraries from bovine kidney, human kid or other suitable sources are commercially available (e. Clontech, Palo Alto CA; Stratagene, La Jolla, CA) . Cloning cDNA into a suitable vector (such as λgtio) and hybridization the probes at various stringencies in three cycles of screeni are performed. Screening for clones of interest by nucleic ac hybridization allows rapid analysis of very large numbers clones. Hybridizing clones are isolated, subcloned into M13mpl8 vector and sequenced using the Sanger method of dideox ediated chain termination (Sanger et al. (1977) Proc. Nat. Aca Sci USA 74.:5463-5467) . Although polymerase chain reaction (PC is mentioned as a means of DNA amplification, other nucleic ac amplification methods are known to those skilled in the art to suitable for use in the methods described herein; for example, t 3SR (Trademark, Baxter Laboratories) method.
Example 6: Expression of TGFe
In order to obtain relatively large amounts of substantial pure TGFe for biological or therapeutic use, the full length TG cDNA is introduced into an appropriate expression syste Nonglycosylated TGFe can be synthesized in a prokaryot expression system. Full-length TGFe cDNA is amplified wit suitable primers (the first primer is based on the cDNA sequence and the second primer is again primer E) using PCR. The PC product is isolated from an agarose gel, digested with EcoRI an Bglll and directionally cloned into the polylinker region of th pFLAG-1 vector (International Biotechnology, Inc. , NewHaven, CT) This vector allows for IPTG-inducible expression of cloned gen products as fusions with an amino-terminal vector-derived peptide Presence of the FLAG peptide facilitates purification of th product by anti-FLAG monoclonal antibody immunoaffinit chromatography. The FLAG fusion protein is purified by affinit chromatography employing anti-FLAG monoclonal antibody coupled agarose. Following purification, the protein is recovered removal of the FLAG peptide with enterokinase.
Grown bacteria are lysed and a pellet fraction contain TGFe is obtained by centrifugation. TGFe is solubilized in urea and further purified as necessary. Because TGFe conta post-translational modifications, including disulfide b formation and glycosylation, a mammalian expression system, s as Chinese hamster ovary (CHO) cells or COS cells is preferred production of glycosylated TGFe.
Full-length TGFe cDNA is inserted into the plasmid V19.8 transfected into COS cells. This approach was recently used express human mast cell growth factor by Mortin et al. (19 supra. For long term production Chinese hamster ovary (CHO) ce are employed. Wong et al. (1985) Science 228:810-815 u cotransformation of dihydrofolate reductase (DHFR)-deficient cells with CSF cDNA cloned into p3A, a plasmid expressing DH The initial transfectants are selected for growth in increas concentrations of methotrexate. Selection of cells resistant methotrexate leads to the amplification of the dhfr gene, w concomitant amplification of extensive regions of DNA flanking dhfr sequence of the cotransferred plasmid. This approach le to cells lines expressing high levels of TGFe. Medium conditio by either cell line is collected and analyzed for the producti of TGFe. TGFe is identified in the antibody capture as (Example 7) . Its biological activity is tested in the soft a assay for stimulation of colony growth of SW-13 cells, and T is purified by a sequencial molecular sieve chromatography, HP and high performance electrophoresis chromatography, as necessar The identity of this recombinant protein with TGFe can be verifi by SDS-PAGE and glycosylation analysis, by amino acid sequenc and, most importantly, by the ability of neutralizing antibody block its biological activity, i.e., stimulation of colo formation of SW-13 cells in soft agar. Example 7
Natural or recombinant TGFe (rTGFe) is used as an antigen intradermal or intraperitoneal immunization to develop monoclo antibodies to TGFe. At least 5 μg to 10 μg of natural TGFe immunization is administered. The relatively greater abunda of recombinant TGFe (TGFe synthesized by transformed ce expressing cDNA or genomic DNA encoding TGFe) makes it possi to use larger immunizing doses of rTGFe. Alternativel intrasplenic immunization is carried out. The advantage intrasplenic immunization is that even smaller amounts of antig are required.
Briefly, female Balb/c mice are immunized with 3 doses TGFe 4-6 weeks apart with several intraperitoneal and intraderm injections administered each time. First dose of TGFe emulsified in complete Freund's adjuvant, the subsequent doses a in incomplete Freund's adjuvant. Alternatively, (using natur or rTGFe, or synthetic peptides) intrasplenic immunization done. Serial dilutions of serum from the mouse to be used f fusion are tested for anti-TGFe activity. The immunized mouse ( mice) are killed, splenocytes are isolated and fused with SP2 myeloma cell line using polyethylene glycol. After screening hybridomas in the antibody capture assays active cell lines a cloned, maintained and frozen so they can be used for futur antibody production.
Many growth factors have highly conserved amino aci sequences between species, making antibody production difficult However, the strategy of developing antibodies to syntheti peptides representing a hydrophilic segment of the full-lengt protein has been successfully used to develop antibodies agains TGFα and TGFB (See Sorvillo et al. (1990) Oncogene 5_:377-386) Flanders et al. (1988) Biochemistry 22:739-746). From the full length sequence of TGFe cDNA, a hydropathy plot of the deduce amino acid sequence is constructed. Highly hydrophilic segment of the TGFe amino acid sequence are compared by computer analysi with known sequences to select one or more unique sequences do not share epitopes with other peptides or proteins. selected peptides of 15-20 amino acids are prepared with flan cysteine or tyrosine residues and coupled to BSA or soy trypsin inhibitor with m-maleimidobenzoyl sulfosuccinimide es as a coupling agent. Alternative carriers, such as keyhole lim hemocyanin, and alternative coupling agents such as glutaraldeh or bisimido esters can be determined experimentally, as descri by van Regenmortel et al. (1988) , Laboratory Techniques Biochemistry and Molecular Biology. Vol 19, (R.H. Burdan and P vanKnoppeberg, eds.) , Elsevier, New York. A number of methods anchoring the synthetic peptide to the carrier are kno Preferably both amino acid carboxy ends are linked to the carr to form a loop of the synthetic peptide that provides maxi exposure of the peptide to the antibody.
Polyclonal chicken and rabbit antibodies have been rai against TGFe after immunization with a peptide of the seque Asp-Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pro-Asp-Asp-Tyr (SEQ NO:51) conjugated to bovine serum albumin.
IgG is precipitated from immune rabbit serum with ammon sulfate. IgY was precipitated from chicken egg yolk w polyethylene glycol. The chicken IgY recognizes the pept (unconjugated) in a Western blot assay and in an ELISA, altho relatively weakly. Both rabbit and chicken antibodies have w TGFe-neutralizing activity, as measured in the SW-13 colony as in soft agar. SW13 colony formation was reduced by 50%.
Because of the partial sequence identity of SEQ ID NO:l, N-terminal sequence of TGFe with epithelins 1 and 2 and w granulin, the following alternate approaches are taken. A pept corresponding in sequence to amino acids 2-20 of SQ ID NO:48 synthesized and aggregated by heat treatment before injecti A shorter peptide, corresponding in sequence to amino acids 2 of SEQ ID NO:48 is synthesized and conjugated to bovine se albumin for use in immunizing for polyclonal antibody producti
Polyclonal antibodies can also be produced. Polyclo antibodies can be raised in the rabbit or in the sheep, or ot species known to elicit an antibody response. It is preferred raise antibodies against conserved mammalian antigens in avi species, such as turkey or chicken. Chickens and rabbits a preferred as animals requiring small amounts of antigen f immunizations. Repeated injections of low doses (2-5 μg) antigen will also allow selection for high affinity antisera. to five adult leghorn chickens are immunized with at least 5 of native bovine TGFe or rTGFe each. Before the fir administration of TGFe 5 ml of control serum is obtained from wing vein. TGFe suspended in a 500 μl water-oil (1:1.5, v/ emulsion of complete Freund's adjuvant is administered throu several pectoral intramuscular sites. Booster doses of 2 to 5 TGFe per chicken suspended in incomplete Freund's adjuvant a administered via the same route 2 to 3 weeks later and at 2 to week intervals thereafter. The animals are bled 10 to 14 da after each boost from one of the wing veins. Production chicken egg yolk antibodies is a particularly efficient and che source of antibodies against a conserved mammalian protei (Gassmann et al (1990) FASEB J. 4.:2528-2532) .
IgY is purified from egg yolks and chicken serum b precipitation with Polyethylene glycol as described by Poison e al. (1980) Immunol. Commun. 9.:495-514. This method achieves 90 pure IgY.
To produce rabbit antibodies, up to five rabbits ar immunized with native TGFe according to the protocol described fo immunizing chickens. Preimmune serum (5 ml) is obtained befor immunization. Antisera are further purified using either combination of ammonium sulfate precipitation and anion exchang chromatography or a protein A bead column (Harlow and Lane (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. An antibody capture assay is preferred for rapid screen of antibodies in production. Ten to twenty ng of pure natural recombinant TGFe are bound to the bottom of the wells o microtiter plate in a phosphate or carbonate buffer overnight 4°C. After extensive washing with PBS and blocking of nonspeci sites, 50 μl of tissue supernatants or small aliquots of sera yolk IgY are added for 1 hr. Horseradish peroxidase-labe rabbit anti-mouse IgG, or suitable anti-rabbit or anti-chicken diluted in 3% BSA/PBS are added fr 1 hr, plates are washed w PBS and antibody bound to TGFe are detected with the addition TMB (3, 3', 5, 5'-tetramethylbenzidine) substrate soluti Positive wells are evaluated by a plate reader at 450 nm (Har and Lane, 1988 supra) .
' SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: University of Georgia Research Foundation, Inc.
(ii) TITLE OF INVENTION: TRANSFORMING GROWTH FACTOR-E (iii) NUMBER OF SEQUENCES: 51
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(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 303/499-8080
(B) TELEFAX: 303/499-8089
(C) TELEX: 823189
(2) INFORMATION FOR SEQ ID NO:l:
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
34 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asp Val Lys Pro Asp Met Glu Val Ser Pro Pro Asp Asp Tyr 1 5 10
Thr 15
(2) INFORMATION FOR SEQ ID NO:2:
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( i) SEQUENCE DESCRIPTION: SEQ ID NO:2
Pro Glu Pro Lys Lys Pro Glu 1 5
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GCGGATCCGA TGTGAAGCCA GACATGGA 28
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SUBSTITUTESHEET (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCGGATCCGA TGTGAAGCCC GACATGGA 28
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GCGGATCCGA TGTGAAGCCA GATATGGA 28
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37
SUBSTITUTESHEET (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Asp Val Lys Pro Asp Met Glu 1 5
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SUBSTITUTESHEET (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GATGTCAAGC CCGATATGGA AGT 23
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SUBSTITUTESHEET ( i) SEQUENCE DESCRIPTION: SEQ ID NO:25: GACGTGAAGC CCGACATGGA AGT 23
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(A) LENGTH: 22 base pairs
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SUBSTITUTESHEE ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCGAATTCTG CAGGATCCAA AC 22
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GCGGATCCAG GAGAAGAARC CHCTSAARCC 30
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
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( i) SEQUENCE DESCRIPTION: SEQ ID NO:47 GCGGATCCGG CTTCAGRGGY TTYTTYTCYT G 31
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(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
48 (ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: <1..2
(D) OTHER INFORMATION: /label= uncertain /note= "Xaa is Ala or Pro"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Xaa Gly Leu Ala Ala Ala Gly Pro Ala Pro Ser Glu Ser Gin Glu 1 5 10 15
Lys Lys Pro Leu Lys Pro Glu Gly Ala
20
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( i) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Asp Val Lys Cys Asp Met Glu Val Ser Cys Pro Asp Gly Tyr Thr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Val Lys Cys Asp Leu Glu Val Ser Cys Pro Asp Gly Tyr Thr 1 5 10
49
SUBSTITUTESHEET (2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( i) SEQUENCE DESCRIPTION: SEQ ID NO:51: sp Val Lys Pro Asp Met Glu Val Ser Pro Pro Asp Asp Tyr 5 10
50

Claims

1. TGFe in substantially purified form, said TGFe being monomeric protein exhibiting an apparent M, of 22,000 t 28,000 d as determined by sodium dodecyl sulfat polyacrylamide gel electrophoresis performed unde nonreducing conditions, said TGFe having biologica activities of stimulating the proliferation of SW-13 cells A431 cells, epithelial cells and epidermal keratinocytes and serving as a mitogen for fibroblasts, said TGFe havin an ED50 of about 0.5 to about 1.5 ng as measured with sof agar growth of SW-13 cells, and said TGFe bein characterized by the N-terminal amino acid sequence N-Asp Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pro-Asp-Asp-Tyr-Thr, a given in SEQ ID NO:l, and internal peptide sequences of Pro Glu-Pro-Lys-Lys-Pro-Glu, as given in SEQ ID NO:2, and Xaa
Gly-Leu-Ala-Ala-Ala-Gly-Pro-Ala-Pro-Ser-Glu-Ser-Gln-Glu-Lys Lys Pro-Leu-Lys-Pro-Glu-Gly-Ala, where Xaa is Ala or Pro, a given in SEQ ID NO:48.
2. A method for purifying TGFe having an ED50 of from about 0. ng to about 1.5 ng, as measured with soft agar growth of SW
13 cells, from pooled active fractions resulting fro molecular sieve chromatography and cation exchang chromatography of an acid-ethanol extract of animal or huma tissue or cells containing TGFe, comprising:
a) fractionating proteins in said pooled active fractions by reverse phase, high performance liqui chromatography to yield TGFe-active fractions;
b) pooling said TGFe-active fractions to yield pooled active fractions; and
c) fractionating proteins of said pooled TGFe-active fractions of step (b) by high performance electrophoresis chromatography to yield subtantially purified TGFe having an ED^, of from about 0.5 ng t about 1.5 ng.
3. The method of claim 2 wherein said cells or tissue are o bovine origin.
4. The method of claim 3 wherein said bovine tissue is bovin tissue.
5. The method of claim 2 wherein said cells or tissue are o human origin.
6. The method of claim 5 wherein said tissue is human kidney.
7. The method of claim 5 wherein said cells of human origin ar cells selected from the group consisting of A431 squamous carcinoma cells, D562 squamous carcinoma cells, T24 bladder carcinoma cells and normal human epidermal keratinocytes.
8. The method of claim 2 wherein the cells are Chinese hamster ovary cells containing and expressing DNA encoding human or bovine TGFe.
9. Cloned DNA encoding a polypeptide having TGFe activity.
10. The cloned DNA of claim 9 encoding bovine TGFe, said TGFe being a monomeric protein having an apparent molecular weight of 22,000 to 28,000 d as determined using SDS- polyacrylamide gel electrophoresis, said TGFe being characterized by the N-terminal amino acid sequence N-Asp- Val-Lys-Pro-Asp-Met-Glu-Val-Ser-Pro-Pro-Asp-Asp-Tyr-Thr, as given in SEQ ID N0:1, and internal peptide sequences of Pro- Glu-Pro-Lys-Lys-Pro-Glu, as given in SEQ ID NO:2, and Xaa-
Gly-Leu-Ala-Ala-Ala-Gly-Pro-Ala-Pro-Ser-Glu-Ser-Gln-Glu-Lys- Lys Pro-Leu-Lys-Pro-Glu-Gly-Ala, where Xaa is Ala or Pro, as given in SEQ ID NO:48, and wherein said TGFe has biological activities including stimulation of soft agar colony formation of SW-13 cells, and stimulation of A431 cells, epithelial cells, fibroblasts and epidermal keratinocytes.
11. The cloned DNA of claim 9 encoding human TGFe.
12. A monoclonal antibody specific for an epitope of human or bovine TGFe.
13. A polyclonal rabbit antibody reactive to human or bovine TGFe.
14. A polyclonal chicken antibody reactive to human or bovine TGFe.
PCT/US1992/008417 1991-10-03 1992-10-02 Transforming growth factor-e WO1993007173A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US77058591A 1991-10-03 1991-10-03
US07/770,585 1991-10-03

Publications (1)

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WO1993007173A1 true WO1993007173A1 (en) 1993-04-15

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ID=25089060

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Application Number Title Priority Date Filing Date
PCT/US1992/008417 WO1993007173A1 (en) 1991-10-03 1992-10-02 Transforming growth factor-e

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WO (1) WO1993007173A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0858808A2 (en) * 1997-01-17 1998-08-19 Johnson &amp; Johnson Medical Ltd. Peptides for use in wound treatment
GB2321191B (en) * 1997-01-17 2000-09-27 Johnson & Johnson Medical Peptides for use in wound treatment
EP1697541B1 (en) * 2003-12-19 2013-03-20 Roche Diagnostics GmbH Oligonucleotides, methods and kits for detecting Neisseria Gonorrhoeae

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, Volume 4, issued November 1986, J. BONNERJEA et al., "Protein Purification: The Right Step at the Right Time", pages 955-958. *
BIOCHEM AND BIOPHYS RES COMM., Volume 173, No. 3, issued 31 December 1990, A. BATEMAN et al., "Granulins, a Novel Class, of Peptide from Leukocytes", pages 1161-1168. *
BIOTECHNIQUES, Volume 1, issued November/December 1983, G. SOFER et al., "Designing an Optimal Chromatographic Purification Scheme for Proteins", pages 198-203. *
CANCER RESEARCH, Volume 47, issued 01 September 1987, J. HALPER et al., "Purification and Characterization of a Novel Transforming Growth Factor", pages 4552-4559. *
EXP CELL RESEARCH, Volume 190, issued 1990, C.A. BROWN et al., "Mitogenic Effects of Transforming Growth Factor Type E on Epithelial and Fibroblastic Cells - Comparison with Other Growth Factors", pages 233-242. *
J. OF CELLULAR BIOCHEMISTRY, Volume 42, issued 1990, P.G. PARNELL, "Purification of Transforming Growth Factor Type E", pages 111-116. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0858808A2 (en) * 1997-01-17 1998-08-19 Johnson &amp; Johnson Medical Ltd. Peptides for use in wound treatment
EP0858808A3 (en) * 1997-01-17 1999-08-25 Johnson &amp; Johnson Medical Ltd. Peptides for use in wound treatment
GB2321191B (en) * 1997-01-17 2000-09-27 Johnson & Johnson Medical Peptides for use in wound treatment
EP1697541B1 (en) * 2003-12-19 2013-03-20 Roche Diagnostics GmbH Oligonucleotides, methods and kits for detecting Neisseria Gonorrhoeae

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