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WO1991005565A1 - CONSTRUCTIONS BIOSYNTHETIQUES DE TGF-$g(b) - Google Patents

CONSTRUCTIONS BIOSYNTHETIQUES DE TGF-$g(b) Download PDF

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Publication number
WO1991005565A1
WO1991005565A1 PCT/US1990/006006 US9006006W WO9105565A1 WO 1991005565 A1 WO1991005565 A1 WO 1991005565A1 US 9006006 W US9006006 W US 9006006W WO 9105565 A1 WO9105565 A1 WO 9105565A1
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WIPO (PCT)
Prior art keywords
protein
amino acid
acid sequence
tgf
sequence
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PCT/US1990/006006
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English (en)
Inventor
Charles M. Cohen
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Creative Biomolecules Inc
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Creative Biomolecules Inc
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Publication of WO1991005565A1 publication Critical patent/WO1991005565A1/fr
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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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to biosynthetic peptide constructs of transforming growth factor-beta, to synthetic genes encoding polypeptides having transforming growth factor-beta-like biological activity, to methods of producing such synthetic genes using recombinant DNA technology, and to the use of such biosynthetic peptide constructs as regulators of cell proliferation and growth.
  • TGF- ⁇ Transforming growth factor-beta
  • This factor has the ability to stimulate cell proliferation in cells of mesenchymal origin, while also being able to inhibit the growth of epithelial cells, embryonic fibroblasts, endothelial cells, and T and B lymphocytes.
  • TGF- ⁇ has a number of other regulatory activitites which appear to be related uniquely to the specialized function of a particular cell type.
  • TGF- ⁇ stimulates the production of matrix components, e.g., inhibiting the synthesis and secretion of proteolytic enzymes which act on tnese components, thereby regulating the synthesis and degradation of extracellular matrix (for a review, see, e.g., Sporn et al. (1987) J. Cell Biol. 1115:1039-1045).
  • TGF- ⁇ -type activities have been identified in many normal fibronectin- and collagen-producing fibroblasts, as well as tissues such as kidney (Roberts et al. (1983) Biochem. 22:5692-5698), placenta (Frolik et al. (1983) Proc. Natl. Acad. Sci.
  • TGF- ⁇ there are four known molecular configurations of TGF- ⁇ , each having an apparent molecular weight of about 25,000 daltons. Three of these species result from homodimeric (TGF- ⁇ l, TGF-B2) and heterodimeric (TGF- ⁇ l.2) combinations of the monomeric subunits, ⁇ l and B2.
  • the fourth species is a homodimer of a B3 subunit. Each subunit is processed from a precursor of about 390 amino acids, and the mature subunit protein includes approximately 112 amino acids of its carboxy terminus.
  • the ⁇ l and ⁇ 2 subunits have about a 70% amino acid sequence homology in their N-termin.al portions, and are highly conserved between species.
  • TGF-B3 The deduced amino acid sequence of TGF-B3 shares about 80% homology with types ⁇ l and ⁇ 2, with many of the differences being conservative substitutions.
  • TGF- ⁇ l was originally isolated from human platelets and placenta (EP 0128849), and bovine kidney (Roberts et al. ibid.).
  • TGF-B2 was originally identified as cartilage-inducing factors (CIF) isolated from bovine bone (US 4,774,228).
  • CIF cartilage-inducing factors isolated from bovine bone
  • TGF- ⁇ l.2 has been found in porcine platelets and other cells which coexpress the ⁇ l and ⁇ 2 chains (Cheifetz et al. (1988) J. Biol. Chem. 211:10783-10789).
  • TGF- ⁇ 3 has been identified in both human and chicken (Duke et al. (1985) Proc. Natl. Acad. Sci. (USA) £5_:4715-4719) .
  • the TGF- ⁇ 's belong to a larger gene family, the members of which encode structurally similar proteins that have similar regulatory activities (reviwed in Massague (1987) Cell 4.1:437-438). Included in this family are: (1) Vgl, a protein involved in mesoderm formation during Xenoous development; (2) decapentaplegic complex (DPP), a polypeptide encoded by a Drosophila gene responsible for development of the dorsoventral pattern in the embryo; (3) OP1, a region of a native osteogenic protein sequence encoded by exons of a genomic DNA sequence retrieved by applicants; (4) cartilage inducing factors (CIFs) isolated from bovine bone (US 4,774,228); (5) mammalian osteogenic bone matrix proteins CBMP-2a, CB P-2b, and CBMP-3, discovered by applicants (see WO89/01453); and (6) ⁇ -inhibin-a and b, gonadal proteins that suppress pituitary secretion of f
  • EP 0200341 discloses nucleic acid sequences encoding native TGF- ⁇ and precursors thereof which can be expressed in a host eukaryotic cell transformed therewith.
  • EP 0150572 discloses the manufacture of structural genes coding for TGF- ⁇ l and analogs thereof and the expression of these genes in microorganisms.
  • the design and expression of consensus protein constructs having considerable sequence homology with a number of the proteins in the TGF- ⁇ family, and displaying TGF- ⁇ -like activity, has heretofore not been contemplated.
  • TGF- ⁇ having TGF- ⁇ biological activities.
  • Another object is to provide an efficient method of producing novel, active TGF- ⁇ analogs.
  • Yet another object is to provide genes encoding novel, non-native, TGF- ⁇ species and methods for their production using recombinant DNA techniques.
  • Another object is to provide novel truncated forms of TGF- ⁇ and structural designs for proteins with TGF- ⁇ biological activity.
  • a further object is to provide metliods of regulating cell proliferation using TGF- ⁇ analogs.
  • TGF- ⁇ forms of native TGF- ⁇ which have been truncated at the N-terminus, and which have fewer than the native number of cysteine residues demonstrate TGF- ⁇ -like biological activity, including the ability to induce an anti-proliferative effect on mammalian epithelial cells in vitro. It has also been unexpectedly discovered that truncated analogs of other structurally similar proteins in the TGF- ⁇ family known to have unrelated biological activities also possess this TGF- ⁇ -like activity. These discoveries enabled the design and construction of DNAs encoding novel, non-native protein constructs which individually and combined are capable of inhibiting the proliferation of mammalian epithelial cells in culture.
  • the invention comprises a truncated TGF- ⁇ analog produced by expression of recombinant DNA in a host cell and capable of inducing an antiproliferative effect in mammalian epithelial cells in vitro.
  • This protein construct includes two polypeptide chains, each including a biologically active domain, and each having fewer than 9, and preferably 6 or 8, cysteine (Cys) residues. It may further be characterized as being unglycosylated.
  • the invention comprises a protein produced by expression of recombinant DNA in a prokaryotic host cell and including a pair of polypeptide chains of fewer than about 112 amino acids each.
  • the sequence of amino acids in each chain is sufficiently duplicative of the sequence of TGF- ⁇ such that the protein is capable of inducing an anti-proliferative effect on mammalian epithelial cells in vitro.
  • the polypeptide chain contains fewer than 9, and more preferably 6 or 8, cysteine residues, and further, may be unglycosylated.
  • cysteine residues are involved in the formation of intra- and inter-chain disulfide bonds (folding), the correct formation of which results in an active construct having TGF- ⁇ -like activity.
  • folding In eucaryotes, the synthesis and proper folding of the protein can occur at least within those cells known to express TGF- ⁇ ; in prokaryotic cells, folding must be performed in vitro, a difficult feat in that any number of combinations of disulfide linkages exist between two polypeptide chains, each having less than 9, and preferably 6 or 8 cysteine residues.
  • An important aspect of this invention is the discovery that truncated constructs of the type described herein may be post-translationally modified and folded (by oxidation) in vitro to produce TGF- ⁇ -like activity.
  • TGF- ⁇ monomers are known in nature, ⁇ l, ⁇ 2, and ⁇ 3. Investigation of the properties and structure of these native forms enabled the development of a rational design for non-native (i.e., not known to be expressed in nature) , truncated protein constructs which also are capable of differentially regulati.ig cell proliferation in various cell types. Further, upon examination of a number of unrelated proteins with some amino acid sequence homology, it was unexpectedly discovered that they, too, possess TGF- ⁇ -like activity.
  • proteins encoded by these consensus sequences include the generic amino acid sequences:
  • each "X" independently represents one of the naturally occurring amino acid or a derivative thereof
  • each M ⁇ " independently represents an amino acid or a peptide bond.
  • the currently preferred active peptide constructs comprise amino acid sequences derived from the three monomeric subunits of TGF- ⁇ (1, 2, and 3), DPP, and OP1.
  • the amino acid sequences of these proteins are set forth in FIGURE 1 relative to the sequence of TGF- ⁇ l.
  • Preferred amino acid sequences within the foregoing generic sequences are:
  • any one of the amino acids shown may be used alternatively in various combinations, and "-" and "—” represent a peptide bond.
  • the numbering of amino acids is selected solely for purposes of facilitating comparisons among alternative sequences.
  • These generic sequences have fewer than 9, and preferably 6-8 cysteine residues where inter- and/or intramolecular disulfide bonds can form, and contain other critical amino acids which influence the tertiary structure of the proteins. Similar structural features are found in the above named known proteins of the TGF- ⁇ family whose amino acid sequences previously have been published.
  • TGF- ⁇ species (1, 2, and 3) have been described as capable of inducing an anti-proliferative effect in mammalian epithelial cells in vitro.
  • Particular useful sequenc3s include analogs having the following amino acid sequences:
  • each of these sequences designates the natural source DNA sequence encoding the amino acid sequence which, as far as applicants are aware, exhibits the most homology with the recited TGF- ⁇ analog.
  • the invention further includes DNA sequences encoding these constructs and a prokaryotic host cell engineered to express these DNA sequences.
  • the prokaryotic host cell e.g. I _ coli
  • the prokaryotic host cell is tranfected with a vector including the TGF- ⁇ -encoding DNA sequence.
  • the transformed cell is cultured to express the protein which is then purified and activated by oxidation in vitro.
  • the protein so treated has the ability to induce an anti-proliferative effect on cultured mammalian epithelial cells.
  • the biosynthetic constructs disclosed herein may be used to regulate cellular activities such as proliferation and growth.
  • these constructs have wide potential clinical applications, for example, by controlling the proliferation of various tumor cell lines, or by enhancing the growth rate of T and B lymphocytes in immunosuppressed (e.g. Acquired Immunodefficiency Syndrome (AIDS) patients.
  • the constructs also may be used in cell cultures to modulate growth of various types of eucaryotic cells.
  • FIGURE 1 is a comparison of the amino acid sequence of various proteins in the TGF- ⁇ family to TGF- ⁇ l.
  • FIGURES 2A and 2B are representations of a DNA sequence and corresponding amino acid sequence of a modified trp-LE leader sequence, two FB domains of protein A, an Asp-Pro cleavage sit3, and (A) the 6 Cys TGF- ⁇ l sequence, and (B) the 8. Cys TGF- ⁇ l sequence.
  • Nucleic acid sequences encoding truncated TGF- ⁇ analogs were designed based on sequence data reported in the literature, codons inferred from known amino acid sequences, and observations of partial homology with known genes of the TGF- ⁇ family. These sequences have been refined by comparison with the sequences present in certain regulatory genes from the TGF- ⁇ family.
  • each X independently represents a naturally occurring amino acid, and ⁇ or ⁇ represents an amino acid or peptide bond.
  • the N-terminal sequence of mature TGF- ⁇ and other related proteins contains a variable number of Cys residues which appear to be crosslinked among each other or with a residue of another amino acid chain, but not to Cys residues in the C-terminal domain. Maturation of the precursor to the mature form of these proteins occurs by trypsin-like cleavages between the precursor and the mature protein, and possibly also within the precursor form as other similar cleavage sites are present therein.
  • All members of the Vgl-related subgroup of the TGF- ⁇ family (including Vgl, DPP, OP1, CBMP-2a, CBMP-2b, and CBMP-3) share the feature of two basic residues (i.e., Lys-Lys, Arg-Arg, ⁇ rg-Lys) following the first Cys in the conserved C-terminal domain.
  • the conserved double basic residues may represent another secondary maturation site. Cleavage by trypsin or related protease releases a C-terminal domain containing only 6 Cys residues.
  • TGF- ⁇ contains up to 5 Cys residues which are not crosslinked to the C-terminal domain, the first of the 7 Cys residues may not be crosslinked to the C-terminal domain either. Therefore 6 Cys residues appear to be sufficient for a properly folded C-terminal domain.
  • genes which encode a number of appropriate amino acid sequences. These genes can be expressed in various types of eucaryotic cells but, for reasons of efficiency are preferably produced in prokaryotic host cells, thereby providing large quantities of active synthetic proteins such as truncated analogs, muteins, fusion proteins, and other constructs, all mimicking the biological activity of native TGF- ⁇ , including the ability to induce an anti-proliferative effect on cultured mammalian epithelial cells. More specifically, the DNA sequences designed according to the above criteria and logic were constructed using known techniques involving assembly of oligonucleotides manufactured in a DNA synthesizer.
  • sequences may be expressed using well established recombinant DNA tschnologies in various prokaryotic host cells, and the expressed proteins may be cleaved from precursors, oxidized, and refolded in vitro for biological activity.
  • This approach has been successful in producing a number of novel protein constructs not found in nature (as far as applicants are aware) which hava TGF- ⁇ -like activity, i.e., the ability to induce an anti-proliferative effect on cultured mammalian epidermal cells.
  • FIGURE 1 compares the amino acid sequences of these proteins with the sequence of TGF- ⁇ l (* denotes a match) , and TABLE 1 summaries the extent of homology.
  • first consensus sequence was designed to preserve 8 of the disulfide crosslinks and the apparent structural homology among the related proteins, while the second more highly truncated consensus sequence was d3signed to preserve 6 disulfide bonds. That sequence is:
  • the synthetic genes designed using the criteria set forth above are produced by assembly of chemically synthesized oligonucleotides. 15-100mer oligonucleotides are synthesized on a Biosearch DNA Model 8600 Synthesizer, and are purified by polyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA buffer (TBE) . The DNA is then electroeluted from the gel. Overlapping oligomers are phosphorylated by T4 polynucleotide kinase, and then ligated into larger blocks which may also be purifed by PAGE. Alternatively, natural gene sequences and cDNAs may be used for expression. The two resulting genes are shown as tne latter portion of the fusion sequences in FIGURES 2A and 2B. The sequence shown in 2A is a truncated form of 2B; five amino acids at the N-terminus have been eliminated.
  • the gene is modified by cassette mutagenesis.
  • the N-terminus is replaced up to the Clal site wish a hinge region that provides for release of the TGF- ⁇ protein from the leader, preceded by a BamHI site for attachment to leader peptides.
  • the modified ⁇ ene is then attached to an FB-dimer leader at che BamHI site.
  • the complete fusion gene is shown in FIGURE 2A.
  • the fusion gene is then inserted as an EcoRI to Pstl fragment into an expression vector based on pBR322 and containing a synthetic tryptophan (trp) promoter/operator and a modified trp-LE (MLE) leader (which is similar to the one described by Huston et al. in Proc. Natl. Acad. Sci. (USA) (1988) 85:5879-5883, but having only a single EcoRI site at the hinge of the MLE leader) .
  • the vector is opened at the EcoRI and PSTI restriction sites, and a FB-FB/TGF- ⁇ gene fragment is then inserted therebetween, where FB is fragment B of Staphylococcal Protein A.
  • the resulting expression vector includes the TGF- ⁇ gene to a fragment encoding FB. 3. Production of Active Analogs
  • the protein constructs are expressed in E. coli host strain JM101 (e.g.) grown in minimal medium (M9) after starvation for trp and induction by indoacrylic acid (IAA) .
  • the cells are lysed and the inclusion bodies collected by differential centrifugation.
  • the fusion proteins are purified from the inclusion bodies by urea or quanidine solubilization.
  • the FB sequence is then chemically cleaved from the TGF- ⁇ protein construct at the hinge region of the fusion protein.
  • the hinge region has the sequence Asp-Pro-Asn-Gly which can be cleaved at the Asp-Pro site with dilute acid, or at the Asn-Gly site with hydroxylamine.
  • the resulting cleavage products are passed through a Sephacryl-200HR column which separates most of the uncleaved fusion products from the TGF- ⁇ analogs.
  • Protein refolding is performed under the conditions of 50 mM Tris-HCl, pH 8.0, 3 M guanidine hydrochloride (GuHCl), 10 mM dithiothreitol (DTT) , and 1-10 mM oxidized glutathione.
  • This assay is based on the ability of TGF- ⁇ to inhibit DNA synthesis in the mink lung epithelial cell line, ATCC no. CCL 64.
  • EMEM Eagle's minimum essential medium
  • FBS fetal bovine serum
  • streptomycin 200 ⁇ g/ml streptomycin
  • the TGF- ⁇ test samples in EMEM containing 0.5% FBS are then added to the wells, and the cells are then incubated for another 18 hours. After incubation, 1.0 ⁇ Ci of 3-j-thymidine in 10 ⁇ l is added to each well. The cells are incubated for 4 hours at 37°C. The media is then removed and the cells washed once with ice-cold phosphate-buffer saline. The DNA is precipitated by adding 0.5 ml of 10% TCA to each well and incubating at room temperature for 15 min. The cells are then washed three times with ice-cold distilled water and lysed with 0.5 ml 0.4 M NaOH. The lysate from each well is then transferred to a scintillation vial and the radioactivity recorded using a scintillation counter (Smith-Kline Beckman) .
  • a scintillation counter Smith-Kline Beckman
  • ED50 50% effective dose

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Abstract

L'invention concerne des constructions du facteur bêta de croissance de transformation tronquée (TGF-β) produites par l'expression d'ADN de recombinaison dans une cellule hôte procaryotique. Ces constructions comprennent au moins une chaîne polypeptide ayant moins de 112 amino-acides environ et moins de 9 restes de cystéine. La séquence d'amino-acides dans ces constructions est suffisamment duplicative de la séquence du facteur-bêta de croissance de transformation natif de sorte qu'elle est capable d'induire un effet antiprolifératif sur des cellules épithéliales mammifères in vitro. L'invention concerne également des procédés de production d'analogues de TGF-bêta en utilisant une technologie d'ADN de recombinaison, ainsi que des procédés d'utilisation de tels analogues.
PCT/US1990/006006 1989-10-18 1990-10-18 CONSTRUCTIONS BIOSYNTHETIQUES DE TGF-$g(b) Ceased WO1991005565A1 (fr)

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US42296289A 1989-10-18 1989-10-18
US422,962 1989-10-18

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WO1991005565A1 true WO1991005565A1 (fr) 1991-05-02

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EP (1) EP0496833A4 (fr)
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AU (1) AU6870791A (fr)
CA (1) CA2070393A1 (fr)
WO (1) WO1991005565A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017099A3 (fr) * 1993-01-26 1995-01-05 Celtrix Pharma PEPTIDES DE TGF-β1 ET TGF-β2 BIOLOGIQUEMENT ACTIFS
US5436228A (en) * 1990-12-12 1995-07-25 Postlethwaite; Arnold E. Chemotactic wound healing peptides
EP0640143A4 (fr) * 1991-12-16 1996-10-23 Baylor College Medicine Inhibiteurs de synthese d'adn derives des cellules senescentes.
WO1996040771A1 (fr) * 1995-06-07 1996-12-19 Creative Biomolecules, Inc. ANALOGUES MONOCATENAIRES DE LA SUPERFAMILLE DU TGF-β (MORPHONS)
US5824647A (en) * 1990-12-12 1998-10-20 Postlethwaite; Arnold E. Chemotactic wound healing peptides
AU697916B2 (en) * 1993-01-26 1998-10-22 Celtrix Pharmaceuticals, Inc. Biologically active TGF-beta1 and TGF-beta2 peptides
WO2000020449A3 (fr) * 1998-10-07 2000-10-12 Stryker Corp PROTEINES MODIFIEES DE LA SUPERFAMILLE DU TGF-$g(B)
US6677432B1 (en) 1998-10-07 2004-01-13 Stryker Corporation Mutations of the C-terminal portion of TGF-β superfamily proteins
WO2007104945A3 (fr) * 2006-03-11 2008-05-15 Renovo Ltd Protéines, acides nucléiques et médicaments
WO2010090523A1 (fr) * 2009-02-06 2010-08-12 Pepscan Systems B.V. Protéines tronquées à noeud cystine
US7893221B2 (en) 2006-03-11 2011-02-22 Renovo Limited Protein folding
US7902150B2 (en) 2006-03-11 2011-03-08 Renovo Limited Medicaments and proteins based on TGF-β monomers for the treatment of wounds

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4572798A (en) * 1984-12-06 1986-02-25 Cetus Corporation Method for promoting disulfide bond formation in recombinant proteins

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IE57724B1 (en) * 1983-05-09 1993-03-24 Todaro George Joseph Biologically active polypeptides
IL78197A (en) * 1985-03-22 1991-07-18 Genentech Inc Nucleic acid encoding tgf-beta and its uses

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US4572798A (en) * 1984-12-06 1986-02-25 Cetus Corporation Method for promoting disulfide bond formation in recombinant proteins

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nature, Volume 316, issued 22 August 1985 DERYNCK et al., "Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells", pages 701-705, see entire document. *
See also references of EP0496833A4 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5436228A (en) * 1990-12-12 1995-07-25 Postlethwaite; Arnold E. Chemotactic wound healing peptides
US5824647A (en) * 1990-12-12 1998-10-20 Postlethwaite; Arnold E. Chemotactic wound healing peptides
EP0640143A4 (fr) * 1991-12-16 1996-10-23 Baylor College Medicine Inhibiteurs de synthese d'adn derives des cellules senescentes.
AU685084B2 (en) * 1993-01-26 1998-01-15 Celtrix Pharmaceuticals, Inc. Biologically active TGF-beta1 and TGF-beta2 peptides
AU697916B2 (en) * 1993-01-26 1998-10-22 Celtrix Pharmaceuticals, Inc. Biologically active TGF-beta1 and TGF-beta2 peptides
WO1994017099A3 (fr) * 1993-01-26 1995-01-05 Celtrix Pharma PEPTIDES DE TGF-β1 ET TGF-β2 BIOLOGIQUEMENT ACTIFS
US6479643B1 (en) 1995-06-07 2002-11-12 Stryker Corporation Single chain analogs of the TGF-β superfamily (morphons)
WO1996040771A1 (fr) * 1995-06-07 1996-12-19 Creative Biomolecules, Inc. ANALOGUES MONOCATENAIRES DE LA SUPERFAMILLE DU TGF-β (MORPHONS)
US6040431A (en) * 1995-06-07 2000-03-21 Stryker Corporation Single chain analogs of the TGF-β superfamily (morphons)
WO2000020449A3 (fr) * 1998-10-07 2000-10-12 Stryker Corp PROTEINES MODIFIEES DE LA SUPERFAMILLE DU TGF-$g(B)
US6677432B1 (en) 1998-10-07 2004-01-13 Stryker Corporation Mutations of the C-terminal portion of TGF-β superfamily proteins
US8518411B2 (en) 1998-10-07 2013-08-27 Stryker Corporation Modified TGF-β superfamily protein
WO2007104945A3 (fr) * 2006-03-11 2008-05-15 Renovo Ltd Protéines, acides nucléiques et médicaments
US7893221B2 (en) 2006-03-11 2011-02-22 Renovo Limited Protein folding
US7902150B2 (en) 2006-03-11 2011-03-08 Renovo Limited Medicaments and proteins based on TGF-β monomers for the treatment of wounds
US7947264B2 (en) 2006-03-11 2011-05-24 Renovo Limited TGF-β3 mutants
WO2010090523A1 (fr) * 2009-02-06 2010-08-12 Pepscan Systems B.V. Protéines tronquées à noeud cystine
US8772236B2 (en) 2009-02-06 2014-07-08 Pepscan Systems B.V. Truncated cystine-knot proteins

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AU6870791A (en) 1991-05-16
EP0496833A1 (fr) 1992-08-05
EP0496833A4 (en) 1992-12-02
JPH05501500A (ja) 1993-03-25
CA2070393A1 (fr) 1991-04-19

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