WO1992016626A1 - Facteur de croissance epidermique equin - Google Patents

Facteur de croissance epidermique equin Download PDF

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Publication number
WO1992016626A1
WO1992016626A1 PCT/GB1992/000416 GB9200416W WO9216626A1 WO 1992016626 A1 WO1992016626 A1 WO 1992016626A1 GB 9200416 W GB9200416 W GB 9200416W WO 9216626 A1 WO9216626 A1 WO 9216626A1
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egf
sequence
protein
equine
polypeptide
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PCT/GB1992/000416
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English (en)
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Richard Mark Edwards
Keith Graham Mccullagh
Christine Anna Grek Power
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British Bio-Technology Limited
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Publication of WO1992016626A1 publication Critical patent/WO1992016626A1/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
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • EGF epidermal growth factor
  • EGF epidermal growth factor
  • epidermal growth factor is a naturally occurring polypeptide of 53 amino acids in length or less that is present in a large number of species including mammalian and avian species. In the mouse, EGF is produced from a larger precursor molecule. In vitro , EGF has been shown to act as a mitogen that can stimulate the proliferation of a range of mesenchymal and epithelial cell types.
  • EGF EGF may play a role in promoting the healing of mucosal ulceration in the gastro-intestinal tract (Wright et al, Nature 343: 82-85 (1990)). Carpenter and Cohen, J . Biol . Chem .
  • Patent Application USSN 506,078 Human EGF (hEGF) was first purified from urine where it is present in a low concentration. Sufficient EGF has now been obtained to allow determination of the amino acid sequence (Gregory et al, Nature 257: 325-327 (1975), Figure 1 and SEQ ID:1). This allowed the design and construction of a synthetic hEGF gene (Smith et al . Nucleic Acids Research 10: 4467-4482 (1982)). Recombinant hEGF has now been produced (see for example Brake et al PNAS 81: 4642-4646 (1984)).
  • hEGF Recombinant hEGF has been shown to have identical physico-chemical properties to natural murine EGF (mEGF, see Fig. 1 and SEQ ID:2).
  • EGF sequences have also been described for pigs (Pascall et al . J . Molecular Endocrinology (in press), the sequence for porcine EGF (pEGF) is given in Fig. 1 and SEQ ID: 3), rats (rEGF sequence is given in Fig. 1 and in SEQ ID: 4) and guinea pigs (gpEGF sequence is given in Fig. 1 and in SEQ ID: 5).
  • Human EGF (or urogastrone as it is sometimes referred to) was identified by its activity when it was found that a component of human urine inhibited gastric acid secretion (J.S. Gray, E. Wieczorowski and A.C. Ivy, Science 89: 489-490 (1939)). Subsequent to this mouse EGF was purified from the submaxillary gland and then sequenced (C.R. Savage, T. Inagami and S.J. Cohen, J. Biol . Chem 247: 7612-7621 (1972)). The primary sequence of human EGF was later determined (H. Gregory and B.M. Preston Int . J . Peptide Protein Res .
  • EGF equine EGF
  • a variant of equine EGF would be more efficacious in promoting epidermal growth than EGF (or variant thereof) from an animal of a different species.
  • the digital flexor tendons and suspensory structures of the equine limb are particularly prone to traumatic injury.
  • the acute and chronic lesions which ensue are described as tendonitis or tenosynovitis (McCullagh, K.G., Goodship, A.E.
  • human EGF or non-equine EGF could be used to treat wounds to the lower limb or tendon injuries.
  • one known assay for detecting EGF using the monkey-derived cell line Vero will detect human, murine and porcine EGF, the cross-reactivity of EGF in heterologous systems can be unpredictable (see Gregory et al "Methods of Hormone Radioimmunoassay” 2nd Edtn. 927-939 (1984)). This is due to the considerable divergence of EGF sequences outside a highly conserved "core" of the molecule, often called the consensus sequence.
  • a protein comprising a polypeptide which is naturally occurring equine EGF or a variant thereof, the variant having up to 13 variations from naturally occurring equine EGF.
  • the protein is a recombinant protein.
  • the protein of the present application is suitable for promoting wound healing such as by promoting epidermal growth, and are particularly suitable for administration to horses.
  • the use of the protein may lead to a decrease, or even avoidance of, scarring and formation of granulation tissue scars of the lower limb.
  • the protein of the present invention may obviate the need for conventional treatments of tendon injuries such as long term rest, "firing" and implant surgery.
  • the application of equine EGF, or a variant thereof, of the present invention, such as to injured tendons may induce the deposition of collagen fibres and thus may lead to the acceleration of healing and the re-establishment of tendon strength.
  • the use of the protein of the present invention is likely to be more efficacious for treating horses than non-equine EGFs and at the same time less likely to induce undesirable immune responses. It has been found that the protein of the present invention has similar properties to murine EGF. Such a protein is useful in the treatment of connective tissue wounds and/or injuries, for example in mammals, and in particular horses.
  • a "variation" in relation to a variant means an addition, deletion or substitution of an amino acid. Substitution is generally preferred.
  • the protein of the present invention can be either non-glycosylated or (preferably) glycosylated. However, it should be noted that naturally occurring eEGF does not contain an N-glycosylation site.
  • the protein of the present invention may thus be provided with a glycosylation site, usually an N-linked glycosylation site. Such a site can be engineered into the protein after expression if desired.
  • a glycosylation site usually an N-linked glycosylation site.
  • Such a site can be engineered into the protein after expression if desired.
  • equine EGF eEGF
  • polypeptide may be the naturally occurring eEGF sequence having a few amino acids (such as up to five) omitted from either the amino or carboxy terminus.
  • a preferred variant has one or more of the five carboxy terminus amino acids omitted (and so preferably does not have the terminal sequence - WELR, i.e. it comprises residues 1 to 48 of natural eEGF).
  • the polypeptide preferably has 6 cysteine residues and 4 glycine residues. In particularly preferred embodiments, the 6 cysteine residues are at residue position nos. 6, 14, 20, 31, 33 and 42 and/or the glycine residues appear at residue position nos.
  • the numbering system employed for the polypeptide is such that the first amino acid is the amino acid at the amino terminus of the polypeptide, which will generally correspond to the amino terminal amino acid in a naturally occurring EGF.
  • the substitution of V for K19 which is a protease site.
  • Others may include M for V21; P for 57; V for E24; and/or H for Y29.
  • the single letter code will often be used to denote the amino acid, instead of the three letter code, and for the sake of reference the single letter and three letter amino acid codes are as follows: Three-letter Single-letter
  • polypeptide possesses the following residues (which, as previously, are indicated by firstly the number to indicate position and secondly a single letter to indicate the amino acid): 13Y, 15L, 16H, 41R, 43Q and/or 47L.
  • polypeptide possesses the residue 21V: this change may assist in purification. It is preferred that the polypeptide possesses one, any combination, or (optimally) all of the following residues:
  • polypeptide possesses one, any combination of, or (optimally) all of the following five amino acids: b) 7S;
  • a particularly preferred polypeptide has the following sequence: X 1-5 C 6 X 7-8 S 9 X 10 DGYCL X 16-17 G 18 X 19 C 20
  • polypeptide i.e. at the carboxy terminus
  • an amino acid sequence may be placed at one end of the polypeptide (e.g. the C-terminus) to aid purification. This may result in a fusion protein.
  • the proteins of the present invention will suitably be less than 60 amino acid residues in length and in preferred embodiments the protein is the polypeptide.
  • the protein of the present invention consists of the amino acid sequence of equine EGF as set out in Figure 1 (SEQ ID: 8).
  • nucleic acid which may be RNA, or preferably, DNA
  • nucleic acid can be isolated nucleic acid or (preferably) recombinant nucleic acid.
  • the nucleic acid may also be chemically synthesised in vitro .
  • the nucleic acid may be in the form of a vector, such as a plasmid, cosmid or virus, and in some embodiments is in an expressible form.
  • a third aspect of the present invention relates to a vector comprising nucleic acid of the second aspect.
  • the vector may be adapted to transform (or transfect: the terms are used interchangeably in this specification) a prokaryotic (e.g.
  • the vector is preferably capable of replication in a prokaryotic, e.g. bacterial, cell such as of the genus Escherichia and/or in a eukaryotic, e.g. yeast, cell such as the genus Saccharomyces .
  • Preferred vectors will be capable of replication in E. coli, Pichia pa s t or i s and/or S a c ch a romy c e s c ere vi s i a e.
  • the vector is provided with a selectable marker for maintenance in the host (e.g.
  • the vector will usually comprise a cloning site and suitably at least one (e.g. selectable) marker gene.
  • the use of one or more marker genes may allow the selection of cells or hosts transformed with the vector and, preferably, to enable the selection of cells harbouring vectors incorporating heterologous DNA. Appropriate transcriptional start and stop signals will generally be present.
  • the vector is intended for expression (and the invention contemplates both expression vectors and cloning vectors) then sufficient regulatory sequences to drive expression will be present.
  • this may be a promoter operatively linked to the sequence to be inserted in the cloning site, and, preferably, a sequence enabling the protein product to be secreted.
  • One suitable promoter is the PGK promoter, which can be obtained from Saccharomyces cerevisiae .
  • a suitable secretion signal for yeast is the yeast ⁇ -factor pre-pro leader sequence.
  • the invention also contemplates host cells, including mammalian or other animals cells such as insect cells, but preferably unicellular organisms, that contain and preferably express, or are capable of expressing, the nucleic acid of the second aspect.
  • a fourth aspect of the present invention relates to a host transformed with a vector of the third aspect.
  • Unicellular hosts may be eukaryotic or prokaryotic and are preferably yeast cells, such as of the genus Saccharomyces , or bacteria, such as of the genus Escherichia .
  • suitable hosts include E . coli and (preferably) Saccharomyces cerevisiae .
  • a process for the preparation of a protein of the first aspect comprising coupling successive amino acid residues and/or oligopeptides and/or polypeptides together. This may be achieved by chemical synthesis, although translation of a nucleic acid of the second aspect in vitro or, particularly, in vivo is preferred.
  • a process for the preparation of a nucleic acid in accordance with the fifth aspect of the invention, the process comprising coupling successive nucleotides and/or ligating oligonucleotides and/or polynucleotides together.
  • nucleic acid may be synthesised chemically, it is preferred to use a nucleic acid-directed polymerase, preferably in vivo .
  • a seventh aspect of the present invention relates to a process for the preparation of a vector of the third aspect, the process comprising coupling successive nucleotides and/or ligating oligonucleotides and/or polynucleotides together.
  • An eighth aspect of the present invention relates to a process for the preparation of a host of the fourth aspect, the process comprising transforming a cell with a vector of the third aspect.
  • a ninth aspect of the present invention relates to a protein of the first aspect for use in human or veterinary (e.g. equine) medicine.
  • a tenth aspect of the present invention relates to the use of a protein of the first aspect in the preparation of any one, or any number of, or (preferably all) the following: a) a cell growth promoting agent or cell growth promoter.
  • promotion of cell growth is to be interpreted as covering induction, stimulation and/or proliferation of cell growth.
  • Preferred cells include epidermal , epithelial and mesenchymal cells ;
  • a lung surfactant production promoter which includes the induction of lung surfactant production
  • sperm production promoter which is to be interpreted as including the restoration of sperm production, for example in sialoadenectomised mammals, such as mice;
  • an anti-ulcer agent such as an anti-mucosal ulcer agent, for example those that exist in the gastro-intestinal tract;
  • a wound healing agent This is to be interpreted as including the promotion, stimulation and/or induction in growth of skin, epidermal or corneal tissue; and h) a connective tissue (e.g. tendon) wound and/or injury healing agent.
  • a connective tissue e.g. tendon
  • the agent may include deposition of collagen fibres, which is desired for injured tendons, such as in the treatment of tendonitis.
  • An eleventh aspect of the present invention relates to a pharmaceutical or veterinary composition comprising one or more proteins of the first aspect of the present invention and a pharmaceutically or veterinarily (for example, equine) acceptable carrier.
  • compositions may be adapted for intravenous administration, and therefore will preferably be sterile.
  • compositions include preparations of one or more sterile proteins of the first aspect in isotonic physiological saline and/or a suitable buffer.
  • the composition may include an anaesthetic to alleviate any pain on injection.
  • the compositions of the present application may be in unit dosage form, for example as a dry powder or water-free concentrate. These may be provided in a hermetically sealed container, such as an ampoule or sachet, which will suitably indicate the quantity of protein in activity units.
  • the amount of protein to be administered will depend upon the effect required, the required speed of action, and the disorder or ailment to be treated.
  • Suitable doses range from 0.01 to 100 ⁇ g/kg, preferably 0.1 to 1.0 ⁇ g/kg body weight.
  • Preferred compositions are injectable compositions. These will suitably be injected at volumes of from 0.1 to 1.0 ml, suitable at regular intervals over 7 to 28 days.
  • the pharmaceutical composition will be administered dose to the site of any injury (e.g. orally), for example to the lower limb if treating tendonitis and/or tenosynovitis. In (e.g. horses) this may be at or dose to the flexor tendons of the (e.g.
  • the proteins of the present invention can thus be useful in a method for the treatment or prophylaxis of ulcers and/or wounds, or additionally for inducing and/or treating any of the effects or disorders in (a) to (h) previously mentioned in the tenth aspect, the method comprising the administration of an effective non-toxic amount of a protein of the first aspect to a human or animal (e.g. a horse).
  • proteins of the present invention may find use in the promotion of cell growth, inhibition of gastric secretion, promotion of lung surfactant production, inhibition of wool growth, promotion of sperm production, the treatment of ulcers and in the healing of wounds and in the treatment of tendonitis and/or tenosynovitis.
  • a twelfth aspect of the present invention relates to nucleic acid comprising: (a) the sequence:
  • ATATGAATTCCTGCATCAANACNGAAGGNGGNTA (b) a sequence (e.g. substantially) complementary to the sequence of (a); (c) a sequence encoding the amino acid sequence
  • Figure 1 shows the amino acid sequences of human, murine, rat, guinea pig and porcine EGF placed above the sequence of equine EGF in accordance with the present invention
  • Figure 2 gives the sequences of the primers that were used to amplify equine EGF cDNA (SEQ ID: 6 and 7)
  • Figure 3 gives the sequence of cDNA for equine EGF (SEQ ID: a ) , and che.
  • FIG. 4 gives the sequences of equine EGF mutagenesis primers and the sequence of synthetic eEGF gene of the present invention in yeast expression vector pTD4-37 , also of the present invention
  • Figure 5 is a map of equine EGF expression vector pTD4-37 of the present invention
  • Figure 6 is a graph of receptor binding analysis of recombinant equine EGF of the present application produced by secretion in a yeast cell.
  • EXAMPLE 1 Design of primers to amplify equine EGF cDNA Although a number of EGF residues are known (see for example I.D. Campbell et al, supra) the identity of the remaining residues cannot usually be predicted based on known sequence homologies. Furthermore, the limited homology between EGF from different species makes it difficult to design oligonucleotide probes that might detect equine EGF cDNA clones. However, use has been made of a small region of high homology between the precursors for mouse and human EGF to design primers that can be used to specifically amplify DNA corresponding to the equine EGF coding sequence.
  • DNA amplification as an in vitro method for the enzymatic synthesis of specific DNA sequences using 2 oligonucleotide primers that hybridise to opposite strands and flank the region of interest in target DNA.
  • a repetitive series of cycles involving template (cDNA) denaturation, primer annealing and the extension of annealed primers by a thermostable DNA polymerase (Taq polymerase) results in the exponential accumulation of a specific DNA fragment whose termini are defined by the 5' end of the primers.
  • the EGF precursor cDNA has been cloned from human kidney and shown to contain approximately 1200 amino acid residues (G.I. Bell et al Nucleic Acids Research. 14: 8427-8446 (1986)).
  • the sequence is highly homologous to the mouse EGF precursor (over 75% homology at the nucleotide level) and includes not only the mature EGF coding sequence but also 8 cysteine rich EGF-like repeats which precede mature EGF and are organised in 2 blocks separated by a 260 amino acid segment.
  • the cloning technique employed in the present application involves the isolation of mRNA from equine kidney. Messenger RNA was reverse transcribed into cDNA (target DNA) using AMV reverse transcriptase.
  • RNA pellets were resuspended in water at a concentration of 1 mg/ml.
  • Poly A + mRNA was isolated from total RNA by 2 rounds cf oligo-dT cellulose chromatography by a modification of the method of Aviv and Leder Proc. Natl . Acad. Sci ., USA 69: 1408-1412 (1972).
  • Total equine RNA (1 mg/1 ml) was heated to 65°C for 5 minutes, adjusted to 0.5 M LiCl, 10 mM Tris-HCl (pH 7.5) and 0.2% SDS and applied to a 1 ml oligo-dT cellulose column equilibrated in HSB (10 mM Tris-HCl (pH 7.5)/0.5 M LiCl/0.2% SDS). The flow through from the column was collected, reheated to 65°C for 5 minutes and reapplied to the column. Non-polyadenylated RNA was eluted with HSB in 15 ⁇ 1 ml fractions.
  • Poly A + mRNA was eluted with LSB (10 mM Tris-HCl (pH 7.5) and 0.2% SDS) in 5 ⁇ 1 ml fractions. The presence of RNA in eluted fractions was determined spectroscopically by absorbance at 260 nm. Poly A + mRNA containing fractions were pooled, the salt concentration adjusted to 0.5 M LiCl, reheated at 65°C and reapplied to a freshly equilibrated oligo-dT cellulose column. Samples were processed as described above. After 2 rounds of oligo-dT cellulose chromatography, poly A + mRNA containing fractions were pooled.
  • EXAMPLE 3 Synthesis of cDNA Template Up to 10 ⁇ g of poly A + mRNA in 120 ⁇ l of sterile water was denatured with 12.5 ⁇ l of 0.1 M MeHgOH for 7 minutes at room temperature. 11 ⁇ l of 0.7 5-mercaptoethanol and a further 25 ⁇ l of H 2 O were added and incubation was continued for 5 minutes at room temperature. The reaction mixture was placed on ice and the following reagents were added: 1 ⁇ l oligo-dT primer (5 ⁇ g/ml)
  • EXAMPLE 4 Amplification of Equine EGF cDNA cDNA encoding equine EGF was amplified from equine kidney cDNA by a modification of the method of Saiki et al, Science 239: 487-491 (1988). 100-300 ng of cDNA was amplified in a reaction volume of 100 ⁇ l containing 10 mM Tris-HCl (pH 8.3) at 25°C, 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM each of dATP, dGTP, dCTP and dTTP. Finally, 1 ⁇ M each of the primers (SEQ. ID Nos.
  • AMPLITAQ Trade Mark, available from Cetus Corporation, 1400 Fifty-Third Street, Emeryville, California 94608, U.S.A.
  • Samples were overlayered with 100 ⁇ l of light paraffin to prevent evaporation.
  • the amplification reaction was performed in a Techne PHC-2 programmable heating block and comprised 40 cycles of the following steps: Heat denaturation (2 minutes at 95°C); annealing (2 minutes at 55°C) and primer extension (2 minutes at 70°C). At the end of the 40th cycle, the samples were further incubated for 10 minutes at 70°C.
  • DNA was precipitated with 2.5 volumes of ethanol, recovered by centrifugation, washed with 70% ethanol and resuspended in 10 ⁇ l of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA).
  • 10 ng of the 300 bp DNA product was ligated into 100 ng of HindIII/EcoRI digested plasmid vector BLUESCRIFT M13KS+ to form the vector pEE11 (prepared as described later) in a 20 ⁇ l ligation reaction containing 10 mM DTT, ImM ATP and 400 units of T4 DNA ligase, incubated for 16 hours at 15°C.
  • Plasmid DNA was sequenced as described in the appendix of this book. A total of 9 independent cDNA clones derived from 3 cDNA preparations and 2 different kidneys were used to confirm the sequence of the cDNA for equine EGF. The sequence deduced from the 9 clones analysed is shown in Figure 3. One of these clones carrying the eEGF sequence was used to clone into a plasmid pEE11 for further use. The CINTEGGY sequence (see Figure 3) of the sense primer is located 41 amino acid residues upstream from the putative EGF start site which occurs at nucleotide 131. The mature EGF comprises 53 amino acids which show 68% homology with the human EGF sequence.
  • EGF-ike molecule Its identity as an EGF-ike molecule is confirmed by the presence of conserved cysteine and glycine residues at positions 6, 12, 14, 18, 20, 31, 33, 36, 39, 42 as well as the presence of a conserved tyrosine at position 37. Altogether, the sequence is 62.5% homologous to human ⁇ GF over residues 1-43. It differs, however, at a number of residues which are otherwise conserved across all known EGF species. These are (equine residues in brackets): P7(S), V19(K), M21(V), E24 (V) and Y29(H).
  • EXAMPLE 5 Expression of Equine EGF in Yeast To confirm that the cloned equine EGF codes for an active molecule, the gene was expressed in yeast. This entailed the fusion of the N-terminus of the mature equine EGF coding residues to the C-terminus of the yeast ⁇ -factor pre-pro sequence (SLDKR, or Ser-Leu-Asp-Lys-Arg, encompassed in SEQ. ID: 13).
  • Mature EGF was then liberated from the pre-pro sequence by the yeast lysine-arginine endopeptidase KEX2 (Julius et al Cell 37: 1075-1089 (1984)) and subsequently secreted into the culture medium.
  • the introduction of the equine EGF coding sequence into the yeast expression vector required the introduction of a HindiII site and five C-terminal coding residues of the ⁇ -factor pre-pro sequence at the N-terminus and a SacI site at the C-terminus of equine EGF. This was achieved by site-directed oligonucleotide mutagenesis using the method of Kunkel et al Methods in Enzymology 154, 367-382(1987).
  • oligonucleotides SEQ. ID: 9 and 10 were designed and synthesised for the mutagenesis as shown in Figure 4 (BB3720 and BB3721). Plasmid pEE11, was digested with EcoRI and HindIII and the resulting 300 bp carrying the eEGF cDNA insert, fragment was then cloned into M13mp18 (M13mp18 is a double-stranded version of the vector BLUESCRIPT M13KS+ and commercially available from New England Biolabs and CP Labs, PO Box 22, Bishops Stortford, Herts, England).
  • the plasmid pEE11 thus comprises the M13KS+ vector into which has been cloned the 297 bp (HindIII to EcoRI) fragment containing the equine EGF sequence (of Figure 3). Recombinant plaques were picked and the presence of the correct insert determined by DNA sequence analysis. The recombinant phage were used to infect Escherichia coli strain RZ1032 (HfrKL16 PO/45 [lysA(61-62)], dutl, ungl, thil, relA1 Zbd-279::Tn10, supE44) and single stranded DNA isolated as described by Kunkel et al supra.
  • Single stranded DNA template was made from the resultant plaques and the sequence of the insert determined to confirm the identity of the desired mutant isolates.
  • mutagenesis techniques were used to obtain a HindIII - SacI fragment by adding the 5aa ⁇ -factor pre-pro sequence at the N-terminus (HinIII end), and introducing a SacI site at the carboxy terminus, of the equine EGF coding sequence.
  • This gene was thus cloned, on a HindIII-SacI fragment, into the yeast expression vector pTD4-18 by replacement of a human EGF gene cloned in this vector being between a Hind and SacI sites, to produce pTD4-37 (see Figures 4 and 5).
  • Plasmid pTD4-37 contains an E. coli replication origin and ampicillin gene derived from pUC18 (Yanisch-Perron et al , Gene 33: 103-119 (1985)).
  • the yeast replication origin and LEU2 gene are derived from the plasmid pJDB219 (Beggs 1978). Transcription is controlled by the Saccharomyces cerevisiae PGK promoter derived from pMA91 (Mellor et al , Gene 24: 1-14 (1983)).
  • the equine EGF coding sequence is fused in-frame with the pre-pro sequence of the yeast alpha factor gene (Kurjan et al Cell 30: 933-943 (1982)) at the ffindlll restriction site and facilitates the secretion of EGF from yeast.
  • Plasmid pTD4-37 was transformed into yeast Saccharomyces cerevisiae strain MD50 (a/ ⁇ leu2-3/leu2-3 Ieu2-112/leu2-112 pep4-3/ +His3-11/+His3-15/+) by the method of J.D. Beggs, Nature 275: 104-109 (1978).
  • coli HW87 containing the plasmid pTD4-37 was deposited under accession number NCIMB 40372 on 5th March 1991 at The National Collections of Industrial and Marine Bacteria Limited, 23 St. Machar Drive, Aberdeen, Scotland AB2 1RY, United Kingdom. All yeast media was as described by Sherman et al , Methods in Genetics, Cold Spring Harbor Laboratory (1986). Yeast containing pTD4-37 were grown in 50 ml of 0.67% synthetic complete medium Yeast Nitrogen Base (1% glucose) and grown for 60 hours at 30°C. The culture was centrifuged and the concentration of EGF in the supernatant measured by a receptor binding assay.
  • Binding Assay A receptor binding assay was used to determine the expression levels of equine EGF.
  • Monkey VERO cells were grown in 24 well plates at a density of 5 ⁇ 10 4 . The growth medium was removed and 150 ⁇ l of receptor binding medium added. Yeast supernatant was diluted at 1 to 10 in receptor binding medium. Samples (40 ⁇ l) were then added and a standard curve of six half log dilutions starting from 316 ng/ml of human EGF was included. 125 I murine EGF 1 uCi (Amersham) was diluted with 500 ⁇ l receptor binding medium and 10 ⁇ l added to each well. The plates were incubated for 1 hour. The medium was removed and the cells washed three times with PBS.
  • the cells were dissolved in 1M NaOH for 1 hour and the samples counted.
  • the EGF concentration in the yeast supernatant expressing pTD4-37 was determined by comparison of the binding of 125 I-EGF to the standard curve.
  • the concentration of EGF in the supernatant was determined to be 218 ng/ml, see Figure 6.
  • Murine EGF mEGF
  • Anti-sense primer used to amplify
  • 19 bp N is C or T
  • Sequence Type Nucleotide with corresponding protein Sequence Length: 34 nucleotides
  • 32 bp N is A or G or T or C
  • Sequence Type Nucleotide with corresponding protein Sequence Length: 297 base pairs
  • Equine EGF mutagenesis oligonucleotide antisense primer to fuse equine EGF in-frame with ⁇ -factor pre-pro sequence from 20 to 25 bp HindIII site
  • Equine EGF mutagenesis oligonucleotide antisense primer to introduce SacI site downstream of equine EGF.
  • Sequence Type Nucleotide with corresponding protein Sequence Length: 286 base pairs

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Abstract

On décrit les séquences d'acides nucléiques et d'acides aminés du facteur de croissance épidermique équin (EGF), ainsi que certaines variantes de celui-ci. On mentionne également des vecteurs et des hôtes pouvant être utilisés pour exprimer par translation l'acide nucléique pour produire l'EGF équin (ainsi que ses variantes). Des compositions pharmaceutiques contenant de l'EGF équin (et ses variantes) sont utiles pour la cicatrisation des plaies et en particulier pour le traitement de la tendonite chez le cheval.
PCT/GB1992/000416 1991-03-14 1992-03-09 Facteur de croissance epidermique equin WO1992016626A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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WO2002095029A2 (fr) * 2001-05-18 2002-11-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Sequences d'acide nucleique et de proteine du facteur de croissance epidermique chez les bovins
US8084042B2 (en) 1995-09-01 2011-12-27 Corixa Corporation Compounds and methods for immunotherapy and diagnosis of tuberculosis

Citations (1)

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JPH06160616A (ja) * 1992-09-07 1994-06-07 Sharp Corp 光学ヘッド

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JPH06160616A (ja) * 1992-09-07 1994-06-07 Sharp Corp 光学ヘッド

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US8084042B2 (en) 1995-09-01 2011-12-27 Corixa Corporation Compounds and methods for immunotherapy and diagnosis of tuberculosis
WO2002095029A2 (fr) * 2001-05-18 2002-11-28 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Sequences d'acide nucleique et de proteine du facteur de croissance epidermique chez les bovins
WO2002095029A3 (fr) * 2001-05-18 2003-05-30 Ca Minister Agriculture & Food Sequences d'acide nucleique et de proteine du facteur de croissance epidermique chez les bovins
US6967090B2 (en) 2001-05-18 2005-11-22 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Nucleic acid and protein sequences of bovine epidermal growth factor

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GB9105446D0 (en) 1991-05-01
IE920768A1 (en) 1992-09-23
ZA921892B (en) 1993-09-13

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