AU2005229155B2 - Nucleotide and amino acid sequences for ruminant epidermal growth factor - Google Patents

Description

WO 2005/095451 PCT/AU2005/000464 NUCLEOTIDE AND AMINO ACID SEQUENCES FOR RUMINANT EPIDERMAL GROWTH FACTOR FIELD OF THE INVENTION The present invention relates to an isolated polynucleotide molecule encoding a ruminant epidermal growth factor (EGF) peptide and to the expression of the encoded EGF peptide for use 5 in, inter alia, de-fleecing of wool-fleece bearing animals, particularly sheep. BACKGROUND OF THE INVENTION The use of chemical agents in the de-fleecing of sheep has been known for many years. Particular examples of agents that have been used or tried, included mimosine and various phthalimidinopentanes (described in the present applicant's Australian Patent Application No 10 59755/80 and in Australian Patent Application No 590971 in the name of Australian Wool Corporation). These agents function by retarding wool growth so that, depending on the dose, a zone of weakness is created in the wool fibres and the fleece is cast. The wool can then be mechanically harvested without the requirement for shearing. However, the use of chemical agents has not been without problems because the minimum dosage level for de-fleecing is close 15 to the levels that may cause serious metabolic disturbances in the animal. One development in this area, aimed at overcoming the problems caused by chemical agents, is described in the present applicant's Australian Patent No 546354, the entire disclosure of which is to be regarded as incorporated herein by reference. In particular, this patent discloses that epidermal growth factor (EGF) can affect wool or hair fibres in a similar way to the 20 abovementioned chemical agents but can be administered with safety to a sheep over a far greater dose range to achieve "biological de-fleecing". Human EGF is a peptide of 53 amino acids which includes three intramolecular disulfide bonds, characteristic of EGF peptides, essential for the peptide's biological activity. The active EGF peptide is translated as a long prepro-protein of 1207 amino acids and the EGF peptide is 25 released after proteolytic processing from position 970-1023 of the prepro-protein. Approximately 70% similarity is observed between human EGF and EGFs that have been previously isolated from other species. For example, human and mouse EGF have 37 amino acids in common across the sequence of 53 amino acids. Six of the common amino acids are WO 2005/095451 PCT/AU2005/000464 2 cysteine residues (amino acids 6, 14, 20, 31, 33 and 42), which have been found to be absolutely conserved (see Figure 1 showing the amino acid sequences for EGF from various different species) and which form the abovementioned three intramolecular disulfide bonds essential for the peptide's biological activity. 5 The gene encoding the EGF prepro-protein from humans is about 110,000 kilobases in length, and consists of 24 exons, with the encoding sequence for the EGF peptide divided between exons 20 and 21. The gene structure for human EGF has been published as has the full length cDNA sequence for mouse and rat EGF, and a partial cDNA sequence for pig and horse EGF. In addition, the Genbank database contains numerous entries for human, mouse and rat EGF 10 cDNAs and the human genome sequence (HGS) contains the full, unannotated gene sequence for the human EGF gene. More recently, the bovine EGF sequence has been cloned, and some precursor sequences for the sheep gene (ie exons 3 to 4 spanning about 400 base pairs of intron sequence) have also been described. The precise biological role of EGF has yet to be fully defined, however it is known to cause a 15 wide range of biological effects. For example, it has been known for some time that EGF is involved in the embryonic and neonatal development of mice, where it can cause premature eyelid opening and premature development of teeth. Also, it has been observed, in a premature lamb model, that injection of EGF can induce elevated maturation of lung alveolar membranes. Further, EGF has been incorporated with beneficial effect in the culture media for bovine and 20 ovine embryos in vitro and is also known to be of value for the in vitro culture, manipulation and survival of embryos from other livestock species. Moreover, it is known that EGF inhibits gastric acid secretion and modulates the synthesis of a number of hormones, including the secretion of prolactin from pituitary tumours and chorion gonadotropin from chorion carcinoma cells. In addition, it is known that, in vitro, EGF is a strong mitogen for many cells of 25 ectodermal, mesodermal and endodermal origin and, in particular, EGF appears to have a role in controlling and stimulating the proliferation of epidermal and epithelial cells, including fibroblasts, kidney epithelial cells, human glial cells, ovary granulosa cells and thyroid cells. EGF also stimulates the proliferation of embryonic cells. Further, EGF is known to act as a differentiation factor for some cell types, and has been shown to strongly affect the synthesis as 30 well as the turnover of various extracellular matrix proteins including fibronectin, collagens, laminin and glycosaminoglycans. Additionally, EGF increases the turnover and release of calcium from bone tissue, thereby promoting bone resorption, and can also act as a mitogen for WO 2005/095451 PCT/AU2005/000464 3 the basal cells of the olfactory epithelium that produce olfactory neurones. Further, due to its mitogenic effect on endothelial cells, promoted by thrombin, EGF can also stimulate angiogenesis. Finally, EGF is a strong chemoattractant for fibroblasts and epithelial cells, and, when either alone or acting with other cytokines, has been found to be an important wound 5 healing factor. A further biological effect of EGF, which is of particular interest to the present applicant, is the inhibition of hair growth, first observed when EGF was injected into new born mice, and which has since been administered in sheep to achieve biological de-fleecing (see the abovementioned Australian Patent No 546354). In particular, recombinant non-ruminant EGF has been used 10 commercially for this purpose, although it is not administered to pregnant ewes, in particular ewes in the third trimester of pregnancy, because research has indicated a risk of adverse effects in such animals. As a consequence, pregnant ewes must be identified within the flock and subjected to standard shearing. The present applicant considers that this problem may be overcome or alleviated by substituting this non-ruminant EGF with an EGF derived from sheep 15 or another ruminant. SUMMARY OF THE INVENTION The present invention is based on the discovery that while ruminant animals such as sheep do not naturally produce an active EGF peptide, these animals do include within their genomes, ancestral EGF-encoding nucleotide sequences (which are now non-functional) which can be used 20 to generate synthetic, functional ruminant EGF genes. In this regard, the present applicants have found through the isolation and sequencing of these remnant nucleotide sequences from sheep, goat, cow and deer and comparing the sequences against those of human and mouse EGF genes, that the sheep, goat and deer sequences for the EGF peptide contain a number of stop codons while the sequence from the cow does not include codons for all of the six cysteine residues that 25 are essential for EGF activity. The present applicant has now been able to modify the isolated sheep sequences to enable the expression of a ruminant EGF. More particularly, the present applicant has now been able to modify an ancestral ovine EGF encoding nucleotide sequence by replacing stop codons present in the sequence with functional codons to enable the expression of an ovine EGF.

4 Accordingly, in a first aspect, the present invention provides an isolated polynucleotide molecule encoding an epidermal growth factor (EGF) or a functional EGF fragment, said molecule comprising a nucleotide sequence showing at least 50% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment, and wherein said EGF or functional EGF 5 fragment comprises an amino acid sequence which includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. In a second aspect, the present invention provides an isolated EGF or functional EGF fragment 0 comprising an amino acid sequence showing at least 50% sequence identity to that set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment, and wherein said amino acid sequence or portion thereof includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. 5 In a third aspect, the present invention provides a method for producing an EGF or a functional EGF fragment, said method comprising introducing a molecule according to the first aspect into a suitable host cell, and culturing said cell under conditions suitable for the expression of said molecule. 0 In a fourth aspect, the present invention provides a composition suitable for administration to an animal, wherein the composition comprises an EGF or functional EGF fragment according to the second aspect and, optionally, a pharmaceutically- or veterinary-acceptable carrier. In a fifth aspect, the present invention provides a method of de-fleecing a wool fleece-bearing animal, 25 said method comprising administering to an animal an EGF or functional EGF fragment according to the second aspect, or a composition of the fourth aspect, in an amount effective to allow for the removal of the wool fleece without the requirement for shearing. BRIEF DESCRIPTION OF THE FIGURES 30 Figure I shows a comparison of the amino acid sequences of the EGF peptides of a number of different non-ruminant species and the amino acid sequences of the EGF peptides "encoded" by isolated ancestral ruminant EGF-encoding nucleotide sequences from sheep, goat, cow, Barbary sheep and deer. Stop codons are designated by an asterisk and conserved cysteine residues are indicated. If stop codons are corrected for conserved residues, then the amino acid sequence of the sheep EGF 35 shows no more than about 47% sequence identity with any of the non-ruminant sequences shown in the figure (nb. shows 25 identical residues with the horse sequence which is the closest non-ruminant sequence). On the other hand, when compared with the goat, cow and WO 2005/095451 PCT/AU2005/000464 5 Barbary sheep, the amino acid sequence of the sheep EGF showed, respectively, about 98%, 81% and 96% sequence identity. Figure 2 shows the sequence of the sheep EGF gene and the deduced peptide sequence where the stop codons have been corrected for the most likely ancestral codon. The first corrected 5 sequence at position 22 is a tyrosine residue (Y) based on the goat EGF-encoding sequence, a cysteine residue was inserted in position 33 based on the conservatism of this residue at this position, and arginine was inserted in position 41 again based on conservatism of this residue at this position. Figure 3 shows an alignment of the nucleotide and inferred amino acid sequence of exons 20 10 and 21 of the EGF gene. Full intron sequence is not included but the positions are indicated. Sequences for the cow, sheep, Barbary sheep and deer have been derived from the present inventors, the sequences for the pig and human are derived from the Genbank database. The location of the final processed sheep EGF peptide sequence is shown in bold. Stop codons are indicated with an asterisk. The nucleotide sequence from Fallow deer contains a deletion in 15 exon 21 indicated by the dashes. Only the nucleotide and amino acid sequences of the goat EGF encoding portion are included in the alignment. The sheep sequence has been found to be identical in Merino sheep, Wiltshire Horn sheep and Rambouillet sheep. DESCRIPTION OF THE SEQUENCES SEQ ID No: 1 nucleotide sequence of the corrected sheep EGF 20 SEQ ID No:2 amino acid sequence of the corrected sheep EGF SEQ ID No:3 nucleotide sequence of the goat EGF SEQ ID No:4 nucleotide sequence of the cow EGF SEQ ID No:5 nucleotide sequence of the deer EGF SEQ ID No:6 nucleotide sequence of the Barbary sheep EGF 25 SEQ ID No:7 amino acid sequence of the native goat EGF SEQ ID No:8 amino acid sequence of the native cow EGF 6 SEQ ID No: 9 amino acid sequence of the native deer EGF SEQ ID No: 10 amino acid sequence of the native Barbary sheep EGF 5 SEQ ID No: 11 nucleotide sequence of the human EGF SEQ ID No: 12 amino acid sequence of the native human EGF SEQ ID No: 13 amino acid sequence of the native rat EGF 0 SEQ ID No: 14 amino acid sequence of the native mouse EGF SEQ ID No: 15 amino acid sequence of the native horse EGF 5 SEQ ID No: 16 nucleotide sequence of the pig EGF SEQ ID No: 17 amino acid sequence of the native pig EGF DETAILED DESCRIPTION OF THE INVENTION 0 By modifying the nucleotide sequence of an ancestral sheep gene for EGF, the present applicant has been able to express a ruminant sheep EGF. It is considered that this EGF will be useful for de fleecing of wool fleece-bearing animals, particularly sheep, and may avoid or reduce the abovementioned problem with recombinant non-ruminant EGF of possibly causing spontaneous abortion in pregnant ewes. More particularly, it is considered that because the ruminant EGF is based 25 on endogenous sequences, the "ruminant EGF" peptide is more likely to be well tolerated by sheep and capable of being administered at substantially lower doses than required for de-fleecing with recombinant non-ruminant EGF. In fact, it is considered that doses as low as 50 to 250 .ig/kg body weight of the ruminant EGF may be suitable for de-fleecing of sheep. 30 The present invention provides an isolated polynucleotide molecule encoding an epidermal growth factor (EGF) or functional EGF fragment, said molecule comprising a nucleotide sequence showing at least 50% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment, and wherein said EGF or functional EGF fragment comprises an amino acid sequence which includes six conserved cysteine residues located at positions corresponding to 35 positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide.

7 Preferably, the molecule comprises a nucleotide sequence showing at least 65% sequence identity, more preferably at least 70% sequence identity, still more preferably at least 85% sequence identity, and even more preferably at least 90%, to that set forth in SEQ ID No: I or a portion thereof which encodes a functional EGF fragment. Most preferably, the molecule comprises a nucleotide sequence 5 showing at least 95% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment, or which substantially corresponds to the sequence set forth in SEQ ID No: I or a portion thereof which encodes a functional EGF fragment. An EGF peptide encoded by a polynucleotide molecule according to the present invention may be a 0 hybrid of EGF peptides from two or more species (eg an EGF peptide comprising a hybrid sequence of the amino acid sequences for sheep EGF and one or more other EGF peptides from other ruminant or non-ruminant species). Such a hybrid EGF peptide would retain one or more biological effects of an EGF peptide in an animal and the hybrid sequence includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human 5 EGF peptide. In other embodiments of the polynucleotide molecule of the present invention, the molecule comprises: a nucleotide sequence substantially corresponding to any one of the sequences set forth in '0 SEQ ID No: 3 (corresponding to goat EGF), SEQ ID No: 4 (corresponding to cow EGF), SEQ ID No: 5 (corresponding to deer EGF), and SEQ ID No: 6 (corresponding to Barbary sheep) but including one or more codon modification such that said nucleotide sequence encodes an epidermal growth factor (EGF) comprising an amino acid sequence which includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human 25 EGF peptide, or a nucleotide sequence substantially corresponding to the sequence of a portion of any one of the sequences set forth in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6 but including one or more codon modification such that said nucleotide sequence encodes a functional EGF fragment comprising an amino acid sequence which includes six conserved cysteine residues 30 located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. The present invention also provides an isolated EGF or functional EGF fragment comprising an amino acid sequence showing at least 50% sequence identity to that set forth in SEQ ID No: 2 or a portion 35 thereof which represents the amino acid sequence of a functional EGF fragment, and wherein said amino acid sequence or portion thereof includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide.

8 Preferably, the EGF or functional EGF fragment comprises an amino acid sequence showing at least 65% sequence identity, at least 70% sequence identity, still more preferably at least 85% sequence identity, and even more preferably at least 90%, sequence identity to that set forth in SEQ ID No: 2 or 5 a portion thereof which represents the amino acid sequence of a functional EGF fragment. Most preferably, the molecule comprises an amino acid sequence showing at least 95% sequence identity to that set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment, or which substantially corresponds to the sequence set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment. 0 In other embodiments of the EGF peptide of the present invention, the EGF peptide comprises: an amino acid sequence substantially corresponding to any one of the sequences set forth in SEQ ID No: 7 (corresponding to goat EGF), SEQ ID No: 8 (corresponding to cow EGF), SEQ ID No: 9 (corresponding to deer EGF), and SEQ ID No: 10 (corresponding to Barbary sheep) but including 5 one or more sequence modification such that said amino acid sequence includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide, or an amino acid sequence substantially corresponding to the sequence of a portion of any one of the sequences set forth in SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10 but including 0 one or more sequence modification such that said amino acid sequence represents the amino acid sequence of a functional EGF fragment and includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. 25 An EGF peptide according to the present invention may be considered to be a hybrid of EGF peptides from two or more species (eg an EGF peptide comprising a hybrid sequence of the amino acid sequences for sheep EGF and one or more other EGF peptides from other ruminant or non-ruminant species). Such a hybrid EGF peptide would retain one or more biological effects of an EGF peptide in an animal. 30 Accordingly, the present invention also provides an isolated EGF peptide or functional EGF fragment comprising an amino acid sequence which is a hybrid sequence, said hybrid comprising a portion of the sequence from SEQ ID No: 2 and a portion of one or more sequences selected from the group consisting of SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, or SEQ ID No: 10 but including one or 35 more codon modification such that said hybrid EGF peptide retains one or more of the biological effects of EGF in an animal and the hybrid sequence includes six conserved cysteine 8a residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide, Sequence identity percentages referred to herein are to be understood as having been calculated by 5 comparing two nucleotide sequences or two amino acid sequences, as the case may be, using aligmnent achieved with the Bestfit program with default settings for determining similarity (nb. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences). From the Bestfit alignment, a determination was made of the number of positions with identical nucleotides or amino 0 acids, divided by the total number of nucleotides or amino acids in the respective sequence (ic. 159 in the case of nucleotide sequences and 53 in the case of amino acid sequences), and expressed as a percentage. The term "substantially corresponding" as used herein in relation to nucleotide sequences is intended 5 to encompass minor variations in the nucleotide sequences which, due to degeneracy in the DNA code, do not result in a change in the encoded peptide. Further, the term is intended to encompass minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded peptide.

WO 2005/095451 PCT/AU2005/000464 9 The term "substantially corresponding" as used herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity of the EGF peptide or functional EGF fragment. These variations may include conservative amino acid substitutions. The substitutions envisaged are: G, A, V, I, 5 L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Na-alkylamino acids. The term "functional EGF fragment" is intended to refer to a fragment of EGF which retains all of the six cysteine residues that are essential for EGF activity and which exhibit activity that is substantially equivalent to the EGF peptide from which it is derived. The EGF peptide or functional EGF fragment of the present invention, may be produced by 10 protein synthesis methods well known to persons skilled in the art or, more preferably, by recombinant techniques well known to persons skilled in the art. Thus, the present invention also provides a method for producing an EGF or functional EGF fragment, said method comprising introducing a molecule according to the first aspect into a suitable host cell, and culturing said cell under conditions suitable for the expression of said 15 molecule. The present invention also relates to a vector, which comprises the isolated polynucleotide molecule of the present invention, and a host cell which contains said vector. The vector may be, for example, a phage, plasmid, viral or retroviral vector. The polynucleotide molecule of the present invention may be ligated to a vector for propagation in a host cell. 20 Generally, a plasmid vector is introduced to a host cell in a precipitate, such as calcium phosphate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell. Within the vector, the polynucleotide molecule should be operatively linked to an appropriate promoter such as phage lambda PL promoter, the E coli lac, trp, phoA and tac promoters, the SV40 early and 25 late promoters and promoters of retroviral LTRs. Other suitable promoters will be well known to the persons skilled in the art. The vector will preferably include at least one selectable marker. Suitable markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E coli and other bacteria.

WO 2005/095451 PCT/AU2005/000464 10 The host cell may be selected from bacterial cells such as E. coli, Streptomyces and S. typhimurium; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as Chinese hamster ovary (CHO), monkey kidney (COS) cells, human embryonic kidney 293 cells and Bowes melanoma cells; and plant cells. Appropriate 5 culture mediums and conditions for host cells of these kinds are well known to persons skilled in the art. The polynucleotide molecule ligated to a vector may comprise a nucleotide sequence encoding an EGF peptide, a functional EGF fragment, or an EGF prepro-protein. In the latter case, the host cell used for expression of the EGF prepro-protein will preferably be one in which post 10 translational processing from position 970-1023 of the translated EGF prepro-protein can occur. The present invention further provides a composition suitable for administration to an animal, wherein the composition comprises an EGF or functional EGF fragment according to the second aspect and, optionally, a pharmaceutically- or veterinary-acceptable carrier. The term "pharmaceutically-or veterinary-acceptable carrier" is intended to refer to any 15 pharmaceutically-or veterinary-acceptable solvent, suspending agent or vehicle for delivering the EGF protein to the subject. The composition of the present invention may be formulated for administration to an animal through any of the routes well known to persons skilled in the art, in particular subcutaneous administration and oral and nasal administration. 20 The present invention also provides a method of de-fleecing a wool fleece-bearing animal, said method comprising administering to an animal an EGF or functional EGF fragment according to the second aspect, or a composition of the fourth aspect, in an amount effective to allow for the removal of the wool fleece without the requirement for shearing. The animal is preferably a sheep, goat, llama or alpaca. More preferably, the animal is a sheep. 25 Most preferably, the animal is a Merino sheep. Preferably, the method involves administering the EGF or functional EGF fragment by subcutaneous administration, for example by injection or through a catheter inserted below the I1 skin. Alternatively, subcutaneous administration may be achieved through sustained release implant compositions or injectable depot-forming compositions. Typically, the EGF or functional EGF fragment will be administered at a dose in the range of 50 to 5 250 ig/kg body weight of the animal, or which is sufficient to establish a plasma concentration of the EGF peptide of at least about 10 ng/ml for at least 15 hours. Following administration of the EGF or functional EGF fragment, the sheep may be fitted with a wool fleece-retention net such as those described in International Patent Specification No WO 02/43474 and .0 US Patent No 5,447, 124 or the present applicant's Australian Patent No 690878. The net retains the wool fleece on the animal until removal is required, and may also assist in the manual removal of the wool fleece. The EGF or functional EGF fragment of the present invention may also suitable for other uses 5 including the promotion of embryo survival in culture. Thus, the present invention also extends to an embryo culture medium comprising an EGF or functional EGF fragment according to the present invention. Such media may further comprise antibiotic compounds and other agents as are well known to persons skilled in the art. The present invention also extends to the in vitro culturing of a ruminant embryo, comprising incubating said embryo in the presence of an embryo culture medium comprising .0 an EGF or functional EGF fragment according to the present invention. It will be further appreciated by persons skilled in the art that the EGF or functional EGF fragment of the present invention may also be suitable for other uses for which EGF peptides are known to be beneficial. Examples in this regard include (i) control of mammary gland maturation and development 25 in research studies, (ii) neonatal gut and lung maturation in research studies, (iii) hormone modulation studies, (iv) human models of wound repair which use ruminant animals, and (v) treatment of wounds. Such uses are to be considered to fall within the scope of the present invention. Still further, it will be appreciated by persons skilled in the art that the EGF or functional EGF 30 fragment of the present invention may be used to derive mimetic compounds suitable for the applications described herein. Thus, the present invention also extends to such mimetic compounds of the EGF and functional EGF fragment of the second aspect.

WO 2005/095451 PCT/AU2005/000464 12 Such mimetic compounds may be designed using any of the methods well known in the art for designing mimetics of peptides based upon peptide sequences in the absence of secondary and tertiary structural information (see Kirshenbaum et al., Curr. Opin. Struct. Biol. (1999) 9:530 535). For example, peptide mimetic compounds may be produced by modifying amino acid side 5 chains to increase the hydrophobicity of defined regions of the peptide (eg substituting hydrogens with methyl groups on aromatic residues of the peptides), substituting amino acid side chains with non-amino acid side chains (eg substituting aromatic residues of the peptides with other aryl groups), and substituting amino- and/or carboxy-termini with various substituents (eg substituting aliphatic groups to increase hydrophobicity). Alternatively, mimetic compounds of 10 the EGF or functional EGF fragment of the present invention may be so-called peptoids (ie non peptides) which include modification of the peptide backbone (ie introducing amide bond surrogates by, for example, replacing the nitrogen atoms in the backbone with carbon atoms), or include N-substituted glycine residues, one or more D-amino acids (in place of L-amino acid(s)) and/or one or more ax-amino acids (in place of p-amino acids or 7-amino acids). Further, 15 mimetic compounds of the EGF or functional EGF fragment of the present invention include "retro-inverso peptides" where the peptide bonds are reversed and D-amino acids assembled in reverse order to the order of the L-amino acids in the peptide sequence upon which they are based, and other non-peptide frameworks such as steroids, saccharides, benzazepinel,3,4 trisubstituted pyrrolidinone, pyridones and pyridopyrazmes. 20 In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples. EXAMPLE 1: GENERATION OF A DNA MOLECULE ENCODING SHEEP EGF Materials and Methods Animals 25 Adult Merino sheep were housed under cover in individual pens or metabolic cages. The animals were fed a maintenance ration of 600g of a pelleted mixture of 60% lucerne and 40% oats as one meal daily. Drinking water was provided. Isolation of a genomic DNA for sheep EGF WO 2005/095451 PCT/AU2005/000464 13 PCR primers for the EGF gene (exons 14 and 15) were constructed from the consensus DNA sequence of human and mouse EGF. These PCR primers were used to amplify a portion of genomic DNA from Merino sheep and the amplified sequence was confirmed, by sequencing, as representing most of exons 14 and 15 and the intervening intron. 5 These primers were then used to screen a sheep and goat genomic DNA BAC library containing 3x coverage of each genome. Several BAC clones were identified in this screen and these were subsequently isolated, purified and characterised. The nucleotide sequence of the clone was confirmed by directly amplifying and sequencing the necessary fragments using a genomic DNA template and PCR primers designed from the BAC DNA sequence according to standard 10 methods. Modification of DNA to allow expression of sheep EGF The native sheep EGF sequence contains stop codons at positions 22, 33 and 41 of the EGFprotein. In order to remove these stop codons, a synthetic gene sequence was generated using an automated synthesiser according to standard methods and the stop codons replaced with 15 functional codons considered to be the most likely ancestral codons. That is, the stop codon at position 22 was replaced with the codon for tyrosine found in the ancestral goat EGF-encoding sequence; the stop codon at position 33 was replaced with a codon for cysteine which is strongly conserved at this position and is essential for EGF activity, and the stop codon at position 41 was replaced with the codon for arginine which is strongly conserved at this position. Figure 2 20 provides the sequence of the sheep EGF gene and deduced peptide sequence where the stop codons have been corrected for the most likely ancestral codon. Results and Discussion One of the BAC clones was found to contain most of a gene encoding a sheep EGF prepro protein and, consequently, major portions of this BAC clone were sequenced, including exons 20 25 and 21 (human gene designation) encoding the putative sheep EGF peptide. Exon sequences were identified by alignment with the human gene sequence and intron-exon boundaries were located likewise (Figure 3). It was found that the sequence included three stop codons at positions 22, 33 and 41, thereby explaining why the sheep EGF gene is non-functional. Otherwise, the translated amino acid sequence showed less than 40% sequence identity with the 30 sequence of human EGF (20 of 53 amino acids in common). During synthesis of the synthetic EGF sequence, it was possible to modify the native sequence by replacing the stop codons present within the sequence with functional codons so as to generate a nucleotide sequence capable of being expressed. The modified nucleotide sequence is WO 2005/095451 PCT/AU2005/000464 14 shown in Figure 2, where it can be seen that the stop codons found were replaced with the most likely ancestral codons to introduce tyrosine at position 22, cysteine at position 33 and arginine at position 41. The translated amino acid sequence of the sheep EGF showed about 45% sequence identity with the sequence of human EGF. 5 Similar procedures, sometimes using consensus or redundant primers, were used to amplify the EGF-encoding portions from genomic DNA of other ruminant species, namely goat, cow, deer, Barbary sheep and Wiltshire Horn sheep. The nucleotide sequences for these EGF-encoding portions are shown in Figure 3. Those of goat, deer and Barbary sheep comprise one or more stop codons (see also Figure 1) and these sequences are similarly amenable to replacement of 10 stop codons with codons that are highly conserved based on sequence alignments. For example, the stop codons at positions 33 and 41 of the EGF nucleotide sequence of Barbary sheep and goat EGF nucleotide sequence can be replaced with the codons for cysteine and arginine respectively. Similarly, the stop codon at position 47 for the deer EGF sequence can be replaced with the codon for leucine which is strongly conserved at this position. A tryptophan codon can 15 also be inserted at position 49 of the deer EGF sequence. EXAMPLE 2: SYNTHESIS OF SHEEP EGF USING A CELL-FREE SYSTEM Materials and Methods Cell-free translation system A Rapid Translation System (RTS 500) E. coli HY Kit (Roche Molecular Biochemicals) was 20 used for the synthesis of three kinds of sheep EGF genes, namely a "native" form (using vector pIVEX 2.3a), a N-terminal Hisx6 form (using vector pIVEX2.4d) and a C-terminal Hisx6 form (using vector pIVEX2.3d) as described in detail in the instruction manual version 2, October 2001, catalogue number 3 269 019 (Roche Molecular Biochemicals). The reaction was performed according to the instruction manual in a 1 ml volume, with 10 gg of vector DNA as 25 template, a reaction temperature of 30'C and a stirrer speed of 120 rpm. Plasmid DNA pIVEX2.3a (NcoI-BamHI) was used as the expression vector for native sheep EGF, pIVEX2.4d (NcoI-BamHI) was used as the expression vector for the N-terminal Hisx6 fusion. pIVEX2.3dMCS (NdeI-XhoI) was used as an expression vector for the C-terminal Hisx6 fusion. 30 Details of the plasmid constructs can be found in the instruction manual version 2, October 2001, WO 2005/095451 PCT/AU2005/000464 15 catalogue number 3 269 019 (Roche Molecular Biochemicals) which can be downloaded at www.roche-applied-science.com/sis/proteinexpression. Localisation test The in vitro reaction mixture was centrifuged at 12000 rpm for 10 min. The supernatant and 5 pellet were regarded as soluble and insoluble fractions, respectively. These fractions were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions according to standard methods with Real Gel Plate (10-20% gradient gel, BioCraft, Japan). The gels were stained with Coomassie Brilliant Blue (CBB) to identify samples that contained recombinant protein. Samples were also analysed by Western Blot and probed with either anti 10 His6 peroxidase (Roche) in the case of His fusion proteins or anti-human EGF antibody in the case of native EGF, and BM Blue POD-substrate precipitating (Roche) was used for colorimetric detection. Pre-stained SDS-PAGE Magic Marker (Invitrogen); M1 and 6xHis Protein Ladder (Qiagen); M2 were used as markers. Purification and Solubilisation of translated EGF peptides 15 Recombinant peptides expressed as insoluble inclusion bodies can be purified under denaturing conditions according to the method described in Pearson R et al., 2000. The recombinant proteins thus purified are in an unfolded state. Prior to use in vaccination, they can be renatured using the Renaturation basic kit for proteins according to the manufacturer's instructions (catalogue Number 96827 by Sigma Aldrich) in view of Tsumoto, K et al., 2003 followed by 20 dialysis in 8M urea, 20mM Tris-HCl, pH 8.5, 500 mM NaCl to remove imidazole as described in Pearson R et al., 2000. Assaying biological activity Cell proliferation assays can be conducted using BaF/3ERX cells, a cell line derived from BaF/3 cells transfected with human EGFR according to the method described in Elleman, T et al., 25 2001. Briefly, sheep EGF may be added to the cells and cell proliferation measured by

[

3 H]Thymidine incorporation determined using an automated beta counter (1205 Betaplate, Wallac, Finland). Results and Discussion Only the N-Hisx6 sheep EGF was synthesised by the cell free system in insoluble form. The 30 size of the protein band was less than 15 kDa, as predicted. The productivity of N-Hisx6 EGF at 24 hours was about 100 pg/ml.

WO 2005/095451 PCT/AU2005/000464 16 EXAMPLE 3: FORMULATING SHEEP EGF FOR DE-FLEECING AND ADMINISTRATION Materials and Methods About 25 mg EGF may be digested in a small volume of 0.05M phosphate buffer, pH 7 with 5 trypsin (50 g, Worthingtom, TPCK-treated) for 4 hours at 37*C. Subsequently, another 50 mg may be added and the incubation repeated. The digestate can then be dialysed overnight at 10*C in Spectro-Por 6 membrane, and the dialysate freeze dried for storage until required. For sub-cutaneous administration, sheep can be injected inside a back leg with between 50 to 250 pg/kg of EGF prepared by resolubilising the freeze dried digestate in saline. 10 Alternatively, sheep may be subcutaneously infused with EGF, through the use of a polythene catheter inserted below the skin so that the orifice lays on the right midside region of the body. The freeze-dried EGF can be resolubilised in sterile saline solution and infused using a Harvard pump delivering a volume of 0.030 ml/min. Throughout this specification the word "comprise", or variations such as "comprises" or 15 "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in 20 the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed anywhere before the priority date of each claim of this application. It will be appreciated by persons skilled in the art that numerous variations and/or modifications 25 may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 2005/095451 PCT/AU2005/000464 17 REFERENCES Elleman, Thomas, C., T. Domagala, N. M. McKern, M. Nerrie, B. Lonnqvist, T. E. Adams, J. Lewis, G. 0. Lovrecz, P. A. Hoyne, K. M. Richards, G. J. Howlett, J. Rothacker, R. N. Jorissen, M. Lou, T. P. J. Garrett, A. W. Burgess, E. C. Nice, and C. W. Ward (2001). 5 Biochemistry 40:8930-8939. Pearson, R., S. Muharsini, G. Wijffels, and T. Vuocolo (2000). Jurnal Ilmu Ternak dan Veteriner 5(3):185-191. Tsumoto, K., D. Ejima, I. Kumagai and T. Arakawa (2003). Protein Expression and Purification 28:1-8

Claims (21)

1. An isolated polynucleotide molecule encoding an epidermal growth factor (EGF) or functional EGF fragment, said molecule comprising a nucleotide sequence showing at least 50% sequence 5 identity to that set forth in SEQ ID No: I or a portion thereof which encodes a functional EGF fragment, and wherein said EGF or functional EGF fragment comprises an amino acid sequence which includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. 0

2- A polynucleotide molecule according to claim 1 comprising a nucleotide sequence showing at least 85% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment.

3. A polynucleotide molecule according to claim 1 comprising a nucleotide sequence showing at 5 least 90% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment.

4. A polynucleotide molecule according to claim 1 comprising a nucleotide sequence showing at least 95% sequence identity to that set forth in SEQ ID No: 1 or a portion thereof which encodes a .0 functional EGF fragment.

5. A polynucleotide molecule according to claim 1 comprising a nucleotide sequence substantially corresponding to the sequence set forth in SEQ ID No: 1 or a portion thereof which encodes a functional EGF fragment. 25

6. An isolated polynucleotide molecule comprising: a nucleotide sequence substantially corresponding to any one of the sequences set forth in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6 but including one or more codon modification such that said nucleotide sequence encodes an epidermal growth factor (EGF) 30 comprising an amino acid sequence which includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide, or a nucleotide sequence substantially corresponding to the sequence of a portion of any one of the sequences set forth in SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, and SEQ ID No: 6 but 35 including one or more codon modification such that said nucleotide sequence encodes a functional EGF fragment comprising an amino acid sequence which includes six conserved cysteine residues 19 located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide.

7. An isolated EGF or functional EGF fragment comprising an amino acid sequence showing at 5 least 50% sequence identity to that set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment, and wherein said amino acid sequence or portion thereof includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. 0

8. An EGF according to claim 7 comprising an amino acid sequence showing at least 85% sequence identity to that set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment.

9. An EGF according to claim 7 comprising an amino acid sequence showing at least 90% 5 sequence identity to that set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment.

10. An EGF according to claim 7 comprising an amino acid sequence showing at least 95% sequence identity to that set forth in SEQ ID No: 2 or a portion thereof which represents the ,0 amino acid sequence of a functional EGF fragment.

11. An EGF according to claim 7 comprising an amino acid sequence substantially corresponding to the sequence set forth in SEQ ID No: 2 or a portion thereof which represents the amino acid sequence of a functional EGF fragment. 25

12. An isolated EGF peptide comprising: an amino acid sequence substantially corresponding to any one of the sequences set forth in SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10 but including one or more sequence modification such that said amino acid sequence includes six conserved cysteine residues located at 30 positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide, or an amino acid sequence substantially corresponding to the sequence of a portion of any one of the sequences set forth in SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10 but including one or more sequence modification such that said amino acid sequence represents the amino acid 35 sequence of a functional EGF fragment and includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide. 20

13. An isolated EGF peptide or functional EGF fragment comprising an amino acid sequence which is a hybrid sequence, said hybrid sequence comprising a portion of the sequence from SEQ ID No: 2 and a portion of one or more sequences selected from the group consisting of SEQ ID No: 7, 5 SEQ ID No: 8, SEQ ID No: 9, or SEQ ID No: 10 but including one or more codon modification such that the hybrid sequence includes six conserved cysteine residues located at positions corresponding to positions 6, 14, 20, 31, 33 and 42 of the amino acid sequence of human EGF peptide, and said hybrid EGF peptide retains one or more of the biological effects of EGF in an animal. 0

14. A method for producing an EGF or functional EGF fragment, said method comprising introducing a molecule according to any one of claims 1 to 6 into a suitable host cell and culturing said cell under conditions suitable for the expression of said molecule.

15. A recombinant vector comprising the polynucleotide molecule according to any one of claims 5 1 to 6.

16. A composition suitable for administration to an animal, wherein the composition comprises an EGF or functional EGF fragment according to any one of claims 7 to 11 and, optionally, a pharmaceutically or veterinary acceptable carrier. 0

17. A method of de-fleecing a wool fleece-bearing animal, said method comprising administering to an animal an EGF or functional EGF fragment according to any one of claims 7 to 11, or a composition according to claim 16, in an amount effective to allow for the removal of the wool fleece without the requirement for shearing. 25

18. A method according to claim 17, wherein the animal is selected from the group consisting of sheep, goat, llama and alpaca.

19. A method according to claim 18, wherein the animal is a Merino sheep. 30

20. A method of promoting ruminant embryo survival in vitro, comprising incubating said embryo in the presence of an embryo culture medium comprising an EGF or functional EGF fragment according to any one of claims 7 to 11. 35

21. Use of an EGF or functional EGF fragment according to any one of claims 7 to 11, said use selected from the group consisting of (i) control of mammary gland and maturation; 21 (ii) control of neonatal gut and lung maturation; (iii) hormone modulation; (iv) human models of wound repair which use ruminant animals; and (v) treatment of wounds. 5