AU741851B2 - Tropoelastin derivatives - Google Patents

Description

WO 99/03886 PCT/AU98/00564 1 TROPOELASTIN DERIVATIVES TECHNICAL FIELD The present invention relates to derivatives of human tropoelastin and variants thereof, to genetic constructs encoding the amino acid sequences of the derivatives and variants and to uses of the derivatives and variants. In particular, the derivatives of the present invention have elastin-like properties or macro-molecular binding properties.

BACKGROUND ART There are various forms of tropoelastin that typically appear to consist of two types of alternating domains: those rich in hydrophobic amino acids (responsible for the elastic properties) and those rich in lysine residues (responsible for cross-link formation).

Hydrophobic and cross-linking domains are encoded in separate exons (Indik et al 1987).

The 26 A region of human tropoelastin is unique amongst tropoelastin domains in that, due to the absence of lysine, this region does not participate in elastin cross-link formation. Furthermore, this region is a serine-rich domain and lacks hydrophobic stretches, indicating that it is unlikely to contribute to the elasticity of tropoelastin. There is otherwise limited information on the structure and functional relationships of the 26 A region (Bedell-Hogan et al., 1993).

The gene for tropoelastin is believed to be present as a single copy in the mammalian genome, and is expressed in the form of multiple transcripts, distinguished by alternative splicing of the pre-mRNA (Indik et al, 1990; Oliver et al, 1987). Modest expression of a natural human tropoelastin sequence has been achieved by Indik et al (1990) using cDNA, providing free polypeptide which unfortunately was unstable.

Expression of substantial amounts of human tropoelastin using synthetic polynucleotides is reported WO 99/03886 PCT/AU98/00564 2 in W094/14958. In particular, a construct, SHEL, providing substantial amounts of full length human tropoelastin is described.

DESCRIPTION OF THE INVENTION In the specification and claims, "derivatives of human tropoelastin" or "tropoelastin derivatives" means novel peptides, polypeptides or proteins which contain amino acid sequences derived from the native amino acid sequences of human tropoelastin molecules. The amino acid sequences of the derivatives of human tropoelastin may be derived from any of the amino acid sequences of the isoforms of human tropoelastin. Derivatives of human tropoelastin are distinguished from human tropoelastin molecules in that the amino acid sequences of derivatives are altered with respect to native tropoelastin sequences by substitution, addition or deletion of residues, or a combination of these alterations, in derivative amino acid sequences.

In a first aspect, the present invention provides derivatives of human tropoelastin which have elastin-like properties. Elastin-like properties are a combination of elastic properties, including the phenomenon of recoil following molecular distention under appropriate conditions, and the ability to be cross-linked to other elastin molecules and/or other elastin-like molecules.

In a second aspect, the present invention provides derivatives of human tropoelastin which have macromolecular binding properties including the ability to bind glycosaminoglycans.

In a third aspect, the present invention provides derivatives of human tropoelastin which have elastin-like properties and macro-molecular binding properties.

The present invention further provides amino acid sequence variants of the derivatives of the invention. In the specification and claims "variants" means amino acid sequences which retain the properties of the corresponding derivative of human tropoelastin, for example, elastin- WO 99/03886 PCT/AU98/00564 3 like properties or macro-molecular binding properties, or a combination of elastin-like properties and macromolecular binding properties, and have an amino acid sequence which is homologous with the amino acid sequence of the corresponding derivative. For the purposes of this description, "homology" between the amino acid sequence of a particular derivative of human tropoelastin and another amino acid sequence connotes a likeness short of identity, indicative of a derivation of one sequence from the other.

In particular, an amino acid sequence is homologous to a derivative of human tropoelastin if the alignment of that amino acid sequence with the sequence of the derivative of human tropoelastin reveals a similarity of about 65% over any 20 amino acid stretch or over any repetitive element of the molecules shorter than 20 amino acids in length.

Such a sequence comparison can be performed via known algorithims, such as that of Lipman and Pearson (1985).

Similarity is observed between amino acids where those amino acids have a side chain which confers a similar chemical property in the same chemical environment. For example, threonine and serine are similar amino acids; aspartic acid and glutamic acid are similar amino acids; valine, leucine and isoleucine are similar amino acids etc. Thus, an amino acid sequence may be considered homologous with the amino acid sequence of a human tropoelastin derivative because the alignment of those sequences reveals a similarity of 65%, although at each amino acid position in the aligned sequences, none of the residues are identical.

Inasmuch as the present invention provides derivatives of human tropoelastin and amino acid sequence variants of those derivatives, the invention thus extends to amino acid sequence variants incorporating amino acid sequences of non-human tropoelastins. Amino acid sequence variants which are non-human tropoelastin derivatives, or are based all, or in part, on non-human tropoelastin derivatives retain properties of the corresponding derivative of non-human tropoelastin, for example, WO 99/03886 PCT/AU98/00564 4 elastin-like properties or macro-molecular binding properties, or a combination of elastin-like properties and macro-molecular binding properties, and have an amino acid sequence which is homologous with the amino acid sequence of the corresponding human derivative. The variants of the invention also include variants of the non-human tropoelastin derivatives, or of derivatives based on the non-human tropoelastin derivatives.

"Homology" between the amino acid sequence of a particular derivative of non-human tropoelastin and another amino acid sequence connotes a likeness short of identity, indicative of a derivation of one sequence from the other.

In particular, an amino acid sequence is homologous to a derivative of non-human tropoelastin if the alignment of that amino acid sequence with the sequence of the derivative of non-human tropoelastin reveals a similarity of about 65% over any 20 amino acid stretch or over any repetitive element of the molecules shorter than 20 amino acids in length. The skilled addressee will understand that species that are substantially phylogenetically related to humans express tropoelastin molecules which consist of amino acid sequences with homology to human tropoelastin amino acid sequences. Indeed, amino acid sequences of non-human tropoelastins have been determined, including the amino acid sequences of chick tropoelastin, bovine tropoelastin and rat tropoelastin (Bressan et al.

1987, Raju et al. 1987, Pierce et al. 1992) and over multiple regions, these are homologous with the human tropoelastin amino acid sequences. The skilled addressee will recognise therefore, that derivatives of human tropoelastin and amino acid sequence variants of those derivatives will necessarily encompass corresponding tropoelastin amino acid sequences from these and other non-human species.

The present invention provides a tropoelastin derivative comprising the amino acid sequence of SHEL6modified (SEQ ID NO:5). The amino acid sequence of WO 99/03886 PCT/AU98/00564 5 SHEL8modified and the alignment of that amino acid sequence with the human tropoelastin sequence is shown in Figure The invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL~modified.

The invention also provides a polynucleotide encoding a tropoelastin derivative comprising the amino acid sequence of SHEL6modified. The nucleotide sequence encoding SHEL~modified is shown in Figure 3 (SEQ ID NO: Preferably the polynucleotide comprises the nucleotide sequence which corresponds to shown in Figure 3.

The invention also provides a polynucleotide encoding an amino acid sequence variant of the derivative SHEL8modified.

The present invention further provides a synthetic polynucleotide encoding a tropoelastin derivative comprising the amino acid sequence of SHEL826A (SEQ ID NO:3). A synthetic polynucleotide is a molecule which comprises a nucleotide sequence that contains silent mutations with respect to the corresponding native polynucleotide molecule. The silent mutations enhance the expression of the synthetic polynucleotide. The amino acid sequence of SHEL626A and the alignment of that amino acid sequence with the human tropoelastin sequence is shown in Figure 2. The SHEL626A derivative excludes the SHEL coding sequence corresponding to exon 26A.

Preferably the synthetic polynucleotide comprises the sequence shown in Figure 1 (SEQ ID NO:1) from nucleotide position 1 to 1676 contiguous with nucleotide position 1775 to 2210.

The invention also provides a polynucleotide encoding an amino acid sequence variant of the derivative SHEL826A.

The invention also provides an amino acid sequence WO 99/03886 PCT/AU98/00564 6 variant of the derivative comprising the amino acid sequence of SHEL626A.

The present inventor has, for the first time, shown that the region encoded by exon 26A (peptide 26A) of the tropoelastin gene binds glycosaminoglycans (GAGs) (Figure 6A and GAGs are macro-molecules particularly associated with the extracellular environment. These molecules play an important role in the architecture and mechanical properties of connective tissues and mediate interactions with and availability of other molecules.

Thus, the present invention provides a tropoelastin derivative comprising the amino acid sequence of peptide 26A. Peptide 26A has the amino acid sequence: GADEGVRRSLSPELREGDPSSSQHLPSTPSSPRV (SEQ ID NO: 12) or GADEGVRRSLSPELREGDPSSSQHLPSTPSSPRF (SEQ ID NO: 13).

The present invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of peptide 26A.

The invention also provides a polynucleotide encoding a tropoelastin derivative comprising the amino acid sequence of peptide 26A. Preferably the polynucleotide comprises the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) from nucleotide position 1687 to 1778.

Preferably the 3' terminal codon is GTT (which encodes V) or TTT (which encodes F).

The invention also provides a polynucleotide encoding an amino acid sequence variant of the derivative comprising the amino acid sequence of peptide 26A.

In appreciating the GAG binding property of peptide 26A, the present inventor envisages the generation of novel subsets of hybrid molecules, comprising biological polymers which are linked to peptide 26A, wherein the peptide 26A imparts GAG binding activity to the polymer.

In particular, the present inventor has recognised that the deletion or insertion of the peptide 26A amino acid sequence, or a variant of that amino acid sequence will alter GAG binding activity. Thus, the present invention relates to tropoelastin derivatives in which full length WO 99/03886 PCT/AU98/00564 7 or partial length tropoelastin molecules have been modified by the addition of one or more exon 26A regions to enhance interactions with GAGs. Moreover, the invention relates to site directed modification of the amino acid sequence of peptide 26A so as to generate variants of the peptide 26A amino acid sequence which have altered affinity or altered specificity for GAGs.

Tropoelastin derivatives or variants of the derivatives which contain altered GAG binding activity may be uncrosslinked or cross-linked.

In another aspect, the invention provides a hybrid molecule. In the specification and claims, "hybrid molecule" means a molecule comprising a biological polymer which is linked to a tropoelastin derivative comprising the amino acid sequence of peptide 26A or an amino acid sequence variant of a derivative comprising the amino acid sequence of peptide 26A. Preferably the biological polymer is a protein. More preferably the protein is selected from the group consisting of growth factors, cytokines and antibodies. Alternatively the biological polymer is selected from the group consisting of lipids, sugars or nucleic acids.

In one embodiment, and where the biological polymer is a protein, the hybrid molecule is produced by recombinant DNA techniques, including for example the construction of a nucleotide sequence which encodes the biological polymer and the tropoelastin derivative comprising the amino acid sequence of peptide 26A, or the amino acid sequence variant of a derivative comprising the amino acid sequence of peptide 26 A, in a single open reading frame. Alternatively, the hybrid molecule may be produced synthetically by solid phase peptide synthesis, including, for example the methods of synthesis disclosed in Merrifield (1963) or Knorr et al. (1989). Examples of peptide synthesis also include the synthesis methods used by peptide synthesisers of Perkin Elmer/Applied Biosystems, CA, US.

In another aspect, the invention provides a WO 99/03886 PCT/AU98/00564 8 polynucleotide sequence encoding a hybrid molecule of the invention.

In another aspect, the invention provides a hybrid molecule which comprises a synthetic polymer which is linked in a tropoelastin derivative comprising the amino acid sequence of peptide 26A, or an amino acid sequence variant of the derivative comprising the amino acid sequence of peptide 26A.

The invention further provides a method of imparting or enhancing GAG binding activity to a biological polymer comprising the step of linking a tropoelastin derivative comprising the amino acid sequence of peptide 26A, or an amino acid sequence variant of peptide 26A with the biological polymer. Preferably the biological polymer is a protein.

The invention further provides a method of deleting or reducing GAG binding activity from a biological polymer comprising the step of deleting a tropoelastin derivative comprising the amino acid sequence of peptide 26A, or an amino acid sequence variant of peptide 26A from the biological polymer. Preferably the biological polymer is a protein.

The present invention also provides a tropoelastin derivative comprising the amino acid sequence of SHELgamma. SHELgamma has the amino acid sequence:

SAMGALVGLGVPGLGVGAGVPGFGAGADEGVRRSLSPELREGDPSSSQHLPSTPSSPR

VPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFG

LVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVA

ARPGFGLSPIFPGGACLGKACGRKRK (SEQ ID NO: 9).

The invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of SHELgamma.

The invention also provides a polynucleotide encoding a tropoelastin derivative, the derivative comprising the amino acid sequence of SHELgamma. The nucleotide sequence of the polynucleotide SHELgamma (SEQ ID NO: 8) is shown in Figure 8. In this nucleotide sequence, the first 9 codons from nucleotide position 948 to 974 are derived WO 99/03886 PCT/AU98/00564 9 -9from the glutathione S-transferase (GST) fusion nucleotide sequence. Preferably the polynucleotide comprises the nucleotide sequence shown in Figure 8. More preferably the polynucleotide comprises the nucleotide sequence shown in Figure 8 from nucleotide sequence position 975 to 1547.

The invention also provides a polynucleotide encoding an amino acid sequence variant of the derivative comprising the amino acid sequence of SHELgamma.

The present invention also provides a polynucleotide encoding a tropoelastin derivative, the derivative comprising the amino acid sequence of SHELgamma excluding exon 26A. The nucleotide sequence of the polynucleotide SHELgamma excluding exon 26A (SEQ ID NO: 6) is shown in Figure 7. In this nucleotide sequence, the first 5 codons from nucleotide position 948 to 962 are derived from the GST nucleotide sequence. SHELgamma excluding exon 26A has the following amino acid sequence:

VPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFG

LVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVA

ARPGFGLSPIFPGGACLGKACGRKRK (SEQ ID NO: 7).

Preferably the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:6. More preferably the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 6 from nucleotide sequence position 15 to 441.

The invention also provides a polynucleotide encoding an amino acid sequence variant of the derivative comprising the amino acid sequence of SHELgamma excluding exon 26A.

The invention also provides a tropoelastin derivative comprising the amino acid sequence of SHELgamma excluding exon 26A.

The invention also provides an amino acid sequence variant of the derivative comprising SHELgamma excluding exon 26A.

The derivatives of the invention based on SHELgamma can also be produced by in vitro biochemical cleavage of tropoelastin products such as SHEL, so as to release a carboxy- terminal fragment. The carboxy-terminal fragment WO 99/03886 PCT/AU98/00564 10 may be purified by reverse phase HPLC.

The present invention also provides a tropoelastin derivative comprising the amino acid sequence of SHEL31- 36. SHEL31-36 has the following amino acid sequence:

GIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACG-

RKRK (SEQ ID NO: SHEL31-36 retains a crosslinking domain. As a consequence of its elastin-like properties, it is envisaged that this and related tropoelastin derivatives can be used to interfere with tropoelastin deposition and formation of unaltered elastic fibre.

The invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL31-36.

The invention also provides a polynucleotide encoding a tropoelastin derivative, the derivative comprising the amino acid sequence of SHEL31-36. Preferably the polynucleotide comprises the nucleotide sequence shown in Figure 1 (SEQ ID NO:1) from nucleotide position 2022 to 2210.

The invention also provides a polynucleotide encoding an amino acid variant of the derivative comprising the amino acid sequence of SHEL31-36.

The present invention also provides a tropoelastin derivative, comprising the amino acid sequence of SHEL32- 36. SHEL32-36 has the following amino acid sequence: GAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK (SEQ ID NO: 11).

The invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL32-36.

The invention also provides a polynucleotide encoding a tropoelastin derivative, the derivative comprising the amino acid sequence of SHEL32-36. Preferably the polynucleotide comprises the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) from nucleotide position 2061 to 2210.

The present invention also provides a polynucleotide WO 99/03886 PCT/AU98/00564 11 encoding an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL32-36.

As a consequence of its elastin-like properties, it is envisaged that SHEL32-36 and related tropoelastin derivatives can be used to interfere with tropoelastin deposition and formation of an unaltered elastic fibre.

The present invention also provides a tropoelastin derivative, comprising the amino acid sequence of SHEL26- 36. SHEL26-36 has the following amino acid sequence:

AAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAGADEGVRRSLSPELREGD

PSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVGAGPAAA

AAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGV

LGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK (SEQ ID NO: 14) The invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL26-36.

The invention also provides a polynucleotide encoding a tropoelastin derivative, the derivative comprising the amino acid sequence of SHEL26-36. Preferably the polynucleotide comprises the nucleotide sequence shown in Figure 1 from nucleotide position 1554-2210.

The present invention also provides a tropoelastin derivative, comprising the amino acid sequence of SHEL26- 36 excluding exon 26A. SHEL26-36 excluding exon 26A has the following amino acid sequence: AAAGLGAG I PGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAVPGALAAAKAAKYGAAVP GVLGGLGALGGVG I PGGWGAGPAAAAAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPG

VGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKA

CGRKRK (SEQ ID NO: The invention also provides an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL26-36 excluding exon 26A.

The invention also provides a polynucleotide encoding a tropoelastin derivative, the derivative comprising the amino acid sequence of SHEL26-36 excluding exon 26A.

Preferably the polynucleotide comprises the nucleotide sequence shown in Figure 1 from nucleotide position 1554 WO 99/03886 PCT/AU98/00564 12 to 1676 contiguous with 1776 to 2210.

The present invention also provides a polynucleotide encoding an amino acid sequence variant of the derivative comprising the amino acid sequence of SHEL26-36.

In another aspect the present invention provides a formulation comprising a tropoelastin derivative, a variant of the derivative or a hybrid molecule of the invention, together with a carrier or diluent.

Formulations of the derivatives, variants or hybrid molecules of the invention can be prepared in accordance with standard techniques appropriate to the field in which they are to be used.

The polynucleotides and synthetic polynucleotides of the invention can be provided in association with other polynucleotide sequences including 5' and 3' untranslated sequences, and 5' upstream and 3' downstream transcriptional regulatory sequences. The polynucleotides and synthetic polynucleotides may be provided as a recombinant DNA molecule including plasmid DNA.

The polynucleotides and synthetic polynucleotides of the invention can be prepared using the techniques of chemical synthesis or recombinant DNA technology, or by a combination of both techniques.

In a further aspect the invention provides a vector comprising a polynucleotide or synthetic polynucleotide encoding a tropoelastin derivative, a variant of the derivative or a hybrid molecule of the invention.

Vectors useful in this invention include plasmids, phages and phagemids. The polynucleotides and synthetic polynucleotides of the present invention can also be used in integrative expression systems or lytic or comparable expression systems.

Suitable vectors will generally contain origins of replication and control sequences which are derived from species compatible with the intended expression host.

Typically these vectors include a promoter located upstream from the polynucleotide, together with a ribosome binding site if intended for prokaryotic expression, and a WO 99/03886 PCT/AU98/00564 13 phenotypic selection gene such as one conferring antibiotic resistance or supplying an auxotrophic requirement. For production vectors, vectors which provide for enhanced stability through partitioning may be chosen. Where integrative vectors are used it is not necessary for the vector to have an origin of replication.

Lytic and other comparable expression systems do not need to have those functions required for maintenance of vectors in hosts.

For E. coli typical vectors include pBR322, pBluescript II SK*, pGEX-2T, pTrc99A, pET series vectors, particularly pET3d, (Studier et al., 1990) and derivatives of these vectors. Derivatives include those plasmids with a modified protease recognition sequence to facilitate purification of a protein domain.

In another aspect the invention provides a cell capable of expressing a polynucleotide or a synthetic polynucleotide which encodes a derivative or variant of the invention, or a polynucleotide which encodes a hybrid molecule of the invention.

A preferred expression system is an E. coli expression system. However, the invention includes within its scope the use of other hosts capable of expressing protein from the polynucleotides designed for use in E.

coli. The invention also includes the use of polynucleotides and synthetic polynucleotides suitable for use in other expression systems such as other microbial expression systems. These other expression systems include yeast, and bacterial expression systems, insect cell expression systems, and expression systems involving other eukaryotic cell lines or whole organisms.

Examples of E. coli hosts include E. coli B strain derivatives (Studier et al, 1990), and K-strain derivatives such as NM522 (Gough and Murray, 1983) and XLl-Blue (Bullock et al, 1987).

In a further aspect the present invention provides an expression product. In the specification and claims, "expression product" means a derivative or variant of the WO 99/03886 PCT/AU98/00564 14 invention expressed by a cell containing a polynucleotide or a synthetic polynucleotide encoding a derivative or variant of the invention.

The expression products of the invention may be fused expression products which include all or part of a protein encoded by the vector in peptide linkage with the derivative or variant. They may also include, for example, an N-terminal methionine or other additional residues which do not permanently impair the elastin-like, or macro-molecular binding properties of the product.

Typically the fusion is to the N-terminus of the expression product. An example of a suitable protein is to the C-terminus of glutathione S-transferase. The fused protein sequence may be chosen in order to cause the expression product to be secreted or expressed as a cell surface protein to simplify purification or expressed as a cytoplasmic protein.

The expressed fusion products may subsequently be treated to remove the fused protein sequences to provide free tropoelastin derivative or variant. Treatment is typically through protease treatment or, in the case of secretion, removal is effected by endogenous host secretion machinery. An example of this is secretion by yeasts.

Non-fused systems include the introduction of or use of a pre-existing methionine codon. An example of this is the use of pET3a or pET3d in E. coli.

In another aspect the invention provides a polynucleotide encoding an expression product of the invention.

In another aspect the present invention provides a formulation comprising an expression product of the invention together with a carrier or diluent. The formulation of the expression product can be prepared in accordance with standard techniques appropriate to the field in which they are to be used.

According to a further aspect of the present invention there is provided a method for producing a WO 99/03886 PCT/AU98/00564 15 tropoelastin derivative or a variant of the derivative comprising providing a vector containing a polynucleotide or a synthetic polynucleotide encoding the derivative or variant; introducing the vector into a suitable host cell; maintaining the cell in conditions suitable for expression of the polynucleotide or synthetic polynucleotide and isolating the derivative or variant of the invention. The method can be applied to the production of the expression products and hybrid molecules (in which the hybrid molecules comprise the peptide 26A or a variant thereof and a further amino acid sequence) of the invention, by providing a vector containing a polynucleotide encoding an expression product or a hybrid molecule; introducing the vector into a suitable host cell; maintaining the cell in conditions suitable for expression of the polynucleotide and isolating the expression product or hybrid molecule.

In one embodiment, the polynucleotide or synthetic polynucleotide encoding the derivative, variant, expression product or hybrid molecule of the invention is expressed in a host cell which is maintained in culture in vitro.

Alternatively, the polynucleotide or synthetic polynucleotide encoding the derivative, variant, expression product or hybrid molecule of the invention is expressed in a host cell which is maintained in vivo.

Thus, in another embodiment, the polynucleotide or synthetic polynucleotide encoding the derivative, variant, expression product or hybrid molecule of the invention is expressed in a transgenic animal. Methods for the generation of transgenic animals are known in the art.

Exemplary methods are described in Slack et al. 1991 and Janne et al. 1992.

The tropoelastin derivatives, variants of the derivatives, and hybrid molecules (in which the hybrid molecules comprise the peptide 26A or a variant thereof and a further amino acid sequence) of the invention may be produced by solid phase peptide synthesis, including, for example, the methods of synthesis disclosed in Merrifield WO 99/03886 PCT/AU98/00564 16 (1963) or Knorr et al (1989). Examples of peptide synthesis also include the synthesis methods used by peptide synthesisers of Perkin Elmer/Applied Biosystems, CA, US. As an alternative to cell synthesis from a polynucleotide or synthetic polynucleotide, the expression products of the invention may be produced by solid phase peptide synthesis.

In a further aspect the present invention provides an implant formed from at least one tropoelastin derivative and/or variant of the derivative of the invention. The implant may alternatively contain at least one expression product and/or at least one hybrid molecule of the invention.

The implants are formed into the required shape by cross-linking the tropoelastin derivative, variant of the derivative, expression product, or hybrid molecule of the invention, in a mould which conforms to the desired shape of the implant. Where the implant is required to be used in sheet form the tropoelastin derivative, variant of the derivative, expression product, or hybrid molecule of the invention can be cross-linked on a flat surface. Relevant methodologies are described in, for example, US Patent No.

4 474 851 and US Patent No. 5 250 516. The elastomeric materials may be exclusively prepared from one or more tropoelastin derivatives, variants of the derivative, expression products, or hybrid molecules of the invention or may be composites prepared from one or more of these constituents together with other materials.

The tropoelastin derivatives or variants of the derivatives can be cross-linked to form elastin or elastin-like material or can be cross-linked in conjunction with other biological or synthetic molecules to form a composite material.

Thus in another aspect the invention provides a cross-linked complex which comprises at least one tropoelastin derivative of the invention and/or at least one variant of a derivative of the invention. The crosslinked complexes may additionally contain at least one WO 99/03886 PCT/AU98/00564 17 expression product and/or at least one hybrid molecule of the invention, which may be cross-linked to the at least one tropoelastin derivative and/or variant of the derivative of the invention.

The cross-linking of the tropoelastin derivatives, variants of the derivatives, hybrid molecules and expression products of the invention can be achieved by chemical oxidation of lysine side chains using processes such as ruthenium tetroxide mediated oxidation and quinone mediated oxidation, or by using homobifunctional chemical cross-linking agents such as dithiobis (succinimidylpropionate), dimethyl adipimidate or dimethyl pimelimidate. Glutaraldehyde cross-linking is an important addition to this repetoire. Another alternative is the cross-linking of lysine and glutamic side chains.

The tropoelastin derivatives, variants of the derivatives, hybrid molecules and expression products of the invention may also be enzymatically cross-linked by methods including lysyl oxidase mediated oxidation or may be cross-linked using gamma irradiation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Nucleotide (SEQ ID NO: 1) and predicted amino acid (SEQ ID NO:2) sequences of synthetic human tropoelastin SHEL. The upper (numbered) nucleotide sequence represents the coding strand.

Figure 2: Alignment of SHEL (SEQ ID NO:2)(upper line) and SHEL626A (SEQ ID NO: 3) amino acid sequences.

Figure 3: Nucleotide (SEQ ID NO: 4) and predicted amino acid (SEQ ID NO: 5) sequences of SHELmodified.

Figure 4: Alignment of SHELimodified (SEQ ID NO: 4) (upper line) and SHEL (SEQ ID NO:1) nucleotide sequences.

Figure 5: Alignment of SHEL8modified (SEQ ID NO: line) and SHEL (SEQ ID NO: 1) amino acid sequences.

Figure 6A: HPLC elution profile of GST-exon 26A fusion protein tropoelastin derivative loaded in from WO 99/03886 PCT/AU98/00564 18 heparin sepharose. 6B: Binding of peptide 26A (SEQ ID NO: 12 and SEQ ID NO: 13) to glycosaminoglycans.

Figure 7: Nucleotide (SEQ ID NO: 6) and predicted amino acid (SEQ ID NO: 7) sequences of SHELgamma excluding exon 26A.

Figure 8: Nucleotide (SEQ ID NO: 8) and predicted amino acid (SEQ ID NO: 9) sequences of SHELgamma.

BEST METHOD OF PERFORMING THE INVENTION The recombinant and synthetic procedures used for the synthesis of the derivatives, variants, expression products and hybrid molecules of the invention are described in standard texts such as Sambrook et al (1989).

Tropoelastin nucleotide sequences may be modified so as to provide derivatives, variants, expression products or hybrid molecules, by conventional site-directed or random mutagenesis. The sequences may also be modified by oligonucleotide-directed mutagenesis, which comprises the following steps: 1. synthesis of an oligonucleotide with a sequence that contains the desired nucleotide substitution (mutation); 2. hybridising the oligonucleotide to a template comprising a structural sequence encoding tropoelastin; and 3. using a DNA polymerase to extend the oligonucleotide as a primer.

Another approach which is particularly suited to situations where a synthetic polynucleotide encoding the tropoelastin derivative is prepared from oligonucleotide blocks bounded by restriction sites, is cassette mutagenesis where entire restriction fragments are replaced.

Purification of the derivatives, variants, expression products or hybrid molecules of the invention is performed using standard techniques including HPLC. The actual sequence of steps in the purification of a particular derivative, variant, expression product or hybrid molecule WO 99/03886 PCT/AU98/00564 19 is limited by the environment from which the molecule is to be purified. By way of example, reference is made to the purification scheme disclosed in W094/14958.

Formulations in accordance with the invention are formulated in accordance with standard techniques.

The amount of derivative, variant, expression product or hybrid molecule that may be combined with a carrier or diluent to produce a single dosage will vary depending on the situation in which the formulation is to be used and the particular mode of administration.

It will be understood also that specific doses for any particular host may be influenced by factors such as the age, sex, weight and general health of the host as well as the particular characteristics of the derivative, variant, expression product or hybrid molecule of the invention being used, and how it is administered.

Injectable preparations, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles or solvents that may be employed are water, Ringer's solution, alcohols and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid and organic solvents find use in the preparation of injectables.

Routes of administration, dosages to be administered as well as frequency of administration are all factors which can be optimised using ordinary skill in the art.

In addition, the derivatives, variants, expression products and hybrid molecules of the invention may be prepared as topical preparations for instance as antiwrinkle and hand lotions using standard techniques for the WO 99/03886 PCT/AU98/00564 20 preparation of such formulations. They may be prepared in aerosol form for, for instance, administration to a patient's lungs, or in the form of surgical implants, foods or industrial products by standard techniques.

SHEL

The preparation of SHEL is described in WO94/14958.

It is directly expressed as a full length human protein with a calculated molecular weight of 64kDa. The full nucleotide sequence and corresponding amino acid sequence of SHEL is shown in Figure 1. The preparation of pSHELF is described in W094/14958.

pSHELF differs from the natural coding sequence(s) in a number of ways. As described in W094/14958, the untranslated regions present in the tropoelastin cDNA sequence were disregarded in designing the synthetic gene, and the nucleotides encoding the signal peptide were removed. Restriction endonuclease recognition sites were incorporated at regular intervals into the gene by typically altering only the third base of the relevant codons, thereby maintaining the primary sequence of the gene product. The facility for silent alteration of the coding sequence was also exploited to change the codon bias of the tropoelastin gene to that commonly found in highly expressed E.coli genes. [Genetics Computer Group (GCG) package version 7-UNIX using Codon Frequency and Gen Run Data: ecohigh-cod]. Two additional stop codons were added to the 3'-end, and an ATG start codon comprising a novel NcoI site was appended to the 5'-end. Bam HI cloning sites were engineered at both ends of the synthetic sequence.

Since the gene contains no internal methionine residues, treatment of the newly-synthesized gene product (expressed directly or as a fusion with another gene) with cyanogen bromide would liberate a protein with the same or similar sequence as one form of natural tropoelastin comprising 731 amino acids. Other forms of processing are envisaged, which may generate tropoelastin species of the same or different lengths.

WO 99/03886 PCT/AU98/00564 21 Two stop codons were added in order to allow the possible use of the construct in suppressor hosts, and also to avoid any potential depletion of termination (release) factors for translation.

As described in the following examples, the derivatives, pSHELF626A, pSHELF8 modified, pSHELgamma, pSHEL31-36, pSHEL32-36 and pSHELgamma626A were derived from the pSHELF nucleotide sequence. These particular derivatives, and indeed the derivaties, variants, expression products and hybrid molecules of the invention can equally be derived from a native human or non -human tropoelastin nucleotide sequence.

Example 1: Construction of pSHELF626A and pSHELF8 modified Mutagenesis was used with pSHELF to remove DNA corresponding to exon 26A. The sequence of the mutagenic primer was: GTT TCG GTG CTG TTC CGG GCG CGC TGG 3' This flanked either side of exon 26A by resulting in its precise deletion. A second selection primer, which mutates a unique restriction site to another restriction site is normally used in the protocol but was not in this case since deletion of exon 26A also resulted in the deletion of a unique restriction site, PmllI. The enzyme PmlI was used to treat the mutation reaction to linearise any unmutated parental plasmid and consequently to enrich for mutant plasmid. The reaction mixture was used to transform competent BMH17-18 mutS E. coli, defective in mismatch repair, by electroporation and the entire transformed culture was grown overnight in LB+ampicillin. Mixed plasmid DNA, containing both mutated and parental plasmids, was isolated from the culture and the plasmid DNA was digested with PmlI to linearise the parental plasmid. The plasmid DNA, now enriched for mutated plasmid, was used to transform E. coli HMS174 by electroporation and transformants selected on LB plates WO 99/03886 PCT/AU98/00564 22 containing 75pgml 1 ampicillin.

Colonies were grown overnight and plasmid minipreparations performed. Constructs were screened using PmlI and those which were insensitive to digestion were further screened by KpnI/PstI double digestion. Candidate clones were sequenced to verify the sequence, named pSHELF6modified.

Sequencing confirmed the region immediately surrounding the deletion was correct. PstI and BssHII restriction sites surrounding the correct region of pSHELF6modified were used to remove the desired segment and re-insert it into the corresponding site of pSHELF.

pSHELF and 7.5jg pSHELF6modified were digested with BssHII, precipitated and digested with PstI. The appropriate three fragments were gel-purified and ligated. DNA was transformed into E. coli XLl-Blue and transformants selected on plates containing 75.gml ampicillin.

Plasmids were isolated by mini-preparations and screened using BglI digestion. A candidate clone was further analysed by restriction enzyme digestion and sequenced, and named pSHELF626A.

Example 2: Synthesis of Exon 26A The region of SHEL corresponding to exon 26A was amplified by PCR, with primers modified to introduce an in-frame BamHl site upstream and a stop codon downstream at the 3' end. Two forms were generated: one terminating in valine (26AV) and the other terminating in phenyalanine (26AF). These forms are as follows: GADEGVRRSLSPELREGDPSSSQHLPSTPSSPRV with properties: Molecular weight 3588.80 Residues 34 Average Residue Weight 105.553 Charge -1 Isoelectric point 5.71 WO 99/03886 PCT/AU98/00564 23 and

GADEGVRRSLSPELREGDPSSSQHLPSTPSSPRF

A 26A coding region was expressed as a glutathione Stransferase (GST) fusion protein.

Example 3: Glycosaminoglycan binding activity of Exon 26A Ultrafiltration assay methodology was developed to examine and quantify interactions occurring in vitro between the 26A region and purified extracellular matrix glcosaminoglycans. GST26A fusion protein and tropoelastin were compared with GST and tropoelastin lacking exon 26A at physiologicaly relevant conditions of pH and ionic strength.

Experimental evidence supports the notion that peptide 26A (26AF and 26AV) binds GAGs. Immobilised heparin column binding shows that GST26A binds more tightly than does GST, and requires a higher sodium chloride concentration for elution (Figure 6B).

Furthermore, GST26A fusion protein binds radioactive heparin with greater efficiencies than GST, and these can be compared with GAGs including chondroitin sulphates and keratin sulphates. An implication of this is that GAGs binding to tropoelastin can be adjusted based upon the content of 26A. Cross-linked tropoelastin would be expected to show differential binding to GAGs based on the relative amounts of SHEL vs. SHEL626A.

In summary, these studies reveal that the 26A region is a functional glycosaminoglycan binding domain, which functions in intact tropoelastin. It is also active when isolated as a fusion entity yet displays no detectable structure in the absence of bound GAG. Furthermore, panel competition studies indicate a preference for those GAGs found in close association with the elastic fibre in the extracellular matrix.

24- Example 4: Construction of pSHELgamma, pSHEL31-36, PSHEL32-36 and pSHELgamma626A pSHELgamma is derived from the pSHELgamma construct disclosed in W094/74958. PSHEL31-36, pSHEL32-36 and pSHELgamma626A were derived from pSHELgamma. pSHELgamma was modified by introducing an oligonucleotide linker at the KpnI site. This encoded a faster Xa cleavage site which could be utilised in subsequent constructs. PCR and site directed mutagenesis was then used to generate further, shorter forms which provided fusions with GST. Constructs were DNA sequenced for verification. Induced protein was isolated as GST-fusion proteins, which were subsequently bound to glutathione agarose. Protease cleavage was optional where fusion proteins were desired; otherwise the 15 cleaved proteins and peptides were further purified by reverse phase HPLC.

INDUSTRIAL APPLICATION The derivatives and expression products of the invention are of use in inter alia the medical, pharmaceutical, veterinary and cosmetic fields.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" or grammatical variations thereof, is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.

WO 99/03886 PCT/AU98/00564 25

REFERENCES

1. Indik Z, Yeh H, Ornstein-Goldstein N, Sheppard P, Anderson N, Rosenbloom JC, Peltonen L and Rosenbloom J (1987) PNAS (USA) 84 5680-5684 2. Indik Z, Abrams Kucich U, Gibson Mecham R.P. and Rosenbloom J (1990) Arch. Biochem Biophys 280 80-86 3. Oliver L, Luvalle PA, Davidson Rosenbloom J, Mathew Betser A.J. and Boyd C.D. (1987) Collagen Rel Res 7 77-89 4. Sambrook Fritsch and Maniatis T. (1989) Molecular cloning: a laboratory manual, second edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 5. Bressan Argos P. and Stanley K.K. (1987) Biochemistry 26 1497-11503 6. Raju K. aand Anwar R.A. (1987) J. Biol Chem 262 5755- 5762 7. Pierce Alatawi A, Deak S.B. Boyd C.D. (1992) Genomics 12 651-658 8. Lipman and Pearson (1985) Science 227,1435.

9. Bedell-Hogan, Trackman, Abrams, W., Rosenbloom, J. and Kagan H. (1993) J. Biol. Chem.

268, 10345-10350 10. Studier, F. Rosenberg, A. Dunn, J. J. and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 11. Gough, and Murray, N. (1983) J. Mol. Biol. 166, WO 99/03886 PCT/AU98/00564 26 1-19 12. Bullock, W. Fernandez, J. M. and Short, J. M.

(1987) BioTechniques 5, 376-379 13. Slack, J. Liska, D. J. and Bornstein P. (1991) Mol. Cell Biol. 11: 2066-2074 14. Janne, Hyttinen, J. Peura, Tolvanen, M., Aihonen, L. And Halmekyto M. (1992) Ann. Med. 24: 273-280.

Merrifield, (1963) J. Am. Chem. Soc. 2149-2154.

16. Knorr Trzeciak, Bannarth Gillessen, D. (1989) Tetrahedron Letters 30: 1927-1930 WO 99/03886 PCT/AU98/00564 27 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: WEISS, ANTHONY S UNIVERSITY, SYDNEY (ii) TITLE OF INVENTION: TROPOELASTIN DERIVATIVES (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: GRIFFITH HACK STREET: 168 WALKER STREET CITY: NORTH SYDNEY STATE: NEW SOUTH WALES COUNTRY: AUSTRALIA ZIP: 2060 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: AU FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: AU P08117 FILING DATE: 18-JUL-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: GUMLEY, THOMAS P REFERENCE/DOCKET NUMBER: 04828ZK (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 61 2 9957 5944 TELEFAX: 61 2 9957 6288 TELEX: 26547 INFORMATION FOR SEQ ID NO:1: WO 99/03886 WO 9903886PCT/AU98/00564 28 SEQUENCE CHARACTERISTICS: LENGTH: 2210 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoinic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GATCCATGGG TGGCGTTCCG GGTGCTATCC CGGGTGGCGT

CAGGCGCGGG

AACCGGTTCC

CGGTTACCTT

AAGCGGCAAA

CTGCTGGCGC

TTGGTCTGCC

GTGTACTGCC

GCGCGTTCGC

TGGGTTACCC

GTAXACTGCC

ACCCAACCGG

CAAAATTCGG

GTGTTCCAGG

TCTGGGTGCA

AGGCGGTCTG

CCCGGGTGCT

GGCAGGTGCG

AGTTGTTCCG

GGGCGTATAC

GGGCGTTCCG

GGGTATCCCG

GATCAAAGCG

GTACGGCTAC

TACTGGTGTT

CGCGGGTGCA

TGCGATCCCG

CTGGGCGGTG

GCAGGTGCTG,

CTGGTTCCGG,

GGTCTGGGCG

CAGCCGGGTG

CCGGGTGGTG

ACCGGTGCAG

GGTGTTGGCC

CCGAAGCTTC

GGTCCGGGTG

GGTCCGCAGG

GCGGGTGTTC

GGCATCGGTG

GTGCGCTGGG,

GTCTGGGTGC

GTGGCGTTGC

GGGTACCAGG

CAGGTGTAAA

TTCTGCCGGG

GTGTTAAACC

CGTTCGGTGG

CAGGTGGCTA

GCGTAGCAGG

CTGCTGCGGC

TGCCGGGCGT

GTATCGCAGG

TCCGGGTGGT

CCCGGGTGGT

AGGTCTGGGC

AGACGCAGCT

TGTTGGCGGT

ACCGGGCAAA

CGCGCGTTTC

GAAGGCACCA

TCCGCAGCCA

CGGTCTGCCG

TGCTGCGGGT

AGCTGCGGCG

AGGTGGTGCT

CGTAGGTACT

GTATTCTACC

AAACCGCTGA

GCGTTCCCGG

GCTGCGTACA

CTGGGTGTAT

GTTCCAGGTG

CCAGGTGTTG

GGTGTAGGCG

GGCGTTCCGC

TACACCACCG

AAAGCAGGCT

AAGGCAGCAG

GGCGTTC!CGG

CCGGCGGCCG

120 180 240 300 360 420 480 540 600 660 720 780 840 WO 99/03886 WO 9903886PCT/AU98/00564 29

CTGCGGCTGC

CGGGTGGTCC

TAGGTGTTCC

TTCCAGGTGT

GAGCTCGTCC

TCCCAGGTTT

GTGGCGTACC

CTGCGGCAGC

CAGCGGCTAA

GCGTAGCACC

CACCAGGTGT

TTGCGGCTGC

CTGGTCTGGG

TAGGTGCAGG

CGGACGAAGG

CCCAGCACCT

AAGCGGCGAA

GTGTTGGTAT

AGGCAGCGGC

GTGTTGGCGG

CAGCTAAAGC

AGTTCCCACT

GCGGTGCATG

GGCAGCTGCG

AGGCTTCGGT

AGGTGCGGGC

TGTATCCCCG

GGGCGTTGGT

CGGCGTTGGT

GGGTGTTGGT

TAAAGCAGCG

AGCAGCGCAG

GGGTGTTGGT

AGGTGTTGCG

TGCGAAATCT

TGCGGGCATC

GGTACCGGGC

TGTACGTCGT

GCCGTCTACC

ATACGGTGCA

CCCGGGCGGT

GAAAGCAGCT

TCTGGGTGTA

GGCTAAATAC

GGGCGGTGTA

CCTGGGTAAA

GCGAAAGCAG

CCGGGTGTTG

ATCCCGGTTG

GAAGCGGCAG

GTTGGTGGCA

GTTGGTGGCA

GGCGTTCCAG

AAGTACGGCG

TTCGGACTAG

GTTGCTCCGG

CCGGGCGTTG

GCTGCGAAGG

CCAGGTCTGG

CTGGGTGTTG

TCCCTGTCTC

CCGTCCTCTC

GCGGTTCCGG

GTTGTAGGTG

CAGTTCGGTC

CCGGGCGTTG

GGTGCAGCAG

GCGGCACGTC

GCTTGCGGCC

CTAAATACGG

TAGGCGTTCC

TACCGGGTGC

CTAAGGCTGC

TCCCGACCTA

TCCCGGGTGT

GTGTAGGTAT

TTGGTACTCC

TTCCGGGCGT

GCGTAGGTCT

GTGTAGCACC

TTGCTGCGAA

GTGTAGGTGT

GTGCAGGCGT

CAGAACTGCG

CACGTGTTCC

GTGTACTGGG

CAGGCCCAGC

TGGTTGGTGC

GTGGTCTGGG

GTCTGGGTGG

CGGGTTTCGG

GTAAACGTALA

TGCGGCAGCA

GGGTGCTGGT

AGGTATCCCG

TGCGAAAGCT

CGGTGTAGGT

AGCTGGTGTT

CTCCCCGGAA

GGCGGCAGCA

AGGTGTTGCG

GGCACCGGGT

GGGTATCGGT

AGCGCAGCTG

TGGTGTTCCG

TCCGGGTTTC

TGAAGGTGAC

GGGCGCGCTG

CGGTCTGGGT

TGCAGCTGCT

AGCAGGTCTG

TGGCATCCCG

CGTTCTGGGT

TCTGTCCCCG

ATAATGATAG

GGCCTGGTTC

GTTCCGGGCG

GGCGCTGCGG

GCGAAATACG

GCAGGCGGTT

CCGTCTGTTG

GCGCAGGCAG

GCTGCTAAAG

CCAGGTGTTG

GTTGGCGTTG

CCGGGTGGCG

CGTGCAGCAG

GGCCTGGGTG

GGTGCTGGCG

CCGTCCTCTT

GCTGCTGCGA

GCTCTGGGCG

GCTGCGGCAA

GGCGGTCTGG

CCGGCGGCGG

GGTGCTGGTC

ATCTTCCCAG

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2210 WO 99/03886 PCT/AU98/00564 30 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 733 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ser Met Gly Gly Val Pro Gly Ala Ile Pro Gly Gly Val Pro 1 Val 10 Ala Gly Gly Phe Tyr Pro Gly Ala Gly Leu Gly 25 Pro Leu Gly Gly Gly Pro Gly Ala Gly Leu Gly Lys Pro Leu Lys Val Pro Gly Gly Ala Leu Leu Ala Gly Thr Phe Pro Gly Ala Gly Gly Ala Phe Pro Ala Gly Ala Leu Val Pro Val Ala Asp Ala Ala Ala Ala Ala Tyr Ala Lys Ala Ala Gly Leu Gly Gly Pro Val Pro Gly Val Gly Gly Leu Gly Val Lys Pro Gly 115 Gly Val Leu Gly Ala Val Val 105 Gly Gin Pro Gly Val Pro Gly Leu Pro Gly Ala Gly Val 110 Tyr Pro Gly Leu Pro Gly Pro Gly Ala 130 Val Pro 145 Arg 135 Val Pro Gly Val Thr Gly Ala Lys Pro Lys Gly Val Gly Gly 160 Ala Phe Ala Gly Ile Pro Gly Val Gly Pro Phe Gly Gly Pro Gin Pro WO 99/03886 PCT/AU98/00564 31 Gly Val Pro Tyr Gly Leu 195 Gly Tyr Pro Ile Ala Pro Lys Leu Pro Gly Gly 190 Tyr Gly Pro Pro Tyr Thr Thr Gly Lys Leu Pro Tyr 200 Gly Gly 210 Val Ala Gly Ala Ala 215 Gly Lys Ala Gly Pro Thr Gly Thr Gly 225 Val Gly Pro Gin Ala Ala Ala Ala Ala Ala Lys Ala Ala 235 Ala 240 Lys Phe Gly Ala Ala Ala Gly Val Leu 250 Pro Gly Val Gly Gly Ala 255 Gly Val Pro Gly Val Gly 275 Val Pro Gly Ala Pro Gly Ile Gly Gly Ile Ala 270 Ala Ala Lys Thr Pro Ala Ala Ala Ala Ala Ala Ala 285 Ala Ala 290 Lys Tyr Gly Ala Ala 295 Ala Gly Leu Val Pro 300 Gly Gly Pro Gly Phe 305 Gly Pro Gly Val Gly Val Pro Gly Gly Val Pro Gly Gly Val Pro Gly Gly Ile Pro Val Val 330 Pro Gly Ala Gly Ile Pro 335 Gly Ala Ala Ala Ala Lys 355 Pro Gly Val Val Ser 345 Pro Glu Ala Ala Ala Lys Ala 350 Gly Val Gly Ala Ala Lys Tyr Ala Arg Pro Gly Gly Ile 370 Pro Thr Tyr Gly Gly Ala Gly Gly Pro Gly Phe Gly Val 385 Gly Val Gly Gly Pro Gly Val Ala Gly 395 Val Pro Ser Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Ile Ser Pro Glu 415 WO 99/03886 PCT/AU98/00564 32 Ala Gin Ala Ala Ala Ala Ala Lys 420 Ala Lys Tyr Gly Val Gly Thr 430 Pro Ala Ala 435 Ala Ala Ala Lys Ala 440 Ala Ala Lys Ala Ala Gin Phe Gly 445 Leu Val 450 Pro Gly Val Gly Ala Pro Gly Val Val Ala Pro Gly Val 465 Gly Val Ala Pro Val Gly Leu Ala Pro 475 Gly Val Gly Val Pro Gly Val Gly Ala Pro Gly Val Val Ala Pro Gly Ile Gly 495 Pro Gly Gly Val Ala Ala Ala Ala 500 Ser Ala Ala Lys Val Ala Ala 510 Lys Ala Gin 515 Leu Arg Ala Ala Ala 520 Gly Leu Gly Ala Gly Ile Pro Gly 525 Leu Gly 530 Val Gly Val Gly Pro Gly Leu Gly Gly Ala Gly Val Gly Leu Gly Val Ala Gly Val Pro Gly 555 Phe Gly Ala Gly Asp Glu Gly Val Arg Ser Leu Ser Glu Leu Arg Glu Gly Asp 575 Pro Ser Ser Pro Gly Ala 595 Ser 580 Gin His Leu Pro Thr Pro Ser Ser Pro Arg Val 590 Ala Ala Val Leu Ala Ala Ala Lys 600 Ala Ala Lys Tyr Pro Gly 610 Val Leu Gly Gly Gly Ala Leu Gly Val Gly Ile Pro Gly 625 Gly Val Val Gly Gly Pro Ala Ala Ala Ala Ala Ala Ala 635 Lys 640 Ala Ala Ala Lys Ala Gin Phe Gly Leu Val Gly Ala Ala Gly Leu 655 WO 99/03886 PCT/AU98/00564 33 Gly Gly Leu Gly Gly Ile 675 Val Gly Gly Leu Gly 665 Val Pro Gly Val Gly Gly Leu 670 Tyr Gly Ala Pro Pro Ala Ala Ala 680 Ala Lys Ala Ala Ala Gly 690 Leu Gly Gly Val Leu 695 Gly Gly Ala Gly Gin 700 Phe Pro Leu Gly Gly 705 Val Ala Ala Arg Gly Phe Gly Leu Ser 715 Pro Ile Phe Pro Gly Ala Cys Leu Lys Ala Cys Gly Arg 730 Lys Arg Lys INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 698 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gly 1 Gly Val Pro Gly Ala Ile Pro Gly 5 Gly 10 Val Pro Gly Gly Val Phe Tyr Pro Gly Gly Gly Lys Ala Gly Leu Gly Ala Leu 25 Gly Gly Gly Ala Leu Gly Pro Gly Ala Gly Pro Leu Lys Pro Val Pro Gly Gly Leu Leu Gly Ala Gly Leu Gly Phe Pro Ala Val Phe Pro Gly Ala Val Pro Gly Gly Ala Asp Ala Ala Ala Ala Tyr Lys Ala Lys Ala Gly Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly WO 99/03886 PCT/AU98/00564 34 Val Ser Ala Ala Val Val Pro Gin Pro Gly Ala Gly 105 Val Lys Pro 110 Gly Gly Val Gly Lys Val Pro Gly Val Gly 115 Pro Gly Val Tyr Leu Pro 130 Gly Ala Arg Phe Gly Val Gly Val Pro Gly Val Pro Thr 145 Gly Ala Gly Val Lys 150 Pro Lys Ala Pro Val Gly Gly Ala Phe 160 Ala Gly Ile Pro Val Gly Pro Phe Gly 170 Gly Pro Gin Pro Gly Val 175 Pro Leu Gly Leu Pro Tyr 195 Pro Ile Lys Ala Pro 185 Lys Leu Pro Gly Gly Tyr Gly 190 Pro Gly Gly Thr Thr Gly Lys Pro Tyr Gly Tyr Val Ala 210 Gly Ala Ala Gly Ala Gly Tyr Pro Gly Thr Gly Val Gly 225 Pro Gin Ala Ala Ala 230 Ala Ala Ala Ala Lys 235 Ala Ala Ala Lys Gly Ala Gly Ala Ala 245 Gly Val Leu Pro Gly Val Gly Gly Ala Gly Val Pro Gly Val Gly Thr Pro 275 Gly Ala Ile Pro Ile Gly Gly Ile Ala Gly Val 270 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr 290 Gly Ala Ala Ala Gly 295 Leu Val Pro Gly Pro Gly Phe Gly Gly Val Val Gly Pro Gly Ala Gly Val Pro Gly Val Gly 315 Pro Gly Ala Gly Ile 325 Pro Val Val Pro Gly 330 Ala Gly Ile Pro Gly Ala 335 WO 99/03886 PCT/AU98/00564 Ala Val Pro Gly Val Val Ser Pro 340 Ala Ala Ala Lys Ala Ala Ala 350 Lys Ala Ala 355 Lys Tyr Gly Ala Arg Pro Gly Val Gly Val Gly Gly Ile Pro Thr 370 Tyr Gly Val Gly Ala 375 Gly Gly Phe Pro Gly 380 Phe Gly Val Gly Val Gly Gly Ile Pro 385 Gly 390 Val Ala Gly Val Ser Val Gly Gly Pro Gly Val Gly Gly 405 Val Pro Gly Val Gly 410 Ile Ser Pro Glu Ala Gin 415 Ala Ala Ala Ala Lys Ala Ala Tyr Gly Val Gly Thr Pro Ala 430 Ala Ala Ala 435 Ala Lys Ala Ala Ala Lys Ala Ala Gin 440 Phe 445 Gly Leu Val Pro Gly 450 Val Gly Val Ala Pro 455 Gly Val Gly Val Ala 460 Pro Gly Val Gly Val 465 Ala Pro Gly Val Gly 470 Leu Ala Pro Gly Gly Val Ala Pro Val Gly Val Ala Gly Val Gly Val Ala 490 Pro Gly Ile Gly Pro Gly 495 Gly Val Ala Ala Ala Lys Ser Ala Lys Val Ala Ala Lys Ala 510 Gly Leu Gly Gin Leu Arg Ala Ala Ala Gly 515 Gly Ala Gly Ile Pro 525 Val Gly 530 Val Gly Val Pro Gly 535 Leu Gly Val Gly Gly Val Pro Gly Gly Val Gly Ala Gly 550 Val Pro Gly Phe Ala Val Pro Gly Leu Ala Ala Ala Ala Ala Lys Tyr Gly 570 Ala Ala Val Pro Gly Val 575 WO 99/03886 PCT/AU98/00564 Leu Gly Gly Val Gly Ala 595 Gly Ala Leu Pro Ala Ala 36 Gly Gly 585 Val Gly Ile Pro Gly Gly Val 590 Ala Ala Ala Ala Ala Ala Ala Lys Ala 610 Gly Val Ala Gin Phe Gly Leu 615 Val Gly Ala Ala Gly 620 Gly Leu Gly Gly Leu Gly Gly Leu Gly 630 Ala Pro Gly Val Leu Gly Gly Pro Ala Ala Ala 645 Gly Lys Ala Ala Gly Ala Ala Gly Leu 655 Val Ala Gly Gly Val Ala Arg Pro 675 Gly Ala Gly Gin 665 Pro Pro Leu Gly Phe Gly Leu Ile Phe Pro Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys 690 695 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 1983 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATGGGTGGCG TTCCGGGTGC TGTTCCGGGT GGCGTTCCGG GTGGTGTATT CTACCCAGGC GCGGGTTTCG GTGCTGTTCC GGGTGGCGTT GCAGACGCAG CTGCTGCGTA CAAAGCGGCA WO 99/03886 WO 9903886PCT/AU98/00564 37

AAGGCAGGTG

GCAGTTGTTC

CCGGGCGTAT

CTGCCGGGCG

TTCGCGGGTA

TACCCGATCA

CTGCCGTACG

ACCGGTACTG

TTCGGCGCGG

GGTGTTCCAG

GCTGCGGCTG

CCGGGTGGTC

GGCGTAGGTG

GCGGGTTTCG

TACGGAGCTC

GGTTTCCCAG

GTTGGTGGCG

GCAGCTGCGG

AAAGCAGCGG

GTTGGCGTAG

GTTGCACCAG

GGCGTTGCGG

CGGGTCTGGG

CGCAGCCGGG

ACCCGGGTTT

TTCCGACCGG

TCCCGGGTGT

AAGCGCCGAA

GCTACGGTCC

GTGTTGGTCC

GTGCAGCGGG

GTGCGATCCC

CGGCAGCTGC

CAGGCTTCGG

TTCCAGGTGC

GTGCTGTATC

GTCCGGGCGT

GTTTCGGCGT

TACCGGGTGT

CAGCTAAAGC

CTAAAGCAGC

CACCGGGTGT

GTGTAGGTGT

CTGCTGCGAA

CGGGGTACCA

TGCAGGTGTA

CGGTrGCTGTT

TGCAGGTGTT

TGGCCCGTTC

GCTTCCAGGT

GGGTGGCGTA

GCAGGCTGCT

TTTCGGTGCT

GGGCATCGGT

GGCGAAAGCA

TCCGGGTGTT

GGGCATCCCG

CCCGGAAGCG

TGGTGTTGGT

TGGTGTTGGT

TGGTGGCGTT

AGCGAAGTAC

GCAGTTCGGA

TGGTGTTGCT

TGCGCCGGGC

ATCTGCTGCG

GGTGTTGGCG

AAACCGGGCA

CCGGGCGCGC

AAACCGAAGG

GGTGGTCCGC

GGCTACGGTC

GCAGGTGCTG

GCGGCAGCTG

GTTCCGGGCG

GGTATCGCAG

GCTAAATACG

GTAGGCGTTC

GTTGTACCGG

GCAGCTAAGG

GGCATCCCGA

GGCATCCCGG

CCAGGTGTAG

GGCGTTGGTA

CTAGTTCCCG

CCGGGCGTAG

GTTGGTGTAG

AAGGTTGCTG

GTCTGGGTGT

AAGTTCCAGG

GTTTCCCAGG

CACCAGGTGT

AGCCAGGCGT

TGCCGTACAC

CGGGTAAAGC

CGGCGAAGGC

TAGGTGGTGC

GCGTAGGTAC

GTGCGGCAGC

CGGGTTTCGG

GTGCAGGTAT

CTGCTGCGAA

CCTACGGTGT

GTGTAGCTGG

GTATCTCCCC

CTCCGGCGGC

GCGTAGGTGT

GTCTGGCACC

CACCGGGTAT

CGAAAGCGCA

ATCTGCTGGC

TGTTGGTCTG

TGTTGGTGTA

AGGCGGCGCG

TCCGCTGGGT

CACCGGTAAA

AGGCTACCCA

AGCAGCAAAA

TGGCGTTCCG

TCCGGCGGCC

AGGCCTGGTT

TGCTGTTCCG

CCCGGCGCT

AGCTGCGAAA

AGGTGCAGGC

TGTTCCGTCT

GGAAGCGCAG

AGCAGCTGCT

TGCGCCAGGT

GGGTGTTGGC

CGGTCCGGGT

GCTGCGTGCA

180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 GCAGCTGGTC TGGGTGCGGG CATCCCAGGT CTGGGTGTAG GTGTTGGTGT TCCGGGCCTG WO 99/03886 WO 9903886PCT/AU98/00564 38 GGTGTAGGTG CAGGGGTACC GGGCCTGGGT GTTGGTGCAG GTTCCGGGCG CGCTGGCTGC TGCGAAAGCG GCGAAATACG GGCGGTCTGG GTGCTCTGGG CGGTGTTGGT ATCCCGGGCG GCTGCAGCTG CTGCTGCGGC AAAGGCAGCG GCGAAAGCAG GCAGCAGGTC TGGGCGGTCT GGGTGTTGGC GGTCTGGGTG GGTGGCATCC CGCCGGCGGC GGCAGCTAAA GCGGCTAA.AT GGCGTTCTGG GTGGTGCTGG TCAGTTCCCA CTGGGCGGTG GGTCTGTCCC CGATCTTCCC AGGCGGTGCA TGCCTGGGTA

AAA

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 660 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein

GCGTTCCGGG

GTGCTGTTCC

GTGTTGTAGG

CTCAGTTCGG

TACCGGGCGT

ACGGTGCAGC

TAGCGGCACG

AAGCTTGCGG

TTTCGGTGCT

GGGTGTACTG

TGCAGGCCCA

TCTGGTTGGT

TGGTGGTCTG

AGGTCTGGGT

TCCGGGTTTC

CCGTAAACGT

1560 1620 1680 1740 1800 1860 1920 1980 1983 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Gly Gly Val Pro Gly Ala Val Pro Gly Gly Val 1 5 10 Phe Tyr Pro Gly Ala Gly Phe Gly Ala Val Pro Gly 25 Ala Ala Ala Ala Tyr Lys Ala Ala Lys Ala Gly Ala 40 Val Pro Gly Val Gly Gly Leu Gly Val Ser Ala Gly 55 Pro Gly Gly Val Gly Gly Ala Val Ala Asp Leu Gly Gly Val Val Pro WO 99/03886 PCT/AU98/00564 39 Gin Pro Gly Ala Gly Val Lys Pro Gly Lys Pro Gly Val Gly Pro Gly Val Tyr Pro Gly Phe Gly Ala Val 90 Pro Gly Ala Arg Phe Pro Gly Val Gly Lys Ala Pro 115 Leu Pro Gly Val Thr Gly Ala Gly Val Lys Pro 110 Gly Val Gly Gly Val Gly Gly Phe Ala Gly Ile Pro Phe 130 Gly Gly Pro Gin Pro 135 Gly Val Pro Leu Gly Tyr Pro Ile 140 Tyr Thr Thr Gly Lys Lys Ala 145 Pro Lys Leu Pro Gly 150 Gly Tyr Gly Leu Pro 155 160 Lys Ala 175 Leu Pro Tyr Gly Gly Pro Gly Gly Val 170 Ala Ala Ala Gly Gly Tyr Pro Ala Ala Lys 195 Gly Thr Gly Val Pro Gin Ala Ala Ala Ala Ala 190 Gly Phe Gly Ala Ala Ala Lys Gly Ala Gly Ala Ala Val Pro Gly Val Gly 210 Gly 215 Ala Gly Val Pro Gly Val Pro Gly Ala 220 Pro Gly Ile Gly Gly 230 Ile Ala Gly Val Gly Thr Pro Ala Ala 235 Ala 240 Ala Ala Ala Ala Ala Ala Lys Ala Ala 250 Lys Tyr Gly Ala Ala Ala 255 Gly Leu Val Pro Gly Phe 275 Gly Gly Pro Gly Gly Pro Gly Val Val Gly Val 270 Ala Gly Ile Gly Ala Val Pro Val Gly Val Pro Pro Val 290 Val Pro Gly Ala Gly 295 Ile Pro Gly Ala Gly Phe Gly Ala Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr WO 99/03886 PCT/AU98/00564 40 Gly Ala Arg Pro Val Gly Val Gly Gly 330 Ile Pro Thr Tyr Gly Val 335 Gly Ala Gly Gly Val Ala 355 Phe 340 Phe Pro Gly Phe Val Gly Val Gly Gly Ile Pro 350 Val Gly Gly Gly Val Pro Ser Val 360 Gly Gly Val Pro Gly 365 Val Pro 370 Gly Val Gly Ile Ser 375 Pro Glu Ala Gin Ala Ala Ala Ala Lys 385 Ala Ala Lys Tyr Val Gly Thr Pro Ala Ala Ala Ala Ala Ala Ala Lys Ala 405 Ala Gin Phe Gly Leu 410 Val Pro Gly Val Gly Val 415 Ala Pro Gly Gly Leu Ala 435 Gly Val Ala Pro Val Gly Val Ala Pro Gly Val 430 Val Ala Pro Pro Gly Val Gly Val Ala Pro Gly Val 440 Gly Val 450 Gly Val Ala Pro Gly 455 Ile Gly Pro Gly Val Ala Ala Ala Ala 465 Lys Ser Ala Ala Lys 470 Val Ala Ala Lys Gin Leu Arg Ala Ala Gly Leu Gly Ala Gly Ile Pro Gly 485 Leu 490 Gly Val Gly Val Gly Val 495 Pro Gly Leu Gly Val Pro 515 Val Gly Ala Gly Pro Gly Leu Gly Val Gly Ala 510 Ala Ala Lys Gly Phe Gly Ala Val 520 Pro Gly Ala Leu Ala Ala 530 Lys Tyr Gly Ala Pro Gly Val Leu Gly Leu Gly Ala Leu Gly Gly Val Gly Ile Pro Gly Gly Val Val Gly Ala Gly Pro Ala 545 550' 555 560 WO 99/03886 PCT/AU98/00564 41 Ala Ala Ala Ala Ala Lys Ala Ala Ala 570 Leu Lys Ala Ala Gin Phe Gly 575 Leu Val Gly Val Pro Gly 595 Lys Ala Ala Gly Leu Gly Gly Val Gly Gly Gly Leu Gly 600 Ile Pro Pro Gly Leu Gly 590 Ala Ala Ala Leu Gly Gly Lys Tyr Gly Ala 615 Gly Ala Gly Leu Gly Ala 625 Leu Gln Phe Pro Gly Val Ala Pro Gly Phe Gly 640 Gly Ser Pro Ile Gly Gly Ala Cys 650 Gly Lys Ala Cys 655 Arg Lys Arg Lys 660 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 441 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TCCGCCATGG GAGGTGTTCC GGGCGCGCTG GCTGCTGCGA AAGCGGCGAA ATACGGTGCA GCGGTTCCGG GTGTACTGGG CGGTCTGGGT GCTCTGGGCG GTGTTGGTAT CCCGGGCGGT GTTGTAGGTG CAGGCCCAGC TGCAGCTGCT GCTGCGGCAA AGGCAGCGGC GAAAGCAGCT WO 99/03886 PCT/AU98/00564 42 CAGTTCGGTC TGGTTGGTGC AGCAGGTGTG GGCGGTCTGG GTGTTGGCGG TCTGGGTGTA CCGGGCGTTG GTGGTCTGGG TGGCATCCCG CCGGCGGCGG CAGCTAAAGC GGCTAAATAC GGTGCAGCAG GTCTGGGTGG CGTTCTGGGT GGTGCTGGTC AGTTCCCACT GGGCGGTGTA GCGGCACGTC CGGGTTTCGG TCTGTCCCCG ATCTTCCCAG GCGGTGCATG CCTGGGTAAA GCTTGCGGCC GTAAACGTAA A INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 147 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ser Ala Met Gly Gly Val Pro Gly Ala Leu Ala Ala Ala Lys Ala Ala 240 300 360 420 441 Ala 10 Leu Tyr Gly Ala Gly Val Gly Val Pro Gly Gly Gly Leu Gly Ala Leu Pro Ala Ala Phe Gly Leu Gly Ile Pro Gly Ala Ala Ala Gly 40 Ala Val Gly Ala G ly Gln Ala Ala Lys Ala Lys Ala Val Gly Ala Gly Ala Ala Gly Pro Leu 70 Leu Gly Leu Gly Val1 Gly Leu Gly Gly Val Gly Gly Gly Ile Pro Ala Ala Ala Ala Lys Gly Ala Ala Ala Lys Tyr 100 Ala Ala Gly Gly Val Leu Gly 110 WO 99/03886 PCT/AU98/00564 43 Gly Gin Phe Pro Leu Gly Gly Val Ala Ala Arg Pro Gly Phe Gly Leu 115 120 125 Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg 130 135 140 Lys Arg Lys 145 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 600 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TCCGCCATGG GAGCTCTGGT AGGCCTGGGC GTACCGGGCC CCGGGTTTCG GTGCTGGCGC GGACGAAGGT GTACGTCGTT GAAGGTGACC CGTCCTCTTC CCAGCACCTG CCGTCTACCC GGCGCGCTGG CTGCTGCGAA AGCGGCGAAA TACGGTGCAG GGTCTGGGTG CTCTGGGCGG TGTTGGTATC CCGGGCGGTG GCAGCTGCTG CTGCGGCAAA GGCAGCGGCG AAAGCAGCTC GCAGGTCTGG GCGGTCTGGG TGTTGGCGGT CTGGGTGTAC GGCATCCCGC CGGCGGCGGC AGCTAAAGCG GCTAAATACG GTTCTGGGTG GTGCTGGTCA GTTCCCACTG GGCGGTGTAG

TGGGTGTTGG

CCCTGTCTCC

CGTCCTCTCC

CGGTTCCGGG

TTGTAGGTGC

AGTTCGGTCT

CGGGCGTTGG

GTGCAGCAGG

CGGCACGTCC

TGCAGGCGTT

AGAACTGCGT

ACGTGTTCCG

TGTACTGGGC

AGGCCCAGCT

GGTTGGTGCA

TGGTCTGGGT

TCTGGGTGGC

GGGTTTCGGT

WO 99/03886 WO 9903886PCT/AU98/00564 44 CTGTCCCCGA TCTTCCCAGG CGGTGCATGC CTGGGTAAAG CTTGCGGCCG TAAACGTAAA INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 200 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ser Ala Met Gly Ala Leu Val Gly Leu Gly Val Pro Gly Leu 1 Gly 10 Gly Gly Val Val Arg Ala Gly Val Pro Gly Phe Gly Ala Asp Glu Arg Ser Leu His Leu Pro Ser Pro Glu Leu Arg 40 Ser Gly Asp Pro Ser Gly Ser Ser Gin Ala Leu Ala Ser Thr Pro Ala Ala Ser 55 Tyr Pro Arg Val Pro Lys Ala Ala Gly Ala Ala Pro Gly Val Leu Gly Gly Gly Leu Gly Ala Gly Val Gly I le Al a Gly Gly Val Val1 Ala Gly Pro Ala Gin Phe 115 Gly Gly Leu 130 Ala Ala Ala Lys Ala Ala Leu Val Gly Ala 120 Val Gly Leu Gly Gly 125 Gly Ala Lys Ala 110 Leu Gly Val Ile Pro Pro Gly Val Pro Gly 135 Gly Gly Leu Gly 140 WO 99/03886 PCT/AU98/00564 45 Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly 145 150 Val Leu Gly Gly Ala Gly Gln Phe Pro Leu 165 170 Ala Ala Gly Leu Gly 155 Gly Gly Val Ala Ala 175 Gly 160 Arg Pro Gly Phe Gly Leu Ser Pro Ile Phe 180 185 Pro Gly Gly Ala Cys Leu Gly 190 Lys Ala Cys Gly Arg Lys Arg Lys 195 200 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 60 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ile Pro Pro Ala Ala Ala Ala Lys Ala 10 Ala Lys Tyr Gly Ala Ala Gly Leu Gly Gly Val Leu Gly Gly Ala Gly 25 Gln Phe Pro Leu Gly Gly Val Ala Ala Arg Pro Gly Phe Gly 40 Leu Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 47 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear WO 99/03886 PCT/AU98/00564 46 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Gly Ala Ala Gly Leu Gly Gly Val Leu Gly Gly Ala Gly Gin Phe Pro 1 5 10 Leu Gly Gly Val Ala Ala Arg Pro Gly Phe Gly Leu Ser Pro Ile Phe 25 Pro Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys 40 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Gly Ala Asp Glu Gly Val Arg Arg Ser Leu Ser Pro Glu Leu Arg Glu 1 5 10 Gly Asp Pro Ser Ser Ser Gln His Leu Pro Ser Thr Pro Ser Ser Pro 25 Arg Val INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid WO 99/03886 PCT/AU98/00564 47

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Gly 1 Gly SEQUENCE DESCRIPTION: SEQ ID NO:13: Ala Asp Glu Gly Val Arg Arg Ser Leu Ser Pro Glu Leu Arg Glu 5 10 Asp Pro Ser Ser Ser Gln His Leu Pro Ser Thr Pro Ser Ser Pro 25 Arg Phe INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 216 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Ala Ala Ala Gly Leu Gly Ala Gly Ile Pro Gly Leu 1 5 10 Gly Val Pro Gly Leu Gly Val Gly Ala Gly Val Pro 25 Gly Ala Gly Val Pro Gly Phe Gly Ala Gly Ala Asp 40 Arg Ser Leu Ser-Pro Glu Leu Arg Glu Gly Asp Pro 55 Gly Val Gly Val Gly Glu Ser Leu Gly Val Gly Val Arg Ser Ser Gln WO 99/03886 PCT/AU98/00564 48 Leu Pro Ser Thr Pro Ser Ser Pro Arg Pro Gly Ala Leu Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Val Pro Gly Val 90 Leu Gly Gly Leu Gly Ala Gly Pro 115 Leu Gly Gly Val Gly 105 Ile Pro Gly Gly Val Val Gly 110 Ala Lys Ala Ala Ala Ala Ala Ala 120 Ala Ala Lys Ala Ala 125 Ala Gln 130 Phe Gly Leu Val Gly Ala Ala Gly Leu 135 Gly 140 Gly Leu Gly Val Gly Leu Gly Val Pro 150 Gly Val Gly Gly Gly Gly Ile Pro Pro 160 Ala Ala Ala Ala Lys Ala Ala Lys Tyr 165 Gly 170 Ala Ala Gly Leu Gly Gly 175 Val Leu Gly Pro Gly Phe 195 Ala Gly Gin Phe Pro 185 Leu Gly Gly Val Ala Ala Arg 190 Cys Leu Gly Gly Leu Ser Pro Ile 200 Phe Pro Gly Gly Lys Ala 210 Cys Gly Arg Lys Arg Lys 215 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 183 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Ala Ala Gly Leu Gly Ala Gly Ile Pro Gly Leu Gly Val Gly Val WO 99/03886 WO 9903886PCT/AU98/00564 49 Gly Val Pro Gly Ala Gly Leu Gly Val Gly Gly Val Pro Gly Leu Gly Val Leu Ala Ala Val Pro Gly Phe Gly Ala Val Pro Gly Ala Lys Ala Ala Lys Tyr Ala Ala Val Pro Val Leu Gly Gly Leu Gly Ala Leu Gly Gly Pro Ala Ala Ala Val Gly Ile Pro Gly Val Val Gly Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Gln Phe Gly Gly Leu Gly 115 Val Gly Ala Ala Gly 105 Leu Gly Gly Leu Gly Val Gly 110 Pro Pro Ala Val Pro Gly Val Gly 120 Gly Leu Gly Gly Ala Ala 130 Ala Lys Ala Ala Tlyr Gly Ala Ala Gly 140 Leu Gly Gly Val Gly Gly Ala Gly Phe Pro Leu Gly Gly 155 Val Ala Ala Arg Gly Phe Gly Leu Pro Ile Phe Pro Gly Gly Ala Cys Leu 170 Gly Lys 175 Ala Cys Gly LyAgLy Lys Arg Lys

Claims (62)

1. A human tropoelastin derivative or an amino acid sequence variant thereof, wherein the derivative or variant has elastin-like properties.

2. A human tropoelastin derivative or an amino acid sequence variant thereof, wherein the derivative or variant has macro-molecular binding properties.

3. A derivative or variant thereof according to claim 2 wherein the macro-molecular binding properties include the ability to bind glycosyaminoglycans.

4. A human tropoelastin derivative or an amino acid sequence variant thereof, wherein the derivative or variant has elastin-like properties and macro-molecular binding properties.

5. A polynucleotide encoding a derivative or variant thereof of any one of claims 1 to 4.

6. A tropoelastin derivative which has the amino acid sequence of SHELimodified. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO:

8. A polynucleotide encoding a tropoelastin derivative according to claims 6 or 7.

9. A polynucleotide which has the nucleotide T Rsequence shown in SEQ ID NO: 4. 0 PCT/AU98/00564 Received 04 November 1999 51 A synthetic polynucleotide encoding a tropoelastin derivative which has the amino acid sequence of SHEL626A.

11. A synthetic polynucleotide which has the nucleotide sequence of from nucleotide position 1 to 1676 contiguous with the sequence of from nucleotide position 1775 to 2210 of SEQ ID NO: 1.

12. A tropoelastin derivative which has the amino acid sequence of SHELgamma.

13. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO: 9.

14. A polynucleotide encoding a tropoelastin derivative according to claim 12 or 13. A polynucleotide which has the nucleotide sequence shown in SEQ ID NO: 8.

16. A tropoelastin derivative which has the amino acid sequence of SHELgamma excluding exon 26A.

17. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO: 7.

18. A polynucleotide encoding a tropoelastin derivative according to claim 16 or 17.

19. A polynucleotide which has the nucleotide sequence shown in SEQ ID NO: 6. RA4: 'Zr PCT/AU98/00564 Received 04 November 1999 52 A tropoelastin derivative which has the amino acid sequence of SHEL31-36.

21. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO:

22. A polynucleotide encoding a tropoelastin derivative according to claim 20 or 21.

23. A polynucleotide which has the nucleotide sequence shown in nucleotide position 2022 to 2210 of SEQ ID NO: 1.

24. A tropoelastin derivative which has the amino acid sequence of SHEL32-36. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO: 11.

26. A polynucleotide encoding a tropoelastin derivative according to claim 23 or 24.

27. A polynucleotide which has the nucleotide sequence shown in nucleotide position 2061 to 2210 of SEQ ID NO: 1.

28. A tropoelastin derivative which has the amino acid sequence of peptide 26A.

29. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13. A polynucleotide encoding a tropoelastin derivative according to claim 28 or 29. PCT/AU98/00564 Received 04 November 1999 53

31. A polynucleotide which has the nucleotide sequence shown in nucleotide position 1667 to 1774 of SEQ ID NO: 1.

32. A tropoelastin derivative which has the amino acid sequence of SHEL26-36.

33. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO: 14.

34. A polynucleotide encoding a tropoelastin derivative according to claim 32 or 33. A polynucleotide which has the nucleotide sequence shown in nucleotide position 1554 to 2210 of SEQ ID NO: 1.

36. A tropoelastin derivative which has the amino acid sequence of SHEL26-36 excluding exon 26A.

37. A tropoelastin derivative which has the amino acid sequence shown in SEQ ID NO:

38. A polynucleotide encoding a tropoelastin derivative according to claim 36 or 37.

39. A polynucleotide which has the nucleotide sequence shown in nucleotide position 1554 to 1676 contiguous with the sequence of from nucleotide position 1776 to 2210 of SEQ ID NO: 1. A vector comprising a polynucleotide according to any one of claims 5, 8, 9, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39, or a synthetic 1 PCT/AU98/00564 Received 04 November 1999 54 polynucleotide according to claim 10 or 11.

41. The vector according to claim 40 wherein the polynucleotide or synthetic polynucleotide is operatively linked to a promoter to enhancer regulatory sequence.

42. The vector according to claim 40 or 41 wherein the polynucleotide or synthetic polynucleotide is operatively linked to a nucleotide sequence, the nucleotide sequence encoding a further amino acid sequence.

43. A cell containing a vector according to any one of claims 40 to 42.

44. A method for producing a derivative of tropoelastin, the method comprising: providing a vector according to any one of claims 40 to 42; introducing the vector into a cell; maintaining the cell in conditions suitable for expression of the vector; and isolating the tropoelastin derivative. A tropoelastin derivative produced by the method of claim 44.

46. A transgenic non-human animal containing a vector according to any one of claims 40 to 42, or a polynucleotide according to any one of claims 5, 8, 9, 14, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39, or a TRAZ synthetic polynucleotide according to claim 10 or 11. -57il.; PCT/AU98/00564 Received 04 November 1999 55

47. A tropoelastin derivative produced by a transgenic animal according to claim 46.

48. A method for producing a tropoelastin derivative according to any one of claims 1-4, 6, 7, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36 or 37, the method comprising producing the tropoelastin derivative by solid-phase peptide synthesis.

49. A tropoelastin derivative produced by the method of claim 48. A formulation comprising at least one tropoelastin derivative according to any one of claims 1-4, 6, 7, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36, 37, 45 or 47, together with a pharmaceutically acceptable carrier or diluent.

51. An expression product comprising a tropoelastin derivative according to any one of claims 1-4, 6, 7, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36, 37, 45 or 47, and a further amino acid sequence.

52. An expression product according to claim 51 wherein the tropoelastin derivative has the amino acid sequence of peptide 26A.

53. A polynucleotide encoding an expression product according to claims 51 or 52.

54. A vector comprising the polynucleotide according to claim 53. PCT/AU98/00564 Received 04 November 1999 -56 A cell containing a vector according to claim 54.

56. A method for producing an expression product according to claim 51 or 52, the method comprising: providing a vector according to claim 54; introducing the vector into a cell; maintaining the cell in conditions suitable for expression of the vector; and isolating the expression product.

57. An expression product produced by the method of claim 56. 58 A transgenic non-human animal containing a vector according to claim 54 or a polynucleotide according to claim 53.

59. An expression product produced by a transgenic animal according to claim 58. A formulation comprising at least one expression product according to any of claims 51, 52, 57 or 59, together with a pharmaceutically acceptable carrier or diluent.

61. A hybrid molecule comprising a biological polymer wherein the polymer is linked to a tropoelastin derivative comprising the amino acid sequence of peptide 26A.

62. A hybrid molecule according to claim 61 r PCT/AU98/00564 Received 04 November 1999 57 wherein the biological polymer is a protein.

63. A hybrid molecule according to claim 62 wherein the protein is selected from the group consisting of cytokines, growth factors and antibodies.

64. A hybrid molecule according to claim 61 wherein the biological polymer is selected from the group consisting of lipids, sugars and nucleic acids. A polynucleotide sequence encoding a hybrid molecule according to claim 62.

66. A vector comprising a polynucleotide sequence according to claim

67. A cell containing a vector according to claim 66.

68. A method for producing a hybrid molecule according to claim 62, the method comprising: providing a vector according to claim 66; introducing the vector into a cell; maintaining the cell in conditions suitable for expression of the vector; and isolating the hybrid molecule.

69. A hybrid molecule produced by the method of claim 68. A transgenic non-human animal containing a vector according to claim 66 or a polynucleotide according -9- S$J PCT/AU98/00564 Received 04 November 1999 58 to claim

71. A hybrid molecule produced by a transgenic animal according to claim

72. A hybrid molecule comprising a synthetic polymer linked to peptide 26A.

73. A formulation comprising at least one hybrid molecule according to any of claims 61-63, 69, 71 and 72, together with a pharmaceutically acceptable carrier or diluent.

74. A cross linked complex, the complex comprising at least one of the following: at least one derivative according to any one of claims 1-4, 6, 7, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32, 33, 36, 37, 45 or 47; (ii) at least expression product according to any one of claims 51, 52, 56 or 59; and (iii) least one hybrid molecule according to any one of claims 61-63, 69, 71 or 72. An implant, the implant comprising at least one of the following: at least one derivative according (ii) to any one of claims 1-4, 6, 7, 12, 13, 16, 17, 20, 21, 24, 28, 29, 32, 33, 36, 37, 45 or 47; at least one expression product according to any one of claims 51, PCT/AU98/00564 Received 04 November 1999 (iii) 59 52, 56 or 59; and at least one hybrid molecule according to any one of claims 61- 63, 69, 71 or 72.

76. A method of imparting glycosaminoglycan binding activity to a biological polymer comprising the step of linking a tropoelastin derivative comprising the amino acid sequence of peptide 26A to the biological polymer.

77. A method of deleting glycosaminoglycan binding activity from a biological polymer comprising the step of deleting a tropoelastin derivative comprising the amino acid sequence of peptide 26A from the biological polymer. 78 The method of claim 64 or 65 wherein the biological polymer is a protein.

79. A formulation comprising a tropoelastin derivative and a synthetic or biological polymer. e -,i ,r