WO2010071938A1 - Novel collagen constructs - Google Patents

Novel collagen constructs Download PDF

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WO2010071938A1
WO2010071938A1 PCT/AU2009/001692 AU2009001692W WO2010071938A1 WO 2010071938 A1 WO2010071938 A1 WO 2010071938A1 AU 2009001692 W AU2009001692 W AU 2009001692W WO 2010071938 A1 WO2010071938 A1 WO 2010071938A1
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type
collagen
sequence
polypeptide
protein
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PCT/AU2009/001692
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French (fr)
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Jerome Werkmeister
John Alan Ramshaw
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Commonwealth Scientific And Industrial Research Organisation
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Abstract

The present invention relates to recombinant chimeric triple helical or collagen-like polypeptides. More particularly, the present invention relates to a recombinant collagen-like polypeptide comprising eukaryotic sequences and wherein the triple helical forming domain of the polypeptide comprises an intrachain chimeric sequence. Also provided are expression vectors and host cells containing the expression vectors for production of the recombinant collagen-like polypeptides. The polypeptides may be used in a wide variety of applications including, but not limited to, medical and/or cosmetic applications.

Description

NOVEL COLLAGEN CONSTRUCTS

This application claims priority from Australian provisional application 2008906652, the contents of which are incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to recombinant chimeric triple helical or collagen-like polypeptides. More particularly, the present invention relates to a recombinant collagen-like polypeptide comprising eukaryotic sequences and wherein the triple helical forming domain of the polypeptide comprises an intrachain chimeric sequence. Also provided are expression vectors and host cells containing the expression vectors for production of the recombinant collagen-like polypeptides. The polypeptides may be used in a wide variety of applications including, but not limited to, medical and/or cosmetic applications.

BACKGROUND OF THE INVENTION

The collagen family of proteins represents the most abundant protein in mammals, forming the major fibrous component of, for example, skin, bone, tendon, cartilage and blood vessels. Each collagen protein consists of three polypeptide chains (α chains) characterised by a (GIy-X-Y)n repeating sequence, which are folded into a triple helical protein conformation. In addition to these natural family of collagens, the repeating triple helical (GIy-X-Y)n may be found in other proteins like CIq, macrophage scavenger receptor and lung surfactant protein. Type I collagen (typically found in skin, tendon, bone) consists of two types of polypeptide chain termed αl(I) and α2(I) [i.e. αl(I)2α2(I)], while other collagen types such as Type II [αl(II)3 ] and Type III [αl (111)3] have three identical polypeptide chains. These collagen proteins can spontaneously aggregate to form fibrils which are incorporated into the extracellular matrix where, in mature tissue, they have a structural role and, in developing tissue, they have a directive role. The collagen fibrils, after cross-linking, are highly insoluble and have great tensile strength.

The ability of collagen to form insoluble fibrils makes them attractive for numerous medical applications including biomedical implant production, soft tissue augmentation and wound/burn dressings. To date, most collagens approved for these applications have been sourced from animal sources, primarily bovine. While such animal-sourced collagens have been successful, there are some concerns with immunogenicity and possible transmission of infective diseases, particularly spongiform encephalopathies (e.g. bovine spongiform encephalopathy (BSE)). Accordingly, there is significant interest in the development of methods of production of collagens or collagen fragments by recombinant DNA technology. Further, the use of recombinant DNA technology is desirable in that it allows for the potential production of synthetic collagens and collagen fragments which may include, for example, exogenous biologically active domains (i.e. to provide additional protein function), collagenase- site modified or resistant helices (i.e. to provide longer resorption/degradation in tissue), chimeric constructs and other useful characteristics (e.g. improved biocompatibility and stability).

SUMMARY OF THE INVENTION

According to one embodiment of the invention there is provided a recombinant collagen- like polypeptide of the formula (1):

Aa-Bb-Cc-Dd-Ee-FrGg-Hh

1 wherein:

A is a biologically active component (BAC), which may include a selection marker for identification or purification;

B is a trimer forming domain which may be selected from, for example, an N- propeptide or fragment or variant thereof, or other sequences, including a coiled-coil trimer forming sequence; C is a cleavage site; D is an N-telopeptide or fragment or variant thereof; E a triple helical forming domain comprising I and J:

(i) wherein I is selected from at least one fragment or variant of at least one triple helical forming sequence; and

J is selected from at least one full length or one fragment or one variant of a triple helical forming sequence, wherein when I and/or J comprises more than one fragment or variant, said fragments or variants may be contiguous or non-contiguous; and wherein I and J are not identical nor derived from an identical triple helical forming sequence; or (ii) wherein I is selected from a full length triple helical forming sequence or a fragment or variant thereof; and J is selected from a full length triple helical forming sequence or a fragment or variant thereof, and wherein I and J are not identical nor derived from an identical triple helical forming sequence; F is a C-telopeptide or a fragment or variant thereof; G is a cleavage site; and

H is a trimer forming domain comprising a C-propeptide or a fragment or variant, or a coiled-coil forming sequence, which may include a selection marker for identification or purification;

and wherein: a = 0 or 1; b = O or 1; c = 0 or 1; d = O or l; e = l; f = 0 or 1, preferably 1; g = 0 or 1 ; and h = 0 or 1 , preferably 1.

In one embodiment of the invention, I and J are eukaryotic sequences. In a further embodiment of the invention, A, B, C, D, E, F, G, H are eukaryotic sequences. The sequences may be human or non-human.

In another embodiment of the invention, I and J are each sequences derived from a polypeptide chain which forms part of a collagen or collagen-like protein. The polypeptide chain may be, for example, an alpha chain polypeptide. The alpha chain polypeptide may be selected from αl[I], cc2[I], αl[II], αl[III], αl[V], cc2[V], cc3[V], αl [XI], cc2[XI].

In another embodiment of the invention, the collagen is selected from the group consisting of human collagen, recombinant collagen, recombinant human collagen, non-human collagen, recombinant non-human collagen.

In another embodiment, I and J are each sequences derived from a polypeptide chain which forms part of a protein selected from the group consisting of collagen type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII, type XXVIII and XXIX preprocollagen, procollagen, a collagen-like protein, a V-domain of a bacterial collagen, V-domain like, foldon or foldon-like, conglutinin or congultinin-like, or any other protein that has this functional characteristic.

In one embodiment of the invention, E is an intrachain chimera comprising I and J, wherein I is derived from an α chain polypeptide sequence of collagen type I (for example, αl[I]) and J is derived from an α chain polypeptide sequence of collagen type III (for example CCl[III]), or vice versa.

While not wishing to be bound by theory, type III collagen has at its C-terminal a Cys- Cys sequence which forms a disulphide bonded structure that holds all three polypeptide chains together and which when introduced into a triple helical domain according to the invention adds thermal stability. The use of type III collagen can also be useful in preparations where the approach is to unwind the collagen and then only the structures with the disulfide linkages rapidly refold. Such uses of type III collagen will be familiar to persons skilled in the art of the present invention.

In another embodiment of the invention, I and/or J is a sequence derived from a protein selected from the group consisting of CIq, macrophage scavenger receptor and lung surfactant protein.

It will be appreciated by persons skilled in the art of the present invention, that I and/or J may be derived from any sequence comprising a repeating triple helical motif (GIy- Xaa-Yaa)n.

For example, I and/or J may be derived from the triple helical structure in the acetyl choline esterase from electric eel which contains 2 short (12 residue) heparin binding domains. Nothing similar is found in interstitial fibril forming collagens. This sequence is useful to add in constructs where the desire is to facilitate interaction with chemokines, cytokines and glycosaminoglycans that play a role in the inflammatory response. In a preferred embodiment of the invention, the combined length of I and J is about 30 - 200% of an interstitial (fibril forming) collagen.

In an embodiment of the invention, the said fragment of a triple helical forming sequence according to the invention is about 30 - 200% of interstitial (fibril forming) collagen.

In another embodiment, the said variant of a triple helical forming sequence includes, for example, deletions, insertions or substitutions of residues within the amino acid sequence of the triple helical sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics, especially a (Gly-Xaa-Yaa)n repeat in fibril forming peptides/proteins.

In another embodiment of the invention, the N-propeptide of the recombinant collagen- like polypeptide of the invention is derived from collagen type I.

In another embodiment, the N and C propeptides are derived from the same or a different collagen source. In one example, the N and C propeptides are both derived from collagen type I. In another example, the N-propeptide is derived from collagen type I and the C-propeptide is derived from collagen type III.

In another embodiment, the N and C telopeptides are derived from collagen type I.

In further embodiments of the invention, the signal sequence may be present or absent. In one example, the signal peptide is derived from collagen type I.

In embodiments of the invention, at least one of a, b, c, d, f, g and h is 1, at least two of a, b, c, d, f, g and h are 1, at least three of a, b, c, d, f, g and h are 1, at least four of a, b, c, d, f, g and h are 1, at least five of a, b, c, d, f, g and h are 1, at least six of a, b, c, d, f, g and h are 1, all of a, b, c, d, f, g and h are 1, or all of a, b, c, d, f, g and h are 0. For example, of a, b, c, d, f and g: at least a is 1; at least a and b are 1; at least a, b and c are 1; at least a, b, c and d are 1; at least a, b, c, d and f are 1; at least a, b, c, d, f and g are 1; at least a, b, c, d and g are 1; at least a, b, c and f are 1; at least a, b, c, f and g are 1; at least a, b, c and g are 1; at least a, b and d are 1; at least a, b, d and f are 1; at least a, b, d, f and g are 1; at least a, b and g are 1; at least a and c are 1; at least a, c and d are 1 ; at least a, c, d and f are 1 ; at least a, c, d, f and g are 1 ; at least a and g are 1 ; at least b are 1; at least b and c are 1; at least b, c and d are 1; at least b, c, d and f are 1; at least b, c, d, f and g are 1; at least b, c, d and g; at least c is 1; at least c and d are 1; at least c, d and f are 1 ; at least c, d, f and g are 1 ; at least d is 1 ; at least d and f are 1 ; at least d, f, g are 1; at least f is 1; at least f and g are 1; or at least g is 1. For further example, of a, b, c, d, f and g: at least a is 0; at least a and b are 0; at least a, b and c are 0; at least a, b, c and d are 0; at least a, b, c, d and f are 0; at least a, b, c, d, f and g are 0; at least a, b, c, d and g are 0; at least a, b, c and f are 0; at least a, b, c, f and g are 0; at least a, b, c and g are 0; at least a, b and d are 0; at least a, b, d and f are 0; at least a, b, d, f and g are 0; at least a, b and g are 0; at least a and c are 0; at least a, c and d are 0; at least a, c, d and f are 0; at least a, c, d, f and g are 0; at least a and g are 0; at least b is 0; at least b and c are 0; at least b, c and d are 0; at least b, c, d and f are 0; at least b, c, d, f and g are 0; at least b, c, d and g; at least c is 0; at least c and d are 0; at least c, d and f are 0; at least c, d, f and g are 0; at least d is 0; at least d and f are 0; at least d, f, g are 0; at least f is 0; at least f and g are 0; or at least g is 0.

In embodiments of the invention where the biologically active component (BAC) is present, the BAC is any entity having biological activity, including, but not limited to, an antibiotic, a fungicide, an anti-viral agent, an anti-tumour agent, a cardiovascular agent, an anti-anxiety agent, a hormone or hormone mimetic, a growth factor, a toxin, an anti-hypertensive, immunomodulating, a cytokine, an opiod-like, an anti-infective, an anti-inflammatory peptide/protein or a peptide/protein comprising a linkage site.

As used herein a "biologically active component" may be a full length polypeptide or a portion of a full-length polypeptide which maintains a defined activity of the full-length polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity or its functional equivalent. In one embodiment of the invention, the biologically active fragment is at least 3 amino acids in length. In one example of the invention, the BAC an RGD peptide. In another example, the BAC is a growth factor. In another example, the BAC is a laminin derived sequence (YIGSR) to facilitate cell binding. In another example the BAC is a VEGF-derived sequence (KLTWQELYQLKYKGI). In another example, the BAC is a sequence which can be used as an immunogen e.g. LHRH in a construct delivered for chemical castration.

In another embodiment of the invention, the collagen-like protein comprises a linkage site. The linkage site is preferably a sulfhydryl group, a hydroxyl group, or an amine group, but could also include tyrosine and histidine groups. In one example, the linkage site is a peptide sequence that contains one or more tyrosine residues that would subsequently allow, in vitro, the chemical addition of another entity by means of cross-linking to the tyr residues.

In another embodiment of the invention, the cleavage sites C and G are independently a chemical cleavage site or an enzyme cleavage site. In yet another embodiment, both are chemical cleavage sites or both are enzyme cleavage sites. In one embodiment, the cleavage site according to the invention is an enzymatic cleavage site.

In yet another embodiment, the cleavage sites are independently natural (endogenous) or introduced (exogenous) or combination thereof. In still another embodiment, the sites are selected from Arg-C proteinase, Asp-N endopeptidase, Asp-N endopeptidase + N-terminal GIu, BNPS-Skatole, Caspasel, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Chymotrypsin, Clostripain (Clostridiopeptidase B), CNBr, Enterokinase, Factor Xa, Formic acid, Glutamyl endopeptidase, GranzymeB, Hydroxylamine, Iodosobenzoic acid, Lys C, Lys N, NTCB ^-nitro-S-thiocyanobenzoic acid), N-protease, C-protease, Pepsin (pH 1.3), Pepsin (pH >2), Proline-endopeptidase, Proteinase K, Staphylococcal peptidase I, Thermolysin, Thrombin, and/or Trypsin sites.

In accordance with another embodiment of the invention there is provided a nucleic acid molecule encoding the recombinant collagen-like polypeptide of the invention. The nucleic acid molecule may be a DNA molecule or RNA molecule, preferably a cDNA molecule.

Preferably, the nucleic acid molecule according to the invention comprises a sequence selected from the group consisting of SEQ ID NOs 1 to 21.

In another embodiment of the invention, the nucleic acid molecule comprises a sequence which encodes a protein or peptide sequence selected from the group consisting of SEQ ID NOs 24 to 35.

In accordance with another embodiment of the invention there is provided an expression vector comprising the nucleic acid molecule according to the invention. The expression vector preferably comprises a sequence selected from the group consisting of SEQ ID NOs 1 to 21. The expression vector may also be depicted in any one of Figures 1 to 17.

In another embodiment of the invention, the expression vector comprises a sequence encoding a protein or peptide selected from the group consisting of SEQ ID NOs 24 to

35.

In accordance with another embodiment of the invention there is provided a host cell comprising the expression vector according to the invention. The host cell may also contain an expression vector as depicted in any one of Figures 1 to 15.

In accordance with another embodiment of the invention there is provided a method of transforming a host cell according to the invention with the nucleic acid molecule according to the invention.

In accordance with another embodiment of the invention there is provided a method of producing a recombinant collagen-like polypeptide according to the invention, the method comprising: (i) introducing into a suitable host cell the expression vector according to the invention; and

(ii) culturing the host cell under conditions suitable to express the recombinant collagen-like polypeptide according to the invention.

Additionally, the method may further comprise purifying the recombinant polypeptide according to the invention.

In accordance with another embodiment of the invention there is provided a biomaterial, medical device, tissue construct, or therapeutic product comprising the recombinant collagen-like polypeptide according to the invention.

In various embodiments, the biomaterial may contain human collagen, recombinant collagen, recombinant human collagen, non-human collagen, recombinant non- human collagen or any combination thereof, respectively. In some embodiments, the collagen is selected from the group consisting of collagen type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII and type XXVIII. The collagen can be collagen of one type free of any other type, or can be a mixture of collagen types, or simply a repetitive triple helical protein.

In another embodiment, the invention provides for the use of a recombinant collagen- like polypeptide according to the invention in the manufacture of a biomaterial, medical device, tissue construct, or therapeutic product.

In another embodiment, the invention provides a method of treating a condition selected from the group consisting of wound repair, tissue augmentation, treatment of burns, cosmetic treatment comprising administering to a subject a recombinant collagen-like polypeptide according to the invention.

It will be appreciated by persons skilled in the art, that the invention also pertains to higher order structures comprising multiple copies of the recombinant collagen-like polypeptide of the invention. For example, the proteins of the invention may be arranged in microfibrils which in turn are arranged in macrofϊbrils (as per collagen fibers). The higher order structures are useful for example, in providing 3 -dimensional scaffolds.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, VoIs. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, VoIs. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press); PCR(Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag), Pichia Protocols (Methods in Molecular Biology) 2nd ed. (Cregg, J.M. ed, 2007, Humana Press).

Throughout this specification the word "comprise", or variations such as "comprises" or "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.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in 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 before the priority date of each claim of this application.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES Figure 1 shows a single chain construct that is a combination of CCl[I] and CCl[III] chains, wherein the majority of the helical domain is derived from the type I collagen CCl[I] chain. This Figure corresponds to constructs 3 & 4.

Figure 2 shows the single chain construct of Figure 1 including a locking (N- propeptide) domain, an N-terminal natural cleavage site and a trimer forming domain. This Figure corresponds to constructs 1 & 2.

Figure 3 shows an alternative construct wherein the type I C-propeptide domain in the construct of Figure 1 is replaced by type III C-propeptide and where the natural cleavage site at the end of the type III C-telopeptide is retained. This Figure corresponds to construct 7.

Figure 4 shows the single chain construct of Figure 3 including an N-terminal locking (N-propeptide) domain and an N-terminal natural cleavage site. This Figure corresponds to construct 8.

Figure 5 shows the construct of Figure 2 including an N-terminal locking domain (N- propeptide) and an introduced C-terminal thrombin cleavage site. The type I C- propeptide is retained. This Figure corresponds to construct 10. Figure 6 shows the construct of Figure 4 including an N-terminal locking domain and an introduced C-terminal thrombin cleavage site. The type III C-propeptide is retained This Figure corresponds to construct 11.

Figure 7 shows the construct of Figure 6 where the N-terminal natural cleavage site has been replaced with an introduced thrombin cleavage site and an N-terminal locking (N- propeptide) domain. This Figure corresponds to construct 12.

Figure 8 shows the construct of Figure 5 where the N-terminal natural cleavage site has been replaced with an introduced cleavage site. This Figure corresponds to construct 14.

Figure 9 shows the construct of Figure 7 where the C-terminal natural cleavage site has been replaced with an introduced thrombin cleavage site. This Figure corresponds to construct 15.

Figure 10 shows construct of Figure 1 including an N-terminal biologically active domain (BAC). This Figure corresponds to construct 16.

Figure 11 shows the construct of Figure 3 including an N-terminal BAC domain. This Figure corresponds to construct 17.

Figure 12 shows the construct of Figure 5 including an N-terminal BAC domain. This Figure corresponds to construct 18.

Figure 13 shows the construct of Figure 6 including an N-terminal BAC domain. This Figure corresponds to construct 19.

Figure 14 shows the construct of Figure 2 including a C-terminal HIS tag. This Figure corresponds to construct 9.

Figure 15 shows the construct of Figure 2 where the N-terminal natural cleavage site has been replaced by an introduced thrombin cleavage site. This Figure corresponds to construct 13. Figure 16 shows the construct of Figure 2 containing the normal length of fibrillar collagen alpha-chain with three elements in the helical domain. This figure corresponds to construct 20.

Figure 17 shows an extended length construct relative to the fibrillar collagen alpha chain with two elements in the helical domain. This figure corresponds to construct 21.

Figure 18 shows expression of the recombinant protein according to one embodiment of the invention in Pichia pastoris.

SUMMARY OF THE SEQUENCE LISTING

SEQ ID NO:1 : construct 1 sequence

SEQ ID NO:2: construct 2 sequence

SEQ ID NO:3: construct 3 sequence SEQ ID NO:4: construct 4 sequence

SEQ ID NO:5: construct 5 sequence

SEQ ID NO:6: construct 6 sequence

SEQ ID NO:7: construct 7 sequence

SEQ ID NO:8: construct 8 sequence SEQ ID NO:9: construct 9 sequence

SEQ ID NO: 10: construct 10 sequence

SEQ ID NO: 11 : construct 11 sequence

SEQ ID NO: 12: construct 12 sequence

SEQ ID NO: 13: construct 13 sequence SEQ ID NO : 14 : construct 14 sequence

SEQ ID NO: 15: construct 15 sequence

SEQ ID NO: 16: construct 16 sequence

SEQ ID NO: 17: construct 17 sequence

SEQ ID NO: 18: construct 18 sequence SEQ ID NO : 19 : construct 19 sequence

SEQ ID NO:20: construct 20 sequence

SEQ ID NO:21 : construct 21 sequence

SEQ ID NO:22: human EGF sequence

SEQ ID NO:23: human Thrombin nucleotide sequence SEQ ID NO:24: human Type I, αl-chain signal peptide, prior to any post-translational modification reactions. SEQ ID NO:25: human Type I, αl-chain iV-propeptide, prior to any post-translational modification reactions

SEQ ID NO:26: human Type I, αl-chain N-telopeptide, prior to any post-translational modification reactions. SEQ ID NO:27: human Type I, αl-chain helix domain, prior to any post-translational modification reactions

SEQ ID NO:28: human Type I, αl-chain C-telopeptide, prior to any post-translational modification reactions

SEQ ID NO:29: human Type I, αl-chain C-propeptide, prior to any post-translational modification reactions

SEQ ID NO:30: human Type III, αl-chain signal peptide, prior to any post-translational modification reactions

SEQ ID NO:31 : human Type III, αl-chain N-propeptide, prior to any post-translational modification reactions SEQ ID NO:32: human Type III, αl-chain N-telopeptide, prior to any post-translational modification reactions

SEQ ID NO:33: human Type III, αl-chain helix domain, prior to any post-translational modification reactions

SEQ ID NO:34: human Type III, αl-chain C-telopeptide, prior to any post-translational modification reactions

SEQ ID NO:35: human Type III, αl-chain C-propeptide, prior to any post-translational modification reactions

SEQ ID NO:36: human thrombin protein sequence.

SEQ ID NO:37: human EGF protein sequence. SEQ ID NO:38: laminin-derived sequence.

SEQ ID NO:39: VEGF-derived sequence.

DEFINITIONS

To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below:

"Biologically active component" ("BAC) when used herein includes any peptide/protein that can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism including, but not limited to, viruses, bacteria, bacteriophages, transposons, prions, fungi, plants, animals and humans. "Chimeric alpha chain" or "chimeric collagen" when used herein refers to a combination of two or more alpha chains, or parts thereof, from different collagens, where the complete chain construct can contain repetitive units of each of these chains and can in total vary in length from 30 - 200% of an interstitial fibril forming collagen.

"Coding region" refers to that portion of a gene which codes for a protein. The term "non-coding region" refers to that portion of a gene that is not a coding region.

"Collagen" refers to any one of the known collagen types, including collagen types I through XXIX, as well as to any other collagens, whether natural, synthetic, semisynthetic, or recombinant. Some collagens (e.g., collagen types I, II, III, etc. ) are, in nature, first produced as precursor molecules, called procollagens, containing N- propeptides and C-propeptides that are subsequently removed during post-translational processing to form collagen. The term "collagen" as used herein specifically encompasses procollagens. The term "collagen" encompasses any single- chain polypeptide, i. e., collagen polypeptide, encoded by a single polynucleotide, as well as any homotrimeric and heterotrimeric assembly of collagen chains. The term "collagen" as used herein specifically encompasses variants and fragments thereof, and functional equivalents and derivatives thereof, which preferably retain at least one structural or functional characteristic of collagen, for example, a (Gly-Xaa-Yaa)n domain. Collagen propeptides and telopeptides are known in the art, and sequences corresponding to these regions are readily accessible, e.g., GenBank Accession Nos. P02452, P02464, P08123 (collagen type I); P02458 (collagen type II); P02461 (collagen type III);P20908 (collagen type V); P12107 and A56371 (collagen type XI); etc. Each of these listings is incorporated herein by reference in its entirety. The above citations are provided merely by way of example, and are not intended to limit application of the present invention.

"Culture" means cell propagation in a medium that leads to their growth and all the consequent subcultures. The term "subculture" refers to cultures of cells grown from cells of other cultures (source culture) or any subculture of the source culture, depending on the number of subcultures that have been developed between the subculture of interest and the source culture. "DNA construct" embraces expression cassettes and also includes DNA fragments that include more than one expression cassette.

"Expression cassette" means a genetic module comprising a gene and the regulatory regions necessary for its expression, which may be incorporated into a vector.

"Expression vector" is understood to include vectors that can express the DNA sequences contained in them, where said sequences are functionally associated with other sequences capable to affect their expression, as for example, promoter sequences. In general, expression vectors normally used in recombinant DNA technologies are in the form of "pi 'asmids", circular double-strand DNAs, which in their form as vectors are not joined to the chromosome. In this description, the terms "vector" and "plasmid" are used indistinctly, however, in the embodiments of the present invention it is understood that other types of expression vectors that may be functionally equivalent can be included.

"Fibrillar collagen" means an interstitial collagen of a type which can normally form collagen fibrils. The fibrillar collagens include collagen types I - III, V, and XI. The collagen monomers that make up the fibrillar collagens contain "telopeptide" regions at the amino (N) and carboxy (C) terminal ends of the monomers which are non-triple- helical in the collagen trimer. These collagens self-assemble into fibrils with the C- terminal end of the helical domain and the C propeptide of one collagen triple helix overlapping with the N telopeptide and the N-terminal end of the triple helical domain of an adjacent collagen molecule. The monomers that make up the fibrillar collagens are made as preproproteins, including an N-terminal secretion signal sequence and N and C-terminal propeptide domains. The signal sequence is normally cleaved by signal peptidase, as with most secreted proteins, and the propeptides are removed by specific proteolytic processing enzymes after association, folding and secretion of trimeric procollagen. The term fibrillar collagen encompasses both native (i.e., naturally occurring) and variant fibrillar collagens (i.e., fibrillar collagens with one or more alterations in the sequence of one or more of the fibrillar collagen monomers). Unless the context clearly indicates otherwise (e.g., the term is modified by the word "monomer") "fibrillar collagen" refers to triple helical fibrillar collagen.

"Foldon" or "foldon domain" refers to the C-terminal amino acid peptide sequence of the bacteriophage T4 fibritin sequence or portions thereof, or fragments thereof having foldon activity. Foldon is capable of forming a trimeric structure. Foldon activity r rpeffperrsβ i tno t tVhipe a aVbniiliittvy n off

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"Fragment" of a molecule such as a protein or nucleic acid is meant to refer to a portion of the native amino acid or nucleotide genetic sequence, and in particular the functional derivatives of the protein of the invention.

"Functional derivative" of a protein or nucleic acid, is a molecule that has been chemically or biochemically derived from (obtained from) such protein or nucleic acid and which retains a biological activity (either functional or structural) that is a characteristic of the native protein or nucleic acid. The term "functional derivative" is intended to include "fragments" , "variants", "analogues" or "chemical derivatives" of a molecule that retain a desired activity of the native molecule.

"Functional equivalent" refers to a polypeptide or polynucleotide that possesses at least one functional and/or structural characteristic of a particular polypeptide or polynucleotide. A functional equivalent may contain modifications that enable the performance of a specific function. The term "functional equivalent" is intended to include fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.

"Heterotrimer" or "heterotrimeric collagen" refer to collagen or procollagen containing a least two different collagen or procollagen α-chains. Heterotrimeric collagen includes collagen or procollagen having two of the same α-chains and one different α-chain (e.g., two αl (I) chains and one α2 (I) chain) as well as collagen or procollagen containing one each of three different a-chains (e.g., one αl (IX) chain, one α2 (IX) chain, and one α3 (IX) chain). Heterotrimeric collagens include, e.g., collagen types I, IV, V, VI, IX, XI, etc. Heterotrimeric collagens additionally include cross-type heterotrimeric collagens, e.g., type V, type XI, etc.

"Homotrimer" or "homotrimeric collagen" refer to collagen or procollagen having three each of the same collagen or procollagen α-chain. Homotrimeric collagens include, e. g, collagen types II and III, etc.

"Hybridization" refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. The conditions employed in the hybridization of two non-identical, but very similar, complementary nucleic acids varies with the degree of complementarity of the two strands and the length of the strands. Thus the term contemplates partial as well as complete hybridization. Such techniques and conditions are well known to practitioners in this field.

"Isolated nucleic acid fragment" is a polymer of RNA or DNA that is single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

"Isolated polynucleotide" means a polynucleotide that is free of one or both of the nucleotide sequences which flank the polynucleotide in the naturally-occurring genome of the organism from which the polynucleotide is derived. The term includes, for example, a polynucleotide or fragment thereof that is incorporated in a vector or expression cassette; into an autonomously replicating plasmid or virus; into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule independent of other polynucleotides. It also includes a recombinant chimeric polynucleotide that is part of a hybrid polynucleotide, for example, one encoding a polypeptide sequence.

"Operably linked" means any linkage, irrespective of orientation or distance, between a regulatory sequence and coding sequence, where the linkage permits the regulatory sequence to control expression of the coding sequence.

"Operative linkage" or "operably associated" refers to the relationship among elements of a DNA construct in which the elements are arranged whereby regulatory sequences of nucleotides that are part of the construct directly or indirectly control expression of the DNA in the construct, including DNA encoding a protein or a peptide.

"Polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric (2 or more monomers) form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Although nucleotides are usually joined by phosphodiester linkages, the term also includes polymeric nucleotides containing neutral amide backbone linkages composed of aminoethyl glycine units. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, labels, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), those containing pendant moieties, such as, for example, proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide. Polynucleotides include both sense and antisense strands.

"Procollagen" refers to a collagen precursor that includes the registration or propeptides, corresponding to any collagen, whether natural, synthetic, semi-synthetic, or recombinant, human or non-human, that possesses additional C-terminal and/or N- terminal propeptides that can be removed, if required. The term procollagen specifically encompasses variants and fragments thereof, and functional equivalents and derivatives thereof, which preferably retain at least one structural or functional characteristic of collagen, for example, a (Gly-Xaa-Yaa)n domain wherein Xaa and Yaa may be any other amino acid residue.

"Regulatory sequence" or "regulatory region" as used in reference to a specific gene refers to the coding or non-coding nucleotide sequences within that gene that are necessary or sufficient to provide for the regulated expression of the coding region of a gene. Thus, the term encompasses promoter sequences, regulatory protein binding sites, upstream activator sequences and the like. Specific nucleotides within a regulatory region may serve multiple functions. For example, a specific nucleotide may be part of a promoter and participate in the binding of a transcriptional activator protein.

"Secretion sequence" or "signal peptide" or "signal sequence" means a sequence that directs newly synthesized proteins to and through membranes of the endoplasmic reticulum, or from the cytoplasm to the periplasm across the inner membrane of bacteria, or from the matrix of mitochondria into the inner space, or from the stroma of chloroplasts into the thylakoid. Fusion of such a sequence to a gene that is to be expressed in a heterologous host ensures secretion of the recombinant protein from the host cell. "Sequence" means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.

"Tήmerization" and "trimer forming refer to the association of three individual polypeptides, e.g., collagen polypeptides, α-chains, etc. Trimerization or trimer forming of collagen can be associated with collagen polypeptide association, registration, and selection such that the three chains are held together in the correct topology.

"Triple helical forming domain" refers to an amino acid sequence, comprising a (GIy- Xaa-Yaa)n motif, wherein Xaa and Yaa are any other amino acid residue, that is capable of folding with two other chains to form a triple helix.

"Variant polynucleotide sequences" include those with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent polypeptide. Included within this definition are sequences displaying polymorphisms that may or may not be readily detectable using particular oligonucleotide probes or through deletion of improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the subject polynucleotide sequence.

"Variant polypeptides" may contain deletions, insertions, or substitutions of amino acid residues which produce a change and result in an equivalent polypeptide. "Variant" polypeptides may also include naturally occurring sequence interruptions. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of the encoded polypeptide is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine. These substitutions can be designed to enhance or reduce the triple helical stability in selected regions. "Variant" or "analog" of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the native molecule, such as that encoded by a functional allele.

DETAILED DISCLOSURE OF PREFERRED EMBODIMENTS

The present invention relates to recombinant chimeric triple helical or collagen-like constructs. More particularly, the present invention relates to a recombinant collagen- like polypeptide comprising eukaryotic sequences and wherein the triple helical forming domain of the polypeptide comprises an intrachain chimeric sequence.

The generation of chimeric intra chain polypeptides according to the invention provides for the inclusion of functional features not present in a single molecule and not typically found combined in a naturally occurring molecule of collagen or collagen-like protein. The recombinant collagen-like polypeptide of the invention preferably also includes propeptide domains which are also present on native collagen. While not wishing to be bound by theory, it is thought that the N-propeptide acts as a steric constraint to control the rate and size of fibril formation. It is has been suggested in the art that there is a feedback mechanism to control synthesis. The C-propeptide appears to be more important. It is the domain that is involved in chain recognition e.g so that a type I chain and a type II chain do not form a hybrid triple helix, it is involved in chain registration, so that the 3 chains are held together in the correct topology, and it is involved in helix initiation so that the 3 chains start to fold into a triple helix, which is in the correct "phase". In some embodiments of the present invention, the pro-domains can be replaced by other entities which can fulfil the same role, for example, by use of foldon or a coiled-coil trimer.

The recombinant collagen-like polypeptides of the invention can be used in a wide variety of applications including medical and cosmetic applications. By including a BAC, the polypeptide can be tailored to specific biological activities. Such activities will be recognised by persons skilled in the art.

Nucleic acid molecule (s)

In accordance with the invention there is also provided a nucleic acid molecule encoding the recombinant collagen-like polypeptide according to the invention. In one embodiment, the nucleic acid molecule is a DNA molecule. In another embodiment, the DNA molecule includes a sequence encoding a post-translation system for hydroxylation of proline and/or lysine. In yet another embodiment, the post-translation system includes the α and/or β subunits of P4H, preferably both.

In yet another embodiment, the nucleic acid molecule also includes a functional selection marker. In still another embodiment, the selection marker is any functional selection marker gene; i.e. any gene that confers a different phenotype and therefore permits it to be identified and grown in a selective way different from the majority of non-transformed cells. Appropriate selection marker genes include, for example, selection marker systems composed of an auxotrophic mutant of P. pastoris strains and a wild type biosynthetic gene that complements the defect in the host cells. For example, for the transformation of P. pastoris His4-strains, the HIS4 gene of S. cerevisiae or P. pastoris can be used, or for the transformation of Arg4-mutants, the ARG4 gene from S. cerevisiae and P. pastoris can be used.

In one embodiment, the nucleic acid molecule contains selection marker genes functional in bacteria. Thus, it is possible to use any gene that confers a phenotype in bacteria that makes it possible to transform them for the purposes of identification and selective cultivation or identifying them from most of the non-transformed cells. This additional selection marker permits that the nucleic acid molecule of the invention to be introduced into bacteria such as E. coli for their amplification.

Appropriate selection marker genes include: the ampicillin (Amp1) resistance gene, the tetracycline (T cr) resistance gene, and alike. When the introduction of the nucleic acid molecule of this invention through bacterial cells is contemplated, a bacterial origin of replication should be included in the construction of the DNA in order to ensure maintenance of the DNA of the invention in the bacteria from generation to generation. Examples of bacterial replication sources include: Fl, colicin, col El and the like.

Expression vector fs) In one embodiment, the nucleic acid of the invention is contained in an expression vector, for example, a circular plasmid. In another embodiment, the expression vector is pHIL-D2, pPIC3.5, pHIL-Sl, pPIC9, pPICZ, pA0815, pBLADE, pBLARG, or pBLURA.

In yet another embodiment, one or more copies of the expression vector are integrated into the same or different loci by additional events of homologous recombination into the interrupted site in the genome of a host organism. A person skilled in the art will appreciate that linearization of the plasmid with restriction enzymes facilitates this type of integration.

In another embodiment, the nucleic acid molecule of the invention is used to transform host cells as a linearized fragment whose ends contain DNA sequences that have sufficient homology with the target gene to be able to carry out the integration of the nucleic acid molecule of the invention within the host genome. In this case, integration takes place by replacement of the DNA in the target site by the nucleic acid molecule of the invention. Alternatively, the nucleic acid molecule of the invention forms part of a circular plasmid that can be linearized to facilitate integration, and will integrate into host chromosome by means of the sequences in the plasmid homologous to those in the host genome.

In still another embodiment of the invention, the expression construct also includes other DNA sequences appropriate for the intended host cell. For example, expression constructs for use in higher eukaryotic cell lines (e.g., vertebrate and insect cell lines) will include a poly-adenylation site and an intron (including signals for processing the intron), as the presence of an intron appears to increase mRNA export from the nucleus in many systems.

Furthermore, in another embodiment, a secretion signal sequence operable in the host cell is included as part of the construct. The secretion signal sequence can be from a collagen monomer gene or from a non-collagen gene. If the secretion signal sequence is derived from a collagen monomer gene, it is preferably from a fibrillar collagen monomer (and can be derived from the same protein as the DNA encoding the fibrillar collagen monomer to be expressed or from a different fibrillar collagen monomer) or a non-fibrillar collagen monomer. Where the expression construct is intended for use in a prokaryotic cell, the expression construct includes a signal sequence which directs transport of the synthesized peptide into the periplasmic space or expression is directed intracellularly.

In yet another embodiment, the expression construct also comprises a means for selecting for host cells which contain the expression construct (a "selectable marker"). Selectable markers are well known in the art. For example, in one embodiment, the selectable marker is a resistance gene, such as an antibiotic resistance gene (e.g., the neor gene which confers resistance to the antibiotic gentamycin), or it is a gene which complements an auxotrophy of the host cell. If the host cell is a yeast cell, the selectable marker is preferably a gene which complements an auxotrophy of the cell (for example, complementing genes useful in S. cerevisiae, P. pastoris and S. pombe include LEU2, TRPl, TRPId, URA3, URA3d, HIS3, HIS4, ARG4, LEU2d), although antibiotic resistance markers such as SH BLE, which confers resistance to ZEOCIN®, may also be used. If the host cell is a prokaryotic or higher eukaryotic cell, the selectable marker is preferably an antibiotic resistance marker (e.g., neor or bla). Alternately, a separate selectable marker gene is not included in the expression vector, and the host cells are screened for the expression protein of the invention (e.g., upon induction or derepression for controllable promoters, or after transfection for a constitutive promoter, fluorescence-activated cell sorting, FACS, may be used to select those cells which express the recombinant collagen).

In yet another embodiment, the expression construct also contains sequences which act as an "ARS" (autonomous replicating sequence) which will allow the expression construct to replicate in the host cell without being integrated into the host cell chromosome. Origins of replication for bacterial plasmids are well known. ARS for use in yeast cells are also well known (the 2μ origin of replication and operative fragments thereof, especially the full length sequence 2μ is preferred, see, for example International Patent Application No. WO 97/14431, although CEN-based plasmids and YACS are also useful in the instant invention) and ARS which act in higher mammalian cells have been recently described (see, for example, Pelletier et al., 1997, J. Cell. Biochem. 66(l):87-97)). Alternately, the expression construct includes DNA sequences which will direct or allow the integration of the construct into a host organism's chromosome by homologous or site-directed recombination.

Where the host cell is a eukaryotic cell, it is advantageous for the expression vector to be a "shuttle vector", because manipulation of DNA is substantially more convenient in bacterial cells. A shuttle vector is one which carries the necessary signals to for manipulations in bacteria as well as the desired host cell. So, for example, the expression construct also comprises an ARS ("ori") which acts in prokaryotic cells as well as a selectable marker which is useful for selection of prokaryotic cells.

In one embodiment, the recombinant collagen-like polypeptide is expressed in Pichia cells, together with the 2 genes that comprise active P4H. This system is characterized by methylotrophic expression in which a strong constitutive promoter (GAP) and a strong inducible promoter (AOXl - alcohol oxidase) are present. Addition of methanol, which can be used as the sole carbon source, allows simple, complete induction. This system uses chromosomal integration of the inserted gene, eliminating the need for continual selection (eg antibiotic) during fermentation. In this system, the product can either be retained within the cell or secreted. High levels of expression of intracellular or secreted proteins in the range of grams/liter have been reported.

Examples of P4H genes suitable for use in the present invention include: Human prolyl 4-hydroxylase α (1) subunit cDNA, ATCC Number MGC-33137; Human prolyl 4-hydroxylase α (2) subunit cDNA, ATCC Number MGC-46171; Human prolyl 4-hydroxylase β subunit cDNA, ATCC Number MGC-9695.

Preferably, the 3 required genes are integrated into the Pichia chromosome for expression. This can be achieved by various approaches. These may include, for example:

(i) A one vector strategy, in which all 3 genes are assembled in a single vector, each with its own promoter and transcription terminator. (ii) A two vector strategy, where the collagen gene is in one vector with its own promoter and terminator and the 2 P4H genes are in a second vector, each with its own promoter and terminator, (iii) A three vector strategy, where each of the collagen and the 2 P4H genes is in and individual vector along with their own promoter and terminator.

The construct may be incorporated into alternative vector systems. For example pKLACl, for use in Kluyveromyces lactis. The yeast K. lactis, a unicellular organism related to Saccharomyces cerevisiae, has been developed as a secretion host employing expression vectors based on the 2μ-like plasmid pKDl. It can also use chromosomal integration of the inserted genes, eliminating the need for continual selection.

A further system is Hansenula polymorpha, (related to Pichia) where there is some endogenous P4H activity, but seemingly not sufficient to achieve full levels of hydroxylation.

Host cells The host cell according to the invention is any convenient host cell, including cells of bacterial, yeast, and eukaryotic origin. Yeast and higher eukaryotic cells are preferred host cells. For yeast host cells, Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Schwanniomyces occidentis, Schizo saccharomyces pombe and Yarrowia lipolytica strains are preferred. Of the higher eukaryotic cells, insect cells such as Sf9 are preferred, as are mammalian cell lines which produce non-fϊbrillar collagens and do not produce any endogenous fibrillar collagens, such as HT-1080, 293, and NSO cells and plant cells that preferably include P4H (prolyl 4-hydroxylase).

Pichia strains which are suitable for use according to the invention include, X-33, KM71, KM71H, GSl 15, GS200, JC300, JC301, JC302, JC303, JC308.

If a host cell is transformed with a linear nucleic acid fragment containing the nucleic acid molecule of the invention under the regulation of, for example, a P. pastoris promoter gene, and if the expression cassette is integrated into the host genome by one of the genetic recombination techniques known in the state of the technique [such as gene replacement by homologous, recombination through a simple crossing over event or by insertion (Cregg, JM 2007. Methods in Molecular Biology 389: Pichia protocols (2nd Edition), then the linear nucleic acid fragment is directed towards the desired locus, and the target gene is interrupted in its reading frame, due principally to the fact that the sequence ends of the nucleic acid fragment have sufficient homology with the target gene to allow integration of the nucleic fragment into said gene.

In vitro expression of an active recombinant P4H from its subunits has been successfully obtained by co-infection of insect cells Spodoptera frugiperda and Trichoplusia ni, with recombinant baculoviruses, or cotransfection with expression vectors in mammalian cell lines COS-I, and HEK293, in yeasts Pichia pastoris and Saccharomyces cerevisiae. The P4H tetramer assembly probably requires molecular chaperones, such as immunoglobulin heavy chain binding protein, BiP.

If the host organism does not have P4H activity (or has insufficient activity as is the case in insect cells), the host cell is altered to produce P4H. This is conveniently accomplished by introducing expression constructs coding for the expression of the subunits of P4H into the host cell. Expression constructs for P4H have been described for yeast and for insect cells. In the case of a bacterial host cell, the expression construct for P4H will preferably incorporate a translocation signal to direct the transport of the subunits of the enzyme to the periplasmic space. Alternately, the P4H expression construct is included in the nucleic acid of the invention construct. In this arrangement, the expression construct may direct the production of separate messages for the collagen-like polypeptide of the invention and the P4H subunits. The nucleotide sequences encoding the P4H α and β subunits can be of any animal origin although they are preferably of avian or mammalian, particularly human, origin. It is also envisaged that the nucleotide sequences may originate from different species. In addition, the nucleotide sequence encoding the P4H α subunit includes a sequence encoding an endoplasmic reticulum (ER) retention signal (e.g. HDEL, KDEL or KEEL) with or without other target signals so as to allow expression of the P4H in the ER, cytoplasm or a target organelle or, alternatively, so as to be secreted.

Alternately, the collagen-like polypeptide of the invention is produced in non- hydroxylated form. Non-hydroxylated collagen- like polypeptide of the invention has reduced thermal stability compared to hydroxylated collagen-like polypeptide of the invention. Collagen-like polypeptide of the invention with reduced thermal stability is desirable for certain uses. Non-hydroxylated as well as hydroxylated collagen- like polypeptide of the invention in one embodiment of the invention, can be modified to increase thermal stability by chemical modification such as, for example, chemical crosslinking.

In a specific embodiment, the present invention provides a Pichia pastoris host cell containing the nucleic acid molecule encoding the recombinant collagen-like polypeptide of the invention. Within the nucleic acid molecule of the invention, codons which are preferably used by yeast cells are used.

Method for the transformation of host cells The nucleotide sequence encoding the recombinant collagen-like polypeptide of the invention is typically provided in the form of an expression vector which is used to transform the host cell. In addition to the expression construct, a nucleotide sequence(s) encoding the P4H α and β subunits is introduced into a host cell lacking endogenous P4H in a manner such that they are borne on one or more DNA molecules that are stably retained and segregated by the host cell during culturing. In this way, all daughter cells will enable stable and efficient expression of a recombinant protein throughout the culturing step and without the use of continuous selection pressure.

The expression construct may be introduced into the host cell by any convenient method known to the art. For example, for yeast host cells, the construct of the invention is introduced by electroporation, lithium acetate/PEG and other methods known in the art. Higher eukaryotes can be transformed by electroporation, microprojectile bombardment, calcium phosphate trans fection, lipofection, or any other method known to the art. Bacterial host cells can be trans fected by electroporation, calcium chloride-mediated transfection, or any other method known in the art. Multiple copies of the nucleic acid molecule of the invention can be introduced into the host cell (e.g. present on a YAC vector or integrated into a host chromosome). It may be particularly advantageous to provide the nucleic acid molecule according to the invention in multicopy and, accordingly, in one embodiment of the invention, it is preferred to introduce the recombinant collagen-like polypeptide encoding nucleotide sequence(s) on a high copy number plasmid (e.g. a YEp plasmid).

After introduction of the expression construct into the host cell, host cells comprising the expression construct are normally selected on the basis of the selectable marker that is included in the expression vector. As will be apparent, the exact details of the selection process will depend on the identity of the selectable marker. If the selectable marker is an antibiotic resistance gene, the transfected host cell population is generally cultured in the presence of an antibiotic to which resistance is conferred by the selectable marker. The antibiotic eliminates those cells which are not resistant (i.e., those cells which do not carry the resistance gene) and allows the propagation of those host cells which carry the resistance gene (and presumably carry the rest of the expression construct as well). If the selectable marker is a gene which complements an auxotrophy of the host cell, then the transfected host cell population is cultured in the absence of the compound for which the host cells are auxotrophic. Those cells which are able to propagate under these conditions carry the complementing gene to supply this compound and thus presumably carry the rest of the expression construct.

Methods for the transformation of methylotrophic yeast, such as Pichia pastoris, as well as the applicable methods for the cultivation of methylotrophic yeast cells containing a gene encoding a heterologous protein in their genome are currently known in the previous art. Preferably, the transformation, positive transformant selection and culturing methods disclosed in U.S. Pat. No. 4,837,148; U.S. Pat. No. 4,855,231; U.S.

Pat. No. 4,882,279; U.S. Pat. No. 4,929,555; U.S. Pat. No. 5,122,465; U.S. Pat. No. 5,324,639 can be used in the present invention. The expression cassette may also be used to transform methylotrophic yeast cells by, for example, the spheroplast technique, by electroporation and by the transformation system of lithium chloride.

Positive transformed organisms can be characterized by the polymerase chain reaction (PCR) or by "Southern blot" analysis to corroborate the integration of the nucleic acid molecule of the invention. In order to analyse the expression of the collagen-like polypeptide of the invention, reverse transcription can be used together with PCR or "Northern blot" technique; and in order to analyse the product, electrophoretic or immunological techniques can be used.

Host cells which pass the selection process may be "cloned" according to any method known in the art that is appropriate for the host cell. For microbial host cells such as yeast and bacteria, the selected cells may be plated on solid media under selection conditions, and single clones are selected for further selection, characterization or use. Higher eukaryotic cells are generally further cloned by limiting dilution (although physical isolation methods such as micromanipulation or "cloning rings" can also be used). This process is carried out several times to ensure the stability of the expression construct within the host cell.

For production of trimeric proteins, the recombinant host cell comprising the expression construct of the invention is generally cultured to expand cell numbers. This expansion process is carried out in any appropriate culturing apparatus known to the art. For yeast and bacterial cells, an apparatus as simple as a shaken culture flask is used, although large scale culture is generally carried out in a fermenter. For insect cells, the culture is generally carried out in " spinner flasks" (culture vessels comprising a means for stirring the cells suspended in a liquid culture medium). For mammalian cell lines, the cells can be grown in simple culture plates or flasks, but as for the yeast and bacterial host cells, large scale culture is generally performed in a specially adapted apparatus, a variety of which are known in the art.

The culture medium used for culture of the recombinant host cells will depend on the identity/origin of the host cells. Culture media for the various host cells used for recombinant culture are well known in the art. The culture medium generally comprises inorganic salts and compounds, amino acids, carbohydrates, vitamins and other compounds which are either necessary for the growth of the host cells or which improve the health and/or growth of the host cells (e.g., protein growth factors and hormones where the host cells are mammalian ).

Where the host cells are yeast cells, media formulations which utilize no animal- derived components, such as casamino acids, are advantageous for the production of the protein in accordance with the invention. Preferred media include media with a defined "base" medium (such as YNB) that is supplemented with specific amino acids. Preferred amino acids for supplementation include arginine, glutamate, lysine, and α- ketoglutarate. Where the defined media is supplemented with α-ketoglutarate, the media is preferably buffered to an initial acid pH, preferably about pH 5.5 to 6.5, more preferably about pH 6.0 as the pH of the media at the beginning of the culture.

If the host cells comprise (either naturally or by introduction of the appropriate expression constructs) P4H, then vitamin C (ascorbic acid or one of its salts) can be added to the culture medium. If ascorbic acid is added, it is generally added to a concentration of between 10-200 μg/ml, preferably about 80 μg/ml. Expression can be driven by constitutive or inducible yeast promoter sequences such as those mentioned above.

The transformed host cells are cultured under conditions appropriate for the expression of the collagen-like polypeptide of the invention. If the expression construct utilises a controllable expression system, the expression of the nucleic acid molecule of the invention is induced or derepressed, as is appropriate for the particular expression construct. The exact method of inducing or derepressing the expression of the nucleic acid molecule of the invention will depend on the properties of the particular expression construct used and the identity of the host cell, as will be apparent to one of skill in the art. Generally, for inducible promoters, a molecule which induces expression is added to the culture medium.

Transformed host cells that possess the phenotype or genotype desired may also be grown in fermenters for large scale production of the protein of the invention.

According to a preferred embodiment of the invention, the heterologous protein expression system used for the expression of the collagen-like polypeptide of the invention uses the promoter derived from the P. pastoris AOXl methanol regulatable gene of P. pastoris, which is very efficiently expressed and accurately regulated. This gene can also be the source of the transcription termination sequence. The expression cassette preferred in this invention consists of the following elements, all functionally associated with one another: the P. pastoris AOXl promoter, the nucleic acid molecule of the invention, and a transcription terminator derived from the P. pastoris AOXl gene, preferably two or more of the expression cassettes mentioned made up of a DNA fragment, in head to tail orientation, in order to generate a multiple expression cassette on one sole DNA fragment. In this invention preference is given to host cells transformed with the expression cassette of which P. pastoris is preferred; there shall be at least one mutation that can be complemented with a selectable marker gene present in the transforming DNA fragment. The fragment containing the expression cassette is inserted into a plasmid containing a marker gene which complements the defective host and can optionally contain additional sequences such as selectable marker genes for bacteria and yeast gene sequences that direct vector integration.

For the development of P. pastoris Mut strains (Mut refers to the phenotype that uses methanol), the DNA that consists of the expression cassette of the invention for transforming yeast is preferably integrated into the yeast genome by a recombination replacement technique. The expression vector of the invention is digested with an appropriate enzyme to produce a linear DNA fragment whose ends are homologous to the AOXl locus. As a result of the gene replacement, Mut strains are obtained. In Mut strains, the AOX 1 gene is replaced by the expression cassette of the invention and therefore the ability to use methanol in this strain is decreased. A slow speed of growth is maintained with methanol due to the expression of the A0X2 gene product. Furthermore, the selected cells are screened for their Mut genotype by growing them in the presence of methanol and recording the speed of growth or by using PCR. In the development of Mut+ strains that express the collagen-like polypeptide of the invention, a fragment containing one or more expression cassettes of the invention is preferably integrated into the genome of the host by transforming the host with a circular or linear plasmid containing the cassette.

A person skilled in the art will be able to select and/or appropriately modify a suitable expression system for the production of a recombinant collagen-like polypeptide according to the invention from systems such as, but not limited to, E. coli, yeast, mammalian cell lines, insect cells, as well as transgenic animals and plants. A person skilled in the art will be cognisant that certain expression systems will have their own disadvantages. For example, E. coli expression system has no post-translational modification, and wild-type yeast expression system lacks P4H activity. Mammalian cell line expression system has low yield and limits to specific tissue types, and insect cell expression system has low P4H activity.

Method for the production of the collagen-like polypeptide of the invention The exact method of recovery of the expressed recombinant collagen-like polypeptide of the invention will depend on the host cell and the expression construct. In many microbial host cells, the collagen will be trapped within the cell wall of the recombinant host cell, even though it has been transported out of the cytoplasm. In this instance, the host cells are preferably disrupted to recover the collagen. Alternately, cell walls may be removed or weakened to release the protein of the invention located in the periplasm. Disruption can be accomplished by any means known in the art, including sonication, microfluidization, lysis in a French Press or similar apparatus, disruption by vigorous agitation/milling with glass beads, or lysis of osmotically fragile mutant yeast strains and the like. Where the collagen-like polypeptide of the invention is recovered by lysis or disruption of the recombinant host cells, the lysis or disruption is preferably carried out in a buffer of sufficient ionic strength to allow the collagen to remain in soluble form (e.g., more than 0.1 M NaCl, and less than 4.0 M total salts including the buffer). Alternately, in higher eukaryotic cells or microbial cells having mutations which render the cell wall "leaky", the protein of the invention is recovered by collection of the culture medium.

Recovered collagen- like polypeptide of the invention may be further purified. As with recovery, the method of purification will depend on the host cell and the expression construct. Generally, recovered solutions can be clarified (if the protein of the invention is recovered by cell disruption or lysis). Clarification is generally accomplished by centrifugation, but can also be accomplished by sedimentation and/or filtration if desired. The collagen-like polypeptide of the invention-containing solution can also be delipidated when the solution contains substantial amounts of lipids (such as when the protein is recovered by cellular lysis or disruption). Delipidation is accomplished by, for example, the use of an adsorbant such as diatomaceous earth or diatomite such as that sold as CELITE 512. When diatomaceous earth or diatomite is utilized for delipidation, it is preferably prewashed before use, and then removed from the delipidated solution by filtration.

Protein purification can be accomplished by any purification technique(s) known in the art. Proteins recovered can be purified by a variety of commonly used methods, including, but not limited to, ammonium sulfate precipitation, differential NaCl precipitation, PEG precipitation, immuno precipitation, ethanol or acetone precipitation, acid extraction, ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography, electrophoresis, and ultra filtration. If required, protein refolding systems can be used to complete the configuration of the protein. Protein solubility can be manipulated by alterations in buffer ionic strength and pH. Collagen produced in accordance with the invention may be induced to: precipitate at high ionic strengths; dissolve in acidic solutions; form fibrils (by assembly of trimeric monomers) in low ionic strength buffers near neutral pH (i.e., about pH 6 to 8), thereby eliminating proteins which do not precipitate at high ionic strength; resolubilize in acidic solutions; and become insoluble in low ionic strength buffers, respectively. Any one of these manipulations may be used, singly or in combination with others to purify collagen of the invention. Additionally, solubilised protein can be purified using any conventional purification techniques known in the art, including gel filtration chromatography, ion exchange chromatography (generally cation exchange chromatography to adsorb the collagen to the matrix, although anion exchange chromatography may also be used to remove a contaminant from the collagen-containing solution), affinity chromatography, hydrophobic interaction chromatography, and high performance liquid chromatography (Miller & Rhodes, 1982, Meth. Enzymol. 82:33-64).

Preferably, collagen-like polypeptide produced in accordance with the present invention will be purified using a combination of purification techniques, such as precipitation, solubilisation and ion exchange chromatography followed by fibril formation. Recombinant collagen-like polypeptide produced in accordance with the method of the invention may also be purified from the host cell culture by techniques including standard chromatographic and precipitation techniques (Miller & Rhodes, 1982 Meth. Enzmol. 82:33-64). For collagens, pepsin treatment and NaCl precipitation at acid and neutral pH may be used (Trelstad RL et al., 1976 Anal Biochem. Mar 71(1): 114-8.). Immunoaffinity chromatography can be used for constructs that contain appropriate recognition sequences, such as the Flag sequence which is recognised by an Ml or M2 monoclonal antibody, His-tagged sequences recognised by antibodies on Ni complexes, or a triple helical epitope, such as that recognised by the antibody 5D8/B1 (Glattauer V et al., 1997 Biochem J. Apr 1;323 (Pt l):45-9.) or 5D8/09 (Werkmeister et al., 1990, Eur. J. Biochem., 187: 439 - 443).

Biomaterial, biomedical devices etc

Preferably, the biomaterial, medical device, tissue construct, or therapeutic product comprising the recombinant collagen- like polypeptide of the invention is a wound repair material, bulking agent, scaffold material, for example the bulking material is a tissue augmentation material or the like. It is specifically contemplated that in some embodiments, the biomaterial is a biomaterial selected from the group consisting of sponges; matrices; membranes; sheets; implants; scaffolds; barriers; stents; grafts, e.g., a tissue graft; sealants, e.g., vascular sealants, tissue sealants, etc.; corneal shields; artificial tissues, e.g., artificial skin; hemostats; bandages; dressings, e.g., wound dressings; coatings, e.g., stent coatings, graft coatings, etc.; adhesives; sutures; and drug delivery devices. It is further contemplated that these biomaterials can be used in various applications and procedures, including, but not limited to, the following: tissue engineering, tissue augmentation, guided tissue regeneration; drug delivery; various surgical procedures including restorative, regenerative, and cosmetic procedures; vascular procedures; osteogenic and chondrogenic procedures, cartilage reconstruction, bone graft substitutes; hemostasis; wound treatment and management; reinforcement and support of tissues; incontinence; etc.

In various embodiments, the biomaterial is human collagen, recombinant collagen, recombinant human collagen, non-human collagen, recombinant non- human collagen or any combination thereof, respectively.

In some embodiments, the collagen is selected from the group consisting of collagen type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII, type XXVIII and type XXIX. The collagen can be collagen of one type free of any other type, or can be a mixture of collagen types.

The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variation in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or other reference were specifically and individually indicated to be incorporated by reference.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not imitative of the remainder of the disclosure in any way whatsoever.

EXAMPLES

Example 1: Construct Preparation

The manipulation of collagen type I and III genes into the Pichia expression plasmid is a standard procedure, as outlined in Molecular Cloning, A Laboratory Manual, Third Edition (Sambrook, Fritsch and Maniatis).

Initially, IOng of c-DNA encoding the collagen I and III genes was transformed into 50μl of the E.coli host strain, ToplO, from Invitrogen, using the heat shock method at 42°C as outlined by Maniatis referenced above (pg 1.118). After heat shock, the cells were left to recover at 37°C in the presence of yeast tryptone media (YT) with gentle agitation for one hour. The transformation mixture was then plated onto ampicillin containing agar plates and grown overnight at 37°C. Colonies resistant to ampicillin were recovered and grown overnight in 15OmIs of YT media. Midi prep plasmid stock preparations were carried out using the HiSpeed Plasmid Midi Kit from Qiagen. Finally the purified DNA was resuspended in 200μl of buffer TE (Tris EDTA) and stored at 4°C.

Restriction digests of the parental clones were carried out in lOμl reaction volumes. 1 μg of parental plasmid DNA was digested using 1 μl of the appropriate New England BioLab (NEB) buffer (1Ox stock), lμl of bovine serum albumin (BSA) (10x stock) and lμl of the specific restriction enzyme (lOunits/μl) from NEB. The digestions took place at 37 0C for one hour and were then analyzed on 1% agarose gel electrophoresis. The gel was stained in an ethidium bromide bath for five minutes and collagen bands were excised using a scalpel blade. The DNA from the gel slices were purified using the Qiagen Mini Elute Reaction Cleanup Kit. This purified insert was then used in the ligation procedure.

Vector plasmid preparations and restriction digests were carried out as described for the parental constructs.

Cloning vectors (e.g. pPICZ-(C)) were digested with the appropriate NEB buffer for one hour at 37°C and were subsequently de-phosphorylated using lμl of Shrimp Alkaline Phosphatase (lunit/μl), from GE Healthcare Bio-Sciences, at 37°C for one hour. The enzyme was inactivated at 65°C for 15 minutes. This vector preparation was then used in the ligation procedure. Vector and purified insert preps were subsequently ligated together using the T4 DNA ligase kit (Invitrogen). Ligations were carried out in lOμl volumes at 12°C overnight. Reactions were set up containing three times the amount of insert to vector along with lμl of ligase buffer (5X) and lμl of enzyme. The ligation mixture was then transformed into Top 10 cells and plated onto ampicillin selective media.

Colony PCR was then used to detect clones that contained the engineered chimeras. Colonies were picked from the selective plates and swirled in a mixture of Go Taq Green PCR Master Mix (Promega) containing 5' and 3' specific oligos, and PCR analyzed using an Applied Biosystems 2720 thermal cycler. The conditions used were 95°C for 5 minutes initially, followed by 25 cycles at 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute/kb of plasmid length. PCR products containing potentially engineered clones were analyzed on 1% agarose electrophoresis.

Human collagen type I, alpha I c-DNA with ATCC accession number 95498 and human collagen type III, alpha I c-DNA with ATCC accession number 95502 were used in the construct design and engineering of the chimeras (constructs 1-21).

The 4.5kb collagen I gene insert was sub-cloned from its parental vector pUC19 using the restriction sites Xbal (5') and Sspl (3') and was cloned into the bacterial shuttle vector pBluescript II KS+ (Stratagene) using sites Xbal (5') and Smal (3'). This cloning allowed the internal BamHI site in collagen I, at base pairs 2929-2934, to act as a unique site in this vector.

For ease of collagen manipulation, two truncations of collagen I (N and C) terminal were constructed. The N terminal truncation contained a 2.7kb fragment of collagen cloned into pBluescript II KS+ at sites Xbal (5') and BamHI (3'), whilst the C terminal truncation, 1.8kb in size was sub-cloned into the shuttle vector pUC19 (New England Biolabs) using restriction sites BamHI (5') and HindIII (3'). To the C terminal truncation, an Nael restriction site (silent mutation) was introduced using the QuickChange II Site -Directed Mutagenesis Kit (Invitrogen) at base pairs 3706-3711 of the C telopeptide, to aid in the cloning of the collagen I-III overlaps. The mutagenesis was carried out in a 50μl reaction volume containing 5μl of Pfu buffer (1Ox) and 5' and 3' mutagenic primers at 125ng/μl. 50μgs of parental DNA was added to the mutagenesis along with lμl of 1OmM dNTPs and Pfu Turbo. 16 cycles were performed in the thermal cycler at temperatures 95°C for 30 seconds, 55°C for one minute and 68°C for lminute/kb of plasmid length. A Hindi site (GTCAAC) was added directly after the collagen I stop codon for ease of cloning of the full length chimeras into the Pichia vector system. To the N terminal truncation, PCR was used to introduce a kozak sequence (ribosome binding site), (ACC) upstream of the initiating methionine residue.

Splice overlap PCR was used to interchange regions of the collagen I α helix with that of the collagen III α helix. The first overlap spanned base pairs 3288-3711 of the Coll I α helix and was interchanged with that of residues 3283-3708 of the Coll III α helix. The 5 ' overlap oligo contained an introduced BamHI site and the 3 ' oligo contained an introduced Nael site. The di-cysteine residues at the end of the Coll III α helix and start of the C-telopeptide were maintained in the overlap fragment. The 423 base pair overlap PCR product was cloned into the pTOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen) and sequence validated. The overlap was digested from pTOPO with BamHI (5') and Nael (3') and was interchanged with the wild type (WT) C terminal clone of collagen I containing the introduced Nael site in pUC19.

The second overlap was shorter and spanned the collagen I helix from residues 3397- 3711 and was interchanged with collagen III helix residues 3370-3708. The 338 base pair PCR overlap fragment was cloned into pTOPO and sequence validated. It too was cloned into pUC19 as above.

The two overlap constructs in pUC19 were digested with BamHI (5') and HindIII (3') and were sub-cloned into these sites using the pBluescript II KS+ collagen (N) construct. This cloning resulted in the full length 4.5kb collagen chimera being cloned into the shuttle vector pBluescript II KS+. The chimeras were sub-cloned from the shuttle vector using Notl (5') and Hindi (3') and cloned into the pPICZ-C (Invitrogen) Pichia expression vector using sites Notl (5') and SnaBI (3')- (constructs 1 and 2). The other chimeric constructs designed were variants of construct 1 and 2.

Removal of the N-propeptide from collagen I, residues 193-609, (constructs 3 and 4) was performed using deletion mutagenesis on the N terminal truncated construct. The gene was sequenced to ensure deletion of the appropriate residues and then cloned into the C terminal sub-fragment of collagen to create the full length gene lacking the N- propeptide which was cloned into pP ICZ-(C). To delete the signal peptide from collagen I, (constructs 5 and 6) residues 129-192 were deleted using site specific mutagenesis on the truncated N terminal fragment of collagen I. Once the signal peptide was confirmed to be deleted via sequencing, the N terminal truncation was ligated to the C terminus using sites Xbal (5') and BamHI (3'). The full length constructs (4.4kb) lacking the signal peptide was then cloned into the pPICZ-(C) expression vector.

Chimeras were also designed to have the collagen I C-propeptide interchanged with that of the collagen III C-propeptide (constructs 7 and 8). Splice overlap PCR was used to introduce the Coll III C-propeptide region into Coll I using the restriction sites BamHI (5') and HindIII (3'). This full length cassette was cloned into pPICZ-(C).

To generate a HIS taged chimera (construct 9), the C terminal truncation was used. The stop codon of collagen I (TAA) was changed to a glycine (GGC) using the mutagenesis strategy. The C terminal truncation was ligated to the N terminal collagen fragment and the full length gene in pBluescript II KS+ was sub cloned into pP ICZ-(C). Mutagenesis of the stop codon allowed utilization of the HIS tag present at the C terminal end of the multiple cloning site of the pPICZ-(C) vector.

Thrombin cleavage sites, either N, C or N and C terminal (constructs 10-15) were also introduced into the chimeras. 18 residues encoding thrombin were introduced at the N or C terminal end of the collagen truncations using mutagenesis. Sequences were confirmed and N and C terminal fragments containing the introduced cleavage sites were ligated together or independently into pBluescript II KS+ and then into pPICZ- (C).

To introduce a biologically active component into the chimeras (constructs 16-19), full length epidermal growth factor, 53 amino acids, were cloned into the construct lacking the N-propeptide. A 159 base pair fragment was PCR'ed up from the pTOPO-EGF construct and cloned in the region of the Coll I N-propeptide using restriction sites Xbal (5') and Avrll (3'). PCR mutagenesis was used to remove the Coll I signal sequence from this construct which was then sub-cloned into the pPICZ-(C) vector.

A chimera was designed to contain two regions of the Coll III α helix integrated into the helical domain of Coll I (construct 20). Construct 1 was used as the backbone for the design of this chimera. Residues 628-1050 of the Coll III α helix were exchanged for residues 660-1039 of the Coll I α helix using sites Agel (5') and Avrll (3'). This construct cassette was sub-cloned into pPICZ-(C).

An extended length chimera was also designed (construct 21) containing an α helical region of 5.2kb. Construct 1 was used as the base for this chimera design. A Coll III helical fragment, base pairs 801-3709 were PCR'ed up containing a BamHI site (5') and an Nael site (3') and this PCR fragment was cloned into construct 1 using these sites at residues 2934 and 3711 of the Coll I α helix. This extended cassette was then cloned into pPICZ-(C).

A summary of the various construct designs are provided in Table I below.

C/)

C DO CΛ

m

C/)

I m m 4^

O

C |— m

Figure imgf000041_0001

N in the N-propeptide column refers to constructs where the N-propeptide of Coll I was deleted.

Constructs 5 & 6 are based on constructs 1 & 2 but lacking a signal peptide sequence.

Construct 9 is based on construct 3 but including a His terminal tag at the C terminal end of the construct

Example 2: Expression of chimera in Pichia pastoris

Construct 9 cloned in the pP ICZ-(C) expression vector was transformed into the Pichia pastoris strain, GS200, along with the enzyme prolyl 4-hydroxylase P4H (α and β subunits). Colonies were grown at 300C in buffered minimal glycerol media until an OD600 of 3 was reached. At this point a T=O sample was taken (ImI). The cells were spun down and induction was started using methanol media. The pPICZ-(C) vector contains an alcohol oxidase (AOXI) promoter that is tightly regulated and induced by methanol. Buffered minimal methanol media containing 0.5% methanol and 80μg/ml of ascorbic acid was replenished daily at 200C. ImI samples were taken (T=I-I l), spun down and the protein was extracted from the cells using lysis buffer (Roche) containing 8M urea and glass beads. The presence of thel65kDa band which was detected via Western analysis using the anti-HG antibody (CSIRO - in house antibody) indicates that the chimeric protein was produced.

The lysates can then be further purified using a HIS column from Qiagen to remove any contaminating proteins from the collagen preparation. The HIS tag could then be enzymatically removed resulting in a final pure prep of synthetic collagen that could potentially be used in medical or cosmetic applications.

Claims

CLAIMS:
1. A recombinant collagen-like polypeptide of the formula (1):
Aa-Bb-Cc-Dd-Ee-F^Gg-Hh
1 wherein:
A is a biologically active component (BAC), which may include a selection marker for identification or purification;
B is a trimer forming domain which may be selected from, for example, an N- propeptide or fragment or variant thereof, or other sequences, including a coiled-coil trimer forming sequence; C is a cleavage site;
D is an N-telopeptide or fragment or variant thereof; E a triple helical forming domain comprising I and J: (i) wherein I is selected from at least one fragment or variant of at least one triple helical forming sequence; and
J is selected from at least one full length or one fragment or one variant of a triple helical forming sequence, wherein when I and/or J comprises more than one fragment or variant, said fragments or variants may be contiguous or non-contiguous; and wherein I and J are not identical nor derived from an identical triple helical forming sequence; or (ii) wherein I is selected from a full length triple helical forming sequence or a fragment or variant thereof; and J is selected from a full length triple helical forming sequence or a fragment or variant thereof, and wherein I and J are not identical nor derived from an identical triple helical forming sequence;
F is a C-telopeptide or a fragment or variant thereof; G is a cleavage site; and
H is a trimer forming domain comprising a C-propeptide or a fragment or variant, or a coiled-coil forming sequence, which may include a selection marker for identification or purification; and wherein: a = 0 or 1; b = 0 or 1; c = 0 or 1; d = 0 or l; e = l; f = 0 or 1, preferably 1; g = 0 or 1 ; and h = 0 or 1 , preferably 1.
2. A polypeptide according to claim 1, wherein I and J are eukaryotic sequences.
3. A polypeptide according to claim 1 or 2, wherein I and J are each sequences derived from a polypeptide chain which forms part of a collagen or collagen-like protein.
4. A polypeptide according to claim 3, wherein the polypeptide chain is an alpha chain polypeptide.
5. A polypeptide according to claim 3, wherein the collagen is selected from the group consisting of human collagen, recombinant collagen, recombinant human collagen, non-human collagen, recombinant non-human collagen.
6. A polypeptide according to any preceding claim, wherein I and J are each sequences derived from a polypeptide chain which forms part of a protein selected from the group consisting of collagen type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII, type XXVIII and XXIX preprocollagen, procollagen, a collagen-like protein, a V-domain of a bacterial collagen, V-domain like, foldon or foldon-like, conglutinin or congultinin-like, or any other protein that has this functional characteristic.
7. A polypeptide according to any preceding claim, wherein E is an intrachain chimera comprising I and J, wherein I is derived from an α chain polypeptide sequence of collagen type I (for example, αl[I]) and J is derived from an α chain polypeptide sequence of collagen type III (for example CCl[III]), or vice versa.
8. A polypeptide according to claim 1, wherein I and/or J is a sequence derived from a protein selected from the group consisting of CIq, macrophage scavenger receptor and lung surfactant protein.
9. A polypeptide according to any preceeding claim, wherein the combined length of I and J is about 30 -200% of an interstitial (fibril forming) collagen.
10. A polypeptide according to claim 1, wherein at least one of a, b, c, d, f, g and h is 1, at least two of a, b, c, d, f, g and h are 1, at least three of a, b, c, d, f, g and h are 1, at least four of a, b, c, d, f, g and h are 1, at least five of a, b, c, d, f, g and h are 1, at least six of a, b, c, d, f, g and h are 1, all of a, b, c, d, f, g and h are 1, or all of a, b, c, d, f, g and h are 0. For example, of a, b, c, d, f and g: at least a is 1; at least a and b are 1; at least a, b and c are 1; at least a, b, c and d are 1; at least a, b, c, d and f are 1; at least a, b, c, d, f and g are 1 ; at least a, b, c, d and g are 1 ; at least a, b, c and f are 1 ; at least a, b, c, f and g are 1; at least a, b, c and g are 1; at least a, b and d are 1; at least a, b, d and f are 1; at least a, b, d, f and g are 1; at least a, b and g are 1; at least a and c are 1; at least a, c and d are 1 ; at least a, c, d and f are 1 ; at least a, c, d, f and g are 1 ; at least a and g are 1; at least b are 1; at least b and c are 1; at least b, c and d are 1; at least b, c, d and f are 1; at least b, c, d, f and g are 1; at least b, c, d and g; at least c is 1; at least c and d are 1 ; at least c, d and f are 1 ; at least c, d, f and g are 1 ; at least d is 1 ; at least d and f are 1 ; at least d, f, g are 1 ; at least f is 1 ; at least f and g are 1 ; or at least g is 1. For further example, of a, b, c, d, f and g: at least a is 0; at least a and b are 0; at least a, b and c are 0; at least a, b, c and d are 0; at least a, b, c, d and f are 0; at least a, b, c, d, f and g are 0; at least a, b, c, d and g are 0; at least a, b, c and f are 0; at least a, b, c, f and g are 0; at least a, b, c and g are 0; at least a, b and d are 0; at least a, b, d and f are 0; at least a, b, d, f and g are 0; at least a, b and g are 0; at least a and c are 0; at least a, c and d are 0; at least a, c, d and f are 0; at least a, c, d, f and g are 0; at least a and g are 0; at least b is 0; at least b and c are 0; at least b, c and d are 0; at least b, c, d and f are 0; at least b, c, d, f and g are 0; at least b, c, d and g; at least c is 0; at least c and d are 0; at least c, d and f are 0; at least c, d, f and g are 0; at least d is 0; at least d and f are 0; at least d, f, g are 0; at least f is 0; at least f and g are 0; or at least g is 0.
11. A polypeptide according to claim 1, wherein the BAC is an antibiotic, a fungicide, an anti-viral agent, an anti-tumour agent, a cardiovascular agent, an antianxiety agent, a hormone or hormone mimetic, a growth factor, a toxin, an antihypertensive, immunomodulating, a cytokine, an opiod-like, an anti-infective, an anti- inflammatory peptide/protein or a peptide/protein comprising a linkage site.
12. A polypeptide according to claim 1, wherein the cleavage site in an enzymatic cleavage site.
13. A nucleic acid molecule encoding the recombinant collagen-like polypeptide of the invention. The nucleic acid molecule may be a DNA molecule or RNA molecule, preferably a cDNA molecule.
14. A nucleic acid according to claim 13, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs 1 to 21.
15. A nucleic acid according to claim 13, wherein the nucleic acid molecule comprises a sequence which encodes a protein or peptide sequence selected from the group consisting of SEQ ID NOs 24 to 35.
16. An expression vector comprising the nucleic acid molecule according to any one of claims 13 to 15.
17. An expression vector comprising a sequence selected from the group consisting of SEQ ID NOs I to 21.
18. An expression vector comprising a sequence encoding a protein or peptide selected from the group consisting of SEQ ID NOs 24 to 35.
19. A host cell comprising the expression vector according to any one of claims 16 to 18.
20. A method of producing a recombinant collagen-like polypeptide according to claim 1, the method comprising: (i) introducing into a suitable host cell the expression vector according to any one of claims 16 to 18; and (ii) culturing the host cell under conditions suitable to express the recombinant collagen- like polypeptide.
21. A biomaterial, medical device, tissue construct, or therapeutic product comprising the recombinant collagen-like polypeptide according to claim 1.
22. Use of a recombinant collagen- like polypeptide according to claim 1 in the manufacture of a biomaterial, medical device, tissue construct, or therapeutic product.
23. A method of treating a condition selected from the group consisting of wound repair, tissue augmentation, treatment of burns, cosmetic treatment comprising administering to a subject a recombinant collagen- like polypeptide according to claim 1.
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WO2014146175A1 (en) * 2013-03-21 2014-09-25 Commonwealth Scientific And Industrial Research Organisation Purification of triple helical proteins
CN105143243A (en) * 2013-03-21 2015-12-09 国家科学和工业研究组织 Purification of triple helical proteins
JP2016514708A (en) * 2013-03-21 2016-05-23 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガナイゼーション Purification of the triple helix protein
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