WO2003087343A1

WO2003087343A1 - Production of stable collagens

Info

Publication number: WO2003087343A1
Application number: PCT/US2003/011252
Authority: WO
Inventors: Kari I. Kivirikko; Johanna Myllyharju
Original assignee: Fibrogen, Inc.
Priority date: 2002-04-11
Filing date: 2003-04-11
Publication date: 2003-10-23
Also published as: AU2003241288A1

Abstract

The present invention relates to stable collagens lacking at least a portion of the C-propetide, to methods of producing these collagens using foldon, and to constructs comprising sequences encoding collagen and foldon.

Description

PRODUCTION OF STABLE COLLAGENS

This application claims the benefit of U.S. Provisional Application Serial No. 60/372,099, filed 11 April 2002, incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION Recombinant proteins

Various recombinant and synthetic techniques provide polypeptides used in diverse research, industrial, pharmaceutical, and other applications. Such production methodologies allow for a reliable source of polypeptides in sufficient quantities for appropriate study and use, especially in cases where obtaining equivalent sources of naturally-derived proteins could be costly, due to the complexity of the extraction or derivation process; could be unethical, such as in the case of certain human proteins; could be associated with certain risks of infectivity or transmission of disease, such as in obtaining animal-derived proteins for use in various medical applications; or could be impractical, due to limited quantities of source material.

Recombinant production can take place in a controlled, stable, and reproducible environment, eliminating much of the variability associated with extraction of these materials from traditional animal sources. Recombinant techniques can reduce the risks of infectivity associated with use of materials extracted from animal sources, which can be due in part to the transmission of infectious agents present in source material, or to the introduction of various pathogens and endotoxins during the lengthy and complex extraction process.

In addition, extraction of proteins from animal sources can result in variability in the physical characteristics of the product material, from animal to animal, and extraction run to extraction run. Variation in the physical properties of extracted material, i.e., in the case of gelatin, for example, variations in molecular weights, etc., can correspond to variation in performance characteristics. This can mean that manufacturers or commercial users often need to blend various production lots and perform additional analysis prior to using the materials as intended. Recombinant production provides the opportunity for consistent production of reproducible material, minimizing variability in the properties and performance of the end product.

However, there are limitations to producing polypeptides in these recombinant and synthetic systems. For example, many polypeptides, including various animal polypeptides, undergo assembly into multimeric structures of varying complexity. Such high-level structure can be critical for essential functional characteristics of a polypeptide, including activity, stability- secretion, etc. Therefore, there is a need in the art for tools, systems, and methodologies that can be used to produce properly assembled and stable protein complexes in substantial amounts.

Collagens

Collagen is widely used in numerous applications in the medical, pharmaceutical, food, and cosmetic industries. Commercially available collagen is typically derived from animal sources using enzymatic and chemical processes. Most material today is isolated from bovine and porcine tissues and bones, and is comprised of a mixture of collagen types, primarily types I and III collagen.

Recombinant production of collagen has been described. (See, e.g., U.S. Patent No. 5,593,859, incorporated by reference herein in its entirety.) Use of recombinant collagens in various applications offers certain advantages over the use of non-recombinant, animal-derived materials in the same applications. The use of recombinant materials minimizes the risk of infectivity associated with use of animal-derived materials, making recombinant collagens attractive choices for use in various pharmaceutical, medical, and cosmetic applications. Furthermore, recombinant biomaterials can be derived from native human sequence, eliminating the risk of immunogenecity that can be associated with the use of bovine- and porcine-derived animal collagens and gelatins in various applications.

In addition to reducing the risks of immunogenecity and infectivity, recombinant collagens exist as a characterizable and reproducible material, distinguished from animal-derived collagens. Recombinant collagen can be produced as a consistent, single type material, or with pre-set percentages of different collagen types, resulting in material optimal for use in specific applications. (See, e.g., International Application No. PCT/USOO/30791, International Publication No. WO 01/34646, incorporated by reference herein in its entirety.)

In its native state, collagen is a triple helical protein composed of three separate α-chains. Collagen appears in heterotrimeric and homotrimeric forms, depending on collagen type. Type I collagen is typically a heterotrimeric complex containing two l(I) and one α2(I) chains, although some tissues contain trace amounts of α 1(1) homotrimer, while type III collagen is a homotrimeric complex containing three αl(III) chains in triple helical formation, etc.

Collagen α-chains possessing N- and C-propeptide regions are expressed and then assembled into trimeric complexes. The propeptide regions are enzymatically cleaved after formation of the triple helix. Collagen C-propeptide regions were long-thought to be essential to both trimeric assembly and triple-helical formation of native collagen. However, recent efforts have shown that trimeric assembly can be achieved to a limited degree in the absence of the C-propeptide domains. (See, e.g., Olsen et al. (2001) J Biol Chem 276:24038-24043.)

Specifically, Olsen et al. showed production of collagen from constructs lacking the C-propeptide region. However, the collagen triple helices produced from C-propeptide-lacking constructs demonstrated reduced stability. In particular, the collagen produced from C-propeptide-lacking constructs had a lower T_m of 30°C as compared to a T_m of 40°C for materials produced from constructs containing a C-propeptide region. (See, e.g., Olsen et ah, supra.) In addition, constructs lacking the C-propeptide region were unable to efficiently drive formation of type I collagen in correct heterotrimeric ratio. (See, e.g., Olsen et al., supra) Therefore, recombinant production of collagen from constructs lacking C-propeptide resulted in triple helical collagen molecules having structural and stability characteristics different from those of native collagen or from recombinant collagen produced from constructs containing C-propeptide.

h summary, collagen is a widely-used material that can be beneficially produced in recombinant form. As native collagen is subject to complex post-translational assembly and processing, there is a need in the art for simplified and direct recombinant production of substantial amounts of properly assembled collagens. There is a further need for production of recombinant collagens stable at biologically relevant temperatures. The present invention answers that need by providing methods for producing stable collagens and collagen-like proteins using foldon. Furthermore, the present invention provides methods for producing recombinant collagens in correct heterotrimeric or homotrimeric form. SUMMARY OF THE INVENTION

The present invention provides stable triple-helical collagens lacking the C-propeptide, methods for producing these collagens, and constructs for use in these methods. In one aspect, the present invention encompasses a method for producing a collagen triple helix, the method comprising introducing into a host cell a polynucleotide comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression, trimerization, and triple helical formation; and recovering the collagen triple helix. In one aspect, the collagen polypeptide is an animal collagen polypeptide. In a preferred aspect, the collagen polypeptide is a human collagen polypeptide. In further aspects, the collagen polypeptide is a bovine or a porcine collagen polypeptide.

In various aspects, the collagen polypeptide is a polypeptide selected from the group consisting of type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XH, 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, and type XXVII collagen.

hi some embodiments, the present methods comprise introducing into a host cell a polynucleotide comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking the entire C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression, trimerization, and triple helical formation; and recovering the collagen triple helix. Methods wherein the collagen polypeptide further lacks at least a portion of the N-propeptide or the entire N-propeptide are also contemplated.

The methods of the present invention can be applied to produce collagen at increased levels, and at an enhanced rate of trimeric assembly and triple helix formation.

In certain embodiments, the present methods comprise introducing into a host cell a polynucleotide comprising a sequence encoding a collagen polypeptide or a fragment or variant tliereof lacking the entire C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression, trimerization, and triple helical formation; removing foldon from the collagen triple helix; and recovering the collagen triple helix, h specific embodiments, pepsin is used to remove foldon.

In various embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In certain embodiments the host cell is selected from the group consisting of a yeast cell, a plant cell, a fungal cell, an insect cell, a bacterial cell, and a mammalian cell.

Methods for producing collagen, the methods comprising introducing into a host cell a polynucleotide comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression and trimerization; and recovering the collagen, are specifically contemplated, h some specific aspects, the collagen is homotrimeric; In other specific aspects, the collagen is heterotrimeric.

In one embodiment, the present invention encompasses a method for producing a collagen polypeptide, the method comprising introducing into a host cell a polynucleotide comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression; and recovering the collagen polypeptide. h one embodiment, the collagen polypeptide is an animal collagen polypeptide. In a preferred embodiment, the collagen polypeptide is a human collagen polypeptide. In further embodiments, the collagen polypeptide is a bovine or a porcine collagen polypeptide. h various embodiments, the collagen polypeptide is a polypeptide selected from the group consisting of type I, type II, type III, type IN, type V, type VI, type VII, type VIII, type LX, 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, and type XXVII collagen.

The present invention further provides methods for producing collagen heterotrimers. In one embodiment, the methods comprise introducing into a host cell a first polynucleotide comprising a first sequence encoding a first collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; introducing into the host cell a second polynucleotide comprising a second sequence encoding a second collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression and trimerization; and recovering the collagen heterotrimer.

In specific embodiments, the collagen heterotrimer is selected from the group consisting of type I collagen, type IV collagen, type V collagen, and type VIII collagen. In certain embodiments, the first polynucleotide encodes a polypeptide selected from the group consisting of α 1(1), αl(IV), αl(V), and αl(VHI) collagens, and the second polynucleotide is selected from the group consisting of α2(I), α2(IV), 2(V), and α2(VIII) collagens.

In a further embodiment, the present invention encompasses a method for producing a collagen heterotrimer, the method comprising introducing into a host cell a first polynucleotide comprising a first sequence encoding a first collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; introducing into the host cell a second polynucleotide comprising a second sequence encoding a second collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; introducing into the host cell a third polynucleotide comprising a third sequence encoding a third collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression and trimerization; and recovering the collagen heterotrimer.

In specific embodiments, the collagen heterotrimer is selected from the group consisting of type V collagen, type VI collagen, type IX collagen, and type XI collagen. In certain embodiments, the first polynucleotide encodes a polypeptide selected from the group consisting of αl(V), αl(VI), αl(IX), and αl(XI) collagens, the second polynucleotide encodes a polypeptide selected from the group consisting of 2(V), α2(VI), α2(IX), and α2(XI) collagens, and the third polynucleotide encodes a polypeptide selected from the group consisting of α3(V), α3(VI), α3(IX), and α3(XI) (i.e., αl(II)) collagens. Cross-type heterotrimers, e.g., type V and type XI heterotrimers are specifically contemplated, as are heterotypic collagens not found in nature. The present invention specifically provides methods for producing type I collagen. In a particular embodiment, the method comprises introducing into a host cell a first polynucleotide comprising a first sequence encoding o ( ) collagen or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; introducing into the host cell a second polynucleotide comprising a second sequence encoding α2(I) collagen or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; culturing the host cell under conditions suitable for expression and trimerization; and recovering the type I collagen. In a further embodiment, the type I collagen has an αl(I)/α2(I) ratio of 2:1.

The invention additionally contemplates a construct comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof. In various aspects, the collagen polypeptide is a polypeptide selected from the group consisting of type I, type II, type III, type IN, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type Xiπ, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXπ, type XXIII, type XX1N, type XXV, type XXVI, and type XXVII collagen. Host cells comprising the constructs of the invention are specifically provided. In various embodiments, the host cell is a eukaryotic cell or a prokaryotic cell, hi certain embodiments, the host cell is selected from the group consisting of a yeast cell, a plant cell, a fungal cell, an insect cell, a bacterial cell, and a mammalian cell. In specific embodiments, the invention contemplates a transgenic plant comprising a host cell of the present invention. Transgenic animals comprising a host cell of the present invention are also contemplated.

In one aspect, the constructs of the present invention express collagen at increased levels, and at an enhanced rate of trimeric assembly and triple helix assembly.

Collagens and collagen polypeptides are provided herein. In one aspect, the collagen polypeptide of the present invention comprises a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, fused to foldon or an active fragment thereof. Collagens having stability at biologically relevant temperatures are specifically provided. In one aspect, the present invention provides a collagen-foldon fusion protein comprising at least one triple helical domain and lacking at least a portion of the C-propeptide, wherein the polypeptide is stable at temperatures above 35°C.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows representation of various recombinant collagen polypeptide chains expressed in

Pichiapastoris.

Figures 2A, 2B, and 2C show western blot (Figures 2A and 2C) and Coomassie Blue staining (Figure 2B) analysis demonstrating expression and pepsin-resistance of various recombinant collagen αl(I) homotrimer polypeptides expressed with or without C-propeptide or foldon.

Figures 3 A and 3B show western blot (Figure 3A) and Coomassie Blue staining (Figure 3B) analysis demonstrating expression and pepsin-sensitivity of various recombinant collagen αl(III) polypeptides expressed with or without C-propeptide or foldon.

Figures 4A, 4B, and 4C set forth data demonstrating thermal stability of recombinant human type I collagen homotrimers expressed with or without C-propeptide or foldon.

Figure 5 sets forth data showing assembly of recombinant human type I collagen heterotrimers expressed with C-propeptides or foldon.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, constructs, and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

It must be noted that as used herein, and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a host cell" is reference to one or more of such host cells and equivalents thereof known to those skilled in the art, and reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the meanings as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies, etc., which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Each reference cited herein is incorporated herein by reference in its entirety.

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, 18^th ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, Vols. 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, 2^nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4^th 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).

Definitions

The term "collagen" refers to any one of the known collagen types, including collagen types I through XXVII, as well as to any other collagens, whether natural, synthetic, semi-synthetic, or recombinant. Some collagens (e.g., collagen types I, II, III, etc.) are, in nature, first produced as precursor molecules, called procoUagens, 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 procoUagens. 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)_π or (Xaa-Pro-Gly)_n domain.

The term "procollagen" refers to a procollagen conesponding to any collagen, whether natural, synthetic, semi-synthetic, or recombinant, that possesses additional C-terminal and/or N-terminal propeptides. 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 or (Xaa-Pro-Gly)_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.

The terms "homotrimer" or "homotrimeric collagen" refer to collagen or procollagen having three each of the same collagen or procollagen -chain. Homotrimeric collagens include, e.g., collagen types I, IL ILL V, VII, X, XII, XIII, XIV, XVI, etc.

The terms "heterotrimer" or "heterotrimeric collagen" refer to collagen or procollagen containing a least two different collagen or procollagen -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 α-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, VIII, IX, XI, etc. Heterotrimeric collagens additionally include cross-type heterotrimeric collagens, e.g., type V, type XI, etc.

The terms "trimerization" and "trimeric assembly" refer to the association of three individual polypeptides, e.g.., collagen polypeptides, α-chains, etc. Trimerization or trimeric assembly of collagen can be associated with collagen polypeptide association, registration, and selection. The term "collagen-like polypeptide" includes any polypeptides having at least one collagen-like or collagenous domain. A "collagen-like domain" or "collagenous domain" encompasses any polypeptide domain having a repeating Gly-Xaa-Yaa or Xaa-Pro-Gly amino acid sequence and that is capable of forming a triple helix. A repeating Gly-Xaa-Yaa or Xaa-Pro-Gly amino acid sequence refers to at least two consecutive Gly-Xaa-Yaa or Xaa-Pro-Gly amino acid sequences. In Gly-Xaa-Yaa, at least one Y-position amino acids is preferably a proline. The collagen domain can be a substrate for prolyl 4-hydroxylase.

The term "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 refers to the ability of foldon to form trimers. In one aspect, foldon refers to the amino acid sequence of SEQ ID NO:l, or the amino acid sequence of SEQ ID NO:7, or any fragments or variants thereof having foldon activity.

"Altered" or "modified" 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 ohgonucleotide 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.

"Altered" or "modified" polypeptides may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent polypeptide. Deliberate amino acid substitutions maybe 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. The term "functional equivalent" as it is used herein 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.

"Amino acid" or "polypeptide" sequences or "polypeptides," as these terms are used herein, refer to oligopeptide, peptide, polypeptide, or protein sequences, and fragments thereof, and to naturally occurring or synthetic molecules. Polypeptide or amino acid fragments are any portion of a polypeptide which retains at least one structural and/or functional characteristic of the polypeptide. In at least one embodiment of the present invention, polypeptide fragments are those retaining at least one (Gly-Xaa-Yaa)_n or (Xaa-Pro-Gly)_π domain.

A "deletion" is a change in an amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

The terms "insertion" or "addition" refer to a change in a polypeptide or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.

The term "derivative" encompasses a molecule containing at least one structural and/or functional characteristic of the molecule from which it is derived.

A "fusion protein" is a protein in which peptide sequences from different proteins are operably linked.

A "substitution" is the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

A polypeptide or amino acid "variant" is an amino acid sequence that is altered by one or more amino acids from a particular amino acid sequence. A polypeptide variant may have conservative changes, wherein a substituted amino acid has similar structural or chemical properties to the amino acid replaced, e.g., replacement of leucine with isoleucine. A variant may also have nonconservative changes, in which the substituted amino acid has physical properties different from those of the replaced amino acid, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Preferably, amino acid variants retain certain structural or functional characteristics of a particular polypeptide. Guidance in detennining which amino acid residues may be substituted, inserted, or deleted may be found, for example, using computer programs well known in the art, such as LASERGENE software (DNASTAR Inc., Madison, WI).

A polynucleotide variant is a variant of a particular polynucleotide sequence that preferably has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence similarity to the particular polynucleotide sequence. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of variant polynucleotide sequences encoding a particular protein, some bearing minimal homology to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard codon triplet genetic code, and all such variations are to be considered as being specifically disclosed.

"Immunogenicity" relates to the ability to evoke an immune response within an organism. An agent displaying the property of immunogenicity is referred to as being immunogenic. Agents can include, but are not limited to, a variety of macromolecules such as, for example, proteins, lipoproteins, polysaccharides, nucleic acids, bacteria and bacterial components, and viruses and viral components. Immunogenic agents often have a fairly high molecular weight (usually greater than 10 kDa).

"Infectivity" refers to the ability to be infective or the ability to produce infection, referring to the invasion and multiplication of microorganisms, such as bacteria or viruses within the body.

The term "isolated" as used herein refers to a molecule separated not only from proteins, etc., that are present in the natural source of the protein, but also from other components in general, and preferably refers to a molecule found in the presence of, if anything, only a solvent, buffer, ion, or other component normally present in a solution of the same. As used herein, the terms "isolated" and "purified" do not encompass molecules present in their natural source. The terms "nucleic acid" or "polynucleotide" sequences or "polynucleotides" refer to oligonucleotides, nucleotides, or polynucleotides, or any fragments thereof, and to DNA or RNA of natural or synthetic origin which maybe single- or double-stranded and may represent the sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. Polynucleotide fragments are any portion of a polynucleotide sequence that retains at least one structural or functional characteristic of the polynucleotide. In one embodiment of the present invention, polynucleotide fragments are those that encode at least one (Gly-Xaa-Yaa)_n or (Xaa-Pro-Gly)_n region. Polynucleotide fragments can be of variable length, for example, greater than 60 nucleotides in length, at least 100 nucleotides in length, at least 1000 nucleotides in length, or at least 10,000 nucleotides in length.

The term "post-translational enzyme" refers to any enzyme that catalyzes post-translational modification of, for example, any collagen or procollagen. The term encompasses, but is not limited to, for example, prolyl hydroxylase, peptidyl prolyl isomerase, collagen galactosyl hydroxylysyl glucosyl transferase, hydroxylysyl galactosyl transferase, C-proteinase, N-proteinase, lysyl hydroxylase, and lysyl oxidase.

The term "purified" as it is used herein denotes that the indicated molecule is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. The term preferably contemplates that the molecule of interest is present in a solution or composition at least 80% by weight; preferably, at least 85% by weight; more preferably, at least 95% by weight; and, most preferably, at least 99.8% by weight. Water, buffers, and other small molecules, especially molecules having a molecular weight of less than about one kDa, can be present.

Invention

Existing art has taught that the C-propeptide region of the collagen molecule is required for correct assembly of trimeric complexes. (See, e.g., McLaughlin and Bulleid (1998) Matrix Biol 16:369-377.) Recent efforts to produce collagen lacking the C-propeptide region resulted in production of collagen helices lacking stability at biologically relevant temperatures. (Olsen et al, supra In Frank et al., synthetic unhydroxylated (GlyProPro)₁₀ peptides fused to the trimerizing agent foldon formed triple helices of higher thermal stability than that of triple helices formed by (ProProGly)₁₀ peptides alone. (See Frank et al. (2001) J Mol Biol 308:1081-1089.) However, the Frank et al. results did not extend to a demonstration that collagen triple helices could be produced and stabilized in reproducible and selective fashion using foldon as a trimerizing agent.

The present invention relates to the discovery that expression of a collagen polypeptide lacking at least a portion of the C-propeptide and fused to foldon resulted in a product capable of forming collagen trimers and triple helices with increased stability. Therefore, the present invention specifically provides collagen polypeptides lacking at least a portion of the C-propeptide domain, and further comprising foldon, which are thereby capable of forming stable triple helices. In a preferred embodiment, the collagens produced are stable at biologically relevant temperatures, i.e., having thermal stability of native collagen, or stable at temperatures appropriate for particular applications of collagen, such as, for example, at temperatures greater than 35°C or about 38-40°C. The foldon domains of the collagen polypeptides can be removed subsequent to trimer formation or triple helix formation without affecting the stability of the collagen triple helix.

In one aspect, the methods of the present invention include providing a construct or constructs encoding a collagen post-translational enzyme or subunit or active fragment thereof. In a preferred embodiment, the collagen post-translational enzyme is prolyl 4-hydroxylase (P4H), the α- or β- subunit of P4H, or any active fragment thereof. The post-translational enzyme can be selected from any species, preferably a mammalian species, and, most preferably, the human species. In certain embodiments, the post-translational enzyme is selected from the same species as the species from which the collagen is derived, e.g., human, h some embodiments, the post- translational enzyme is selected from the same species as that of the host cells/expression system containing the constructs of the present invention, e.g., plant.

The present invention further relates to the production of heterotrimeric collagen. The C-propeptide region of collagen is believed to be involved in the proper selection of α-chains for assembly into collagen triple helices. Previous efforts to produce heterotrimeric collagen from constructs lacking C-propeptide sequence resulted in formation of trimeric collagen having an unpredictable mixture of homotrimeric or heterotrimeric forms. For example, production of collagen from C-propeptide lacking constructs resulted in collagen type I triple helices having an overall αl(I)/α2(I) ratio of 5:1, as compared to the αl(I)/α2(I) ratio of 2:1 found in nature. (See, e.g., Olsen et al., supra, describing presence of type I heterotrimeric and homotrimeric (αl)₃ collagen, page 24041.) The present inventors have discovered that the use of foldon as a trimerizing agent led to the production of heterotrimeric type I collagen having an αl(I)/α2(I) ratio of 2:1. (See, e.g., Example 6.)

In one aspect, the present invention relates to the production of collagen lacking at least a portion of the N-telopeptide region or the C-telopeptide region. In a particular aspect, the present invention relates to the production of atelopeptide collagen, lacking both the N- and the C-telopeptide regions. It has been reported that the telopeptide regions of collagen are responsible at least in part for the immunogenicity of collagen used in certain medical and pharmaceutical applications. (See, e.g., Lindsley et al. (1971) J Exp Med 133:1309-1324.) Therefore, the atelopeptide collagens of the present invention could be particularly advantageous for use in minimizing the immunogenicity of the collagen material in applications such as injectable collagens for tissue augmentation, e.g., for cosmetic use, etc.

The present invention provides methods for producing collagens in which the sizable C-propeptide domain is replaced in part or in full with foldon or an active fragment thereof, dramatically reducing the size of the protein product. This can lead to increased yields in production, as well as increased secretability of the protein product. Further, the present invention provides for the production of modified collagens. In previous attempts to produce modified collagens, i.e., collagens derived from full-length collagen but different in sequence from native collagen, it was thought that the desired portions of the molecule must be expressed in concert with the propeptide and telopeptide regions in order to produce polypeptides capable of forming a triple helical molecule. (See, e.g., Arnold et al. (1998) J Biol Chem 273:31822- 31828.) As discussed, supra, the art has long held that — in order to get expression of a desired triple helical region of collagen — the encoding polynucleotide sequence must additionally comprise C-propeptide sequence, believed to provide nucleation/registration domains or activity necessary for initation of trimerization and triple helical formation. While Olsen et al. showed production of collagen from polynucleotides lacking the C-propeptide sequence, the resulting collagen product demonstrated reduced stability. The present methods, in contrast, can be applied to production of modified collagens or collagen cassettes capable of trimerization and formation of stable triple helices. In one embodiment, the present invention provides for the production of modified collagens in which specific regions of collagen exist in stable triple helical form. In particular, the present invention contemplates the production of collagens modified to reduce or increase specific activities, e.g., as desired to achieve specific biological effects. For example, modified collagens could be produced from collagen constructs encoding a modified collagen polypeptide lacking at least a portion of the C-propeptide in combination with foldon or an active fragment thereof. Regions of the collagen molecule associated with specific activities and/or functions could be deleted or copied in full or in part to produce a collagen lacking or with reduced specific activity, or additional copies of the specific region could be added to produce a collagen with enhanced activity, e.g., suitable to specific medical and pharmaceutical applications.

The present methods of producing collagens are advantageous in providing increased yields and enhanced trimeric assembly and triple helix formation. Specifically, the collagens of the present invention are able to assemble more rapidly post-expression, and are thus more quickly stabilized, e.g., within a host cell, expression system, etc. The present collagens are thus less sensitive to cellular proteases which degrade unassembled collagen chains. In particular, production of collagen using collagen-foldon constructs of the present invention resulted in levels of expression which were increased three-fold compared to levels of expression obtained with constructs encoding collagen containing the C-propeptide. (See, e.g., Example 4.)

Foldon is derived from the C-terminus of the polypeptide chains in the T4 bacteriophage protein fϊbritin, a three-stranded α-helical coiled-coil protein. Foldon appears essential for assembly of the fibritin molecule, and has been used as a trimerizing agent in the production of complex proteins such as, for example, human immunodeficiency virus type I gpl40. (See, e.g., Yang et al. (2002) J Virol 76:4634-4642.) The invention specifically contemplates the use of foldon or any active fragment thereof. Sequence corresponding to foldon is known (see, e.g., GenBank Accession Nos. AF158101 and AAD42679) and active fragments thereof can be identified using any of the number of conventional techniques available in the art. To date, about twenty-seven types of collagen have been identified. (See, e.g., Myllyharju and Kivirikko (2001) Ann Med 33:7-21; Pace et al. (2003) Matrix Biology (internet pre-publication); and The Extracellular Matrix FactsBook, 2^nd edition, (1998) Ayad et al, eds, Academic Press, San Diego, CA, incorporated by reference herein in its entirety). The collagens of the present invention can be derived from any species including, but not limited to, human, bovine, porcine, equine, rodent, chicken, ovine, and piscine species, or from non-vertebrate species. In a preferred embodiment, the collagen is mammalian collagen, e.g., human, bovine, porcine, ovine, equine, murine, rat, etc. In a most preferred embodiment, the collagen of the present invention is human collagen.

The methods of the present invention can be practiced, and the constructs of the present invention expressed, in any of the expression systems well-known in the art. Use of prokaryotic and eukaryotic expression systems is specifically contemplated herein. Such expression systems include, but are not limited to, bacteria, fungi, yeast, plant, and animal mammalian and non- mammalian expression systems. Use of the present methods and constructs in transgenic plants and transgenic animals is specifically contemplated. Recombinant expression systems and methods for recombinant expression are well known in the art. (See, e.g., Walden, R. (1999) Chapter 13, pp. 335-355, in Plant Biochemistry and Molecular Biology, 2^nd edition, Lea and Leegood, eds., John Wiley and Sons, Inc., Chichester, England; Short Protocols in Molecular Biology, 4^th edition (1999) Chapter 16, Protein Expression, pp. 16-1 to 16-108, Ausubel et al, eds., John Wiley and Sons, Inc., New York; and Sambrook et al. (1989) Chapter 16 and 17, Molecular Cloning, A Laboratory Manual, 2^nd edition, Irwin et al, eds., Cold Spring Harbor Laboratory Press, New York.)

The following examples explain the invention in more detail. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods that are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. EXAMPLES

Unless otherwise stated, the following materials and methods were used in the examples of the present invention.

Example 1. Collagen aid) Expression Constructs

In the present Examples, various collagen expression constructs were prepared and expressed in Pichia. pastoris yeast cells. P. pastoris host strain yJC300 (his4, arg4, adel) and expression vectors pBLADESX and pBLARGIX have been previously described. (See Cereghino et al. (2001) Gene 263:159-169.) The plasmid vectors pPIC3K, pPICZB, and pPICZαA were from INVITROGEN ™ Life Technologies (Carlsbad, CA). Recombinant P. pastoris yeast strains having a methanol utilization plus phenotype were generated by electroporation of the expression constructs according to the manufacturer's instructions.

P. pastoris yeast strains expressing the α- and β-subunits of human prolyl 4-hydroxylase were prepared as follows. A cDNA coding for the β-subunit of human prolyl 4-hydroxylase lacking the signal sequence and flanked by EcoRI restriction sites was cloned into pPICZαA in-frame with the Saccharomyces cerevisiae α mating factor (αMF) pre-pro sequence. (See Vuorela et al. (1997) EMBO J. 16:6702-6712.) The expression cassette encoding the αMF-β polypeptide was excised from pPICZαAβ by digestion with BamHI-BglϊI and subsequently cloned into pBLADESX. To generate a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase α β tetramers, pBLARGIXα (previously described inNokelainen et al. (2001) Yeast 18:797-806) and pBLADESXβ were linearized with Hindi and Spel, respectively, and co- transformed into P. pastoris yJC300 strain. The resulting P. pastoris strain was referred to as ArgαAdeβ,his-.

The C-propeptide of the αl type I chain of human procollagen (pCαl(I)) (GenBank Accession No. Z74615) was replaced with foldon-encoding sequence as described below. The amino acid sequence (Y1TEAPRDGQAYVRKDGEWVFLSTFLSPA) (SEQ ID NO.:7) comprises the C-terminal 29 amino acid residues of fibritin, encoded by nucleotides 92129-93592 of the enterobacteria phage T4 genome. (See GenBank Accession Nos. AF158101 and AAD42679). A P. pastoris expression cassette encoding human type I pC l chains (procollagen chains lacking the N-propeptide) was excised from pPICZBpCαl(I) (Vuorela et al., supra) by digestion with Pmel-Noil and cloned into pPIC3K to generate pPIC3KpCαl(I). A cDNA coding for the 29-amino-acid foldon domain of fibritin (see, e.g., Tao et al (1997) Structure 5:789-798; Letarov et al. (1999) Biochemistry (Moscow) 64:403-434; and Boudko et al. (2002) Eur J Biochem 269:833-841) was generated by annealing the ohgonucleotide Foldon5'-l (5 '-AGCTTTATATTCCTGAAGCTCCAAGAGATGGGCAAGCTTACGTTCGTAA-3 ') (SEQ ID NO.:2) with the ohgonucleotide Foldon5'-2

(5'-CCATCTTTACGAACGTAAGCTTGCCCATCTCTTGGAGCTTCAGGAATATAA-3') (SEQ ID NO.:3), and by annealing the ohgonucleotide Foldon3'-l (5'-AGATGGCGAATGGGTATTCCTTTCTACCTTTTTATCACCAGCATAAGC-3') (SEQ ID NO.:4) with the ohgonucleotide Foldon3'-2

(5'-GGCCGCTTATGCTGGTGATAAAAAGGTAGAAAGGAATACCCATTCG-3') (SEQ ID NO.:5). These oligonucleotides (obtained from INVITROGEN ™ Life Technologies) were designed such that HindTTl and Notl overhangs (underlined in the polynucleotide sequences above) were created at the 5'- and 3 '-ends, and cohesive overhangs at the 3 '- and 5 '-ends of the annealed Foldon5' and Foldon3' fragments, respectively. The resulting foldon DΝA fragments were co-ligated into pBlueScript® phagemid vectors (Stratagene, La Jolla, CA) previously digested with HindTTl-Notl to generate pBSfoldon.

To replace the polynucleotide sequence coding for the C-propeptide of the pCαl(I) chain with that coding for foldon, two DΝA fragments were generated by PCR. The first DΝA fragment extended from an internal BamTTl site of the pC l(I) cDΝA to the codon for the last amino acid of the C-telopeptide, and the second DΝA fragment extended from the first codon of the foldon cDΝA to the Notl site following the stop codon. These fragments were co-ligated into pPIC3KpCαl(I) previously digested with i?αmHI-NotI to generate pPIC3Kαl(I)foldon. To delete the C-propeptide, a DΝA fragment extending from the internal BamΗI site of pCαl(I) cDΝA to the codon for the last amino acid of the C-telopeptide followed by a stop codon and a Notl site was created by PCR and ligated into pPIC3KpCαl(I) previously digested with BamEI- Notl to generate pPIC3Kαl(I). The pPIC3KpCαl(I), pPIC3Kαl(I)foldon, and pPIC3Kαl(I) constructs were subsequently linearized with Sail and transformed into the ArgαAdeβ,his- P. pastoris strain expressing recombinant human prolyl 4-hydroxylase α₂β₂ tetramers. Schematic representations of the structural domains included in proαl(I) collagen chain, pCαl(I), αl(I)foldon, and αl(I) chains are shown in Figures 1A, IB, 1C, and ID, respectively.

Example 2. Collagen α2(D Expression Constructs

The C-propeptide of the α2 type I chain of human procollagen (pCα2(I)) (GenBank Accession No. Z74616) was replaced with foldon sequence as follows. A P. pastoris expression cassette encoding human ρCα2(I) chains was excised from ρBLADEIXpCα2(I) (Nokelainen et al, supra) by digestion with Pmel-Notl, and subsequently cloned into pPICZB to generate pPICZBpCα2(I). To replace the C-propeptide of pCα2(I) with foldon, two DNA fragments were generated by PCR. The first DNA fragment extended from an internal AvrU site of the pCα2(I) cDNA to the codon for the last amino acid of the C-telopeptide, and the second DNA fragment encoded foldon, and was prepared as described above in Example 1. These DNA fragments were co-ligated into pPICZBpCα2(I) previously digested with Avrll-Notl to generate pPICZBα2(I)foldon. The constructs were linearized with Pmel and transformed into the ArgαAdeβ,his- P. pastoris yeast strain to generate strains pCα2(I) and α2(I)foldon. In experiments to study the expression of type I collagen heterotrimers, the linearized pPICZBpCα2(I) and pPICZBα2(I)foldon constructs were transformed into the P. pastoris yeast strains expressing pCαl(I) and αl(I)foldon chains, respectively, as described above.

Example 3. Collagen α 1(111 Expression Constructs

Expression constructs for human type III collagen were prepared as follows. The DNA sequence encoding the N-propeptide of proαl(III) chains was deleted from the DNA sequence encoding αl type III chain of human procollagen (pCαl(III); GenBank Accession No. X14420) as follows. A PCR DNA fragment extending from the Pmel site of the alcohol oxidase 5' sequence of pPICZB to the end of the signal sequence of proαl(III) was generated and ligated directly to the sequence coding for the N-telopeptide of proαl(III). An internal Ndeϊ site was amplified using pPICZBproαl (III) as a template (See, Vuorela et al, supra) and the oligonucloetide

S'-GCCCJΓ^ΓG^ΓCJΓ^CΓGTGCCAAAATAATAGTGGGATGAAGCA-S' (SEQ ID

NO.:6) as the reverse primer. In the ohgonucleotide sequence shown above, nucleotides corresponding to the signal sequence and N-telopeptide are shown in bold and italics, respectively; nucleotides corresponding to the Ndel site are underlined. The resulting PCR fragment was cloned into pPICZBproαl(lTI) previously digested with Pmel-Ndel to generate pPICZBpCαl(III). To change the expression vectors, the proαl(III) and pCαl(III) expression cassettes were excised from the pPICZB vectors by digestion with Pmel-Notϊ and cloned into pPIC3Kto generate pPIC3Kproαl(III) and pPIC3KpCαl(III).

The C-propeptide of the αl type III chain of human procollagen (pCαl(III)) was replaced by foldon sequence as follows. Two DNA fragments were generated by PCR amplification: the first DNA fragment extended from an internal _^4vrII site of the pCα 1(111) cDNA to the 3' end of the C-telopeptide sequence; the second DNA fragment is the foldon-encoding fragment described above in Example 1. The DNA fragments were co-ligated into pPIC3KpCα 1(111) previously digested with ^vrll-Notl to generate pPIC3Kαl (III)foldon. To delete the C-propeptide, a fragment extending from the internal AvrT site to the 3' end of the C-telopeptide sequence followed by a stop codon and a Notl site was created by PCR and ligated into pPIC3KpCαl(HI) previously digested with vrll-NotI to generate pPIC3Kαl(III). The pPIC3Kproαl(πi), pPIC3KpCαl(III), pPIC3Kαl(III)foldon, and pPIC3Kαl(III) constructs were linearized with Stwl and transformed into the ArgαAdeβ,his- P. pastoris yeast strain.

Example 4. Assembly of Stable Type I and Type III Collagen Homotrimers In order to study the effect of replacement of the C-propeptide with foldon and deletion of the C-propeptide on the assembly of type I and III collagen homotrimers, expression constructs encoding pCαl(I), αl(I)foldon, αl(I) chains, and expression constructs encoding proαl(III), pCαl(III), αl(III)foldon, and αl(III) chains were transformed by electroporation into a P. pastoris strain expressing human prolyl 4-hydroxylase.

Cells were cultured in 25 ml shaker flasks in a buffered glycerol complex medium (BMGY, pH 6.0) with 1 g/1 yeast extract and 2 g/1 peptone. Expression was induced in a buffered minimal methanol medium (BMM, pH 6.0), and methanol was added every 12 h to a final concentration of 0.5%. Amino acids were added up to 100 μg/1 as required.

Cells were harvested after a 60 hour methanol induction at 30°C, washed once, and suspended in cold (4°C) 5% glycerol, 1 mM PEFABLOC SC protease inhibitor, and 50 mM sodium phosphate buffer, pH 7.4. The cells were broken by vortexing with glass beads, and the lysate was centrifuged at 10,000 x g for 30 min. Aliquots of the soluble fractions were analyzed by SDS- PAGE under reducing conditions, followed by western blotting with polyclonal antibodies to type I or type III human collagen (Rockland). Further aliquots were digested with pepsin for 2 hours at 22°C, the thermal stability of the pepsin-resistant recombinant collagens was studied by digestion with a mixture of trypsin and chymotrypsin for 2 min at various temperatures as described (Bruckner and Prockop (1981) Anal Biochem 110:360-368.), and the samples were analyzed by SDS-PAGE under reducing conditions followed by Coomassie Blue staining or western blotting as described, supra. The amounts of collagen chains were estimated by densitometry of the Coomassie Blue-stained bands using a GS-710 Calibrated Imaging Densitometer (BioRad Laboratories).

Collagen chain expression is shown in Figures 2A and 3. Immunoblots in Figure 2 A show protein bands corresponding to pCαl(I), αl(I)foldon, and αl(I) chains. (Figure 2A, lanes 1-3, respectively.) Immunoblots showed protein bands corresponding to proαl(III), pC l(III), αl(III)foldon, and al(III) chains. (Figure 3A, lanes 1-4, respectively.) These data indicated that collagen αl(I) and αl(III) chains were expressed using the expression constructs described, supra.

Assembly of the collagen chains into triple-helical molecules was analyzed by digesting aliquots of the cell extracts with pepsin. The triple helix of collagens is resistant to pepsin; non-triple- helical collagen chains and collagen propeptides of triple-helical molecules are sensitive to proteases and, therefore, digested by pepsin. (See Bruckner and Prockop, supra) Pepsin- resistant polypeptides conesponding to the αl(I) chains were seen in samples from all three strains expressing pCαl(I), αl(I)foldon, and αl(I) chains in Coomassie Blue stained SDS-PAGE (Figure 2B, lanes 1-3, respectively) and in the corresponding immunoblots (Figure 2C, lanes 1-3, respectively). Likewise, pepsin-resistant polypeptides conesponding to αl(III) chains were seen in pepsin-digested samples obtained from the yeast strains expressing proαl(III), pCαl(III), αl(III)foldon, and α 1(111) chains in Coomassie Blue-stained SDS-PAGE (Figure 3B, lanes 1-4, respectively). Expression of α2(I)foldon chains (data not shown) alone did not lead to pepsin- resistant molecules, indicating that non-propeptide α2(I) collagen domains contain restrictions that limit formation of stable α2(I) homotrimers.

These data indicated that the foldon domain allowed for efficient collagen chain assembly, as evidenced by increased amounts of pepsin-resistant αl(I) and αl(III) chains seen in samples from cells expressing αl(I)foldon and αl(ffl)foldon chains, respectively. (See Figures 2B, 2C, 3 A, and 3B.) Densitometric analysis of the polypeptide bands obtained from 11 individual samples demonstrated that the increase in the expression level of type I collagen homotrimers in yeast strains expressing αl(I)foldon chains was approximately 3.0-fold higher relative to that observed in yeast strains expressing pCαl(I) chains and showed the highest expression level. Similar experiments showed a 2.4-fold increase in the expression level of type III collagen homotrimers in yeast strains expressing αl(III)foldon chains above that of control strains. The l(I) and αl(III) chains lacking any oligomerization domain also assembled into homotrimeric triple-helical molecules (Figures 2B and 2C, lanes 3; Figures 3 A and 3B, lanes 4), but their assembly levels were lower than those of the pCαl(I), pCαl(I)foldon, pCal(III), and αl(UI)foldon chains (Figures 2B, 2C, 3A, and 3B).

Stability of type I collagen homotrimers expressed and assembled from the pCαl(I), αl(I)foldon, and αl(I) chains was analyzed by digestion of the polypeptides with a mixture of trypsin and chymotrypsin after heating to various temperatures, using methods previously described. (See Bruckner and Prockop, supra) The T_m of recombinant type I collagen homotrimers expressed in yeast strains was between 38°C and 40°C (Figures 4A, 4B, and 4C), similar to that reported previously for human type III collagen produced in P. pastoris in shaker flasks (Vuorela et al, supra).

Example 5. Assembly of Stable Type I Collagen Heterotrimers The C-propeptides are believed to have an essential role in the selective association of procollagen α-chains in a type-specific manner. (Prockop and Kivirikko (1995) Annu Rev Biochem 64:403-434; McLaughlin and Bulleid (1998) Matrix Biol 16:369-377; and Lees et al. (1997) EMBO J 16:908-916.) The effect of replacement of the C-propeptides of bothpCαl(I) and pCα2(I) chains with foldon on the assembly of type I collagen heterotrimers was studied by transforming expression constructs encoding pCα2(I) and α2(I)foldon chains into the above- described recombinant P. pastoris strains expressing pCαl(I) and αl(I)foldon strains, respectively. The strains were cultured and harvested as described above. SDS-PAGE and Coomassie Blue staining of the expressed proteins showed polypeptide bands corresponding to the pCαl(I) and pCα2(I) chains (Figure 5, lane 1). Two pepsin-resistant polypeptides conesponding to the αl(I) and α2(I) chains were seen in samples from the two strains coexpressing pCαl(I) and pCα2(I) chains (Figure 5, lane 1) or αl(I)foldon and α2(I)foldon chains (Figure 5, lane 2).

Example 6. Assembly of Stable Type I Collagen Heterotrimers With 2:1 Ratio Densitometric analysis of the pepsin-resistant αl(I) and α2(I) polypeptide bands from 10 individual strains coexpressing αl(I)foldon and α2(I)foldon chains indicated that the αl(I) to α2(I) chain ratio was 1.91 ± 0.31 (SD) (Figure 5, lane 2), and an increased amount of pepsin resistant αl(I) and α2(I) chains was also seen in these samples (Figure 5). Densitometric analysis of the polypeptide bands in seven individual samples indicated that the increase in expression level of type I collagen heterotrimers in these strains was 2.1 -fold relative to that obtained with the strains coexpressing pCαl(I) and pCα2(I) chains. The T_m of the recombinant type I collagen heterotrimers produced in the strains coexpressing pCαl(I) and pCα2(I) chains or αl(I)foldon and α2(I)foldon chains was between 38°C and 40°C (data not shown).

Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention. Although the invention has been described in connection with specific prefened embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Various modifications of the described modes for carrying out the invention which are obvious to those skilled in the present art and related fields are intended to be within the scope of the following claims. All references cited herein are incorporated by reference herein in their entirety.

Claims

What is claimed is:

1. A method for producing a collagen triple helix, the method comprising:

(a) introducing into a host cell a polynucleotide comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof;

(b) culturing the host cell under conditions suitable for expression, trimerization, and formation of a triple helix; and

(c) recovering the collagen triple helix.

2. The method of claim 1 , wherein the collagen polypeptide is a human collagen polypeptide.

3. The method of claim 1 , wherein the collagen polypeptide is selected from the group consisting of a bovine collagen polypeptide and a porcine collagen polypeptide.

4. The method of claim 1, wherein the collagen polypeptide lacks the entire C-propeptide.

5. The method of claim 1, wherein the collagen polypeptide lacks at least a portion of the N-propeptide.

6. The method of claim 1, wherein the collagen polypeptide lacks the entire N-propeptide.

7. The method of claim 1, wherein the method further comprises removing the foldon from the collagen polypeptide.

8. A method for producing a collagen, the method comprising:

(b) culturing the host cell under conditions suitable for expression and trimerization; and (c) recovering the collagen.

9. The method of claim 8, wherein the collagen is homotrimeric.

10. The method of claim 8, wherein the collagen is heterotrimeric.

11. A method for producing a collagen polypeptide, the method comprising:

(b) culturing the host cell under conditions suitable for expression; and

(c) recovering the collagen polypeptide.

12. A method for producing a collagen heterotrimer, the method comprising:

(a) introducing into a host cell a first polynucleotide comprising a first sequence encoding a first collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof;

(b) introducing into the host cell a second polynucleotide comprising a second sequence encoding a second collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof;

(c) culturing the host cell under conditions suitable for expression and trimerization; and

(d) recovering the collagen heterotrimer.

13. A method for producing a collagen heterotrimer, the method comprising:

(c) introducing into the host cell a third polynucleotide comprising a third sequence encoding a third collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof;

(d) culturing the host cell under conditions suitable for expression and trimerization; and

(e) recovering the collagen heterotrimer.

14. The method of claim 12, wherein the collagen heterotrimer is selected from the group consisting of type I collagen, type JV collagen, type V collagen, and type VIII collagen.

15. The method of claim 13, wherein the collagen heterotrimer is selected from the group consisting of type V collagen, type VI collagen, type IX collagen, and type XI collagen.

16. The method of claim 1, wherein the host cell is a eukaryotic host cell.

17. The method of claim 1, wherein the host cell is a prokaryotic host cell.

18. The method of claim 1, wherein the host cell is selected from the group consisting of a yeast cell, a plant cell, a fungal cell, an insect cell, a bacterial cell, and a mammalian cell.

19. A method for producing type I collagen, the method comprising:

(a) introducing into a host cell a first polynucleotide comprising a first sequence encoding αl(I) collagen or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof;

(b) introducing into the host cell a second polynucleotide comprising a second sequence encoding α2(I) collagen or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof; (c) culturing the host cell under conditions suitable for expression and trimerization; and (e) recovering the type I collagen.

20. The method of claim 19, wherein the type I collagen has an αl(I)/α2(I) ratio of 2: 1.

21. A construct comprising a sequence encoding a collagen polypeptide or a fragment or variant thereof lacking at least a portion of the C-propeptide, and further comprising a sequence encoding foldon or an active fragment thereof.

22. A host cell comprising the construct of claim 21.

23. A collagen-foldon fusion protein comprising at least one triple helical domain and lacking at least a portion of the C-propeptide, wherein the polypeptide is stable at temperatures above 35°C.