Novel recombinant collagen polypeptides and molecules

Abstract

The present invention relates to novel recombinant collagen polypeptides and collagen molecules, polynucleotides encoding them, methods for their preparation, and compositions and uses thereof.The novel recombinant collagen polypeptides or collagen molecules can be used in cosmetics, personal care, dietary supplements, and biomedical applications due to their multiple benefits for the skin, including preventive anti-aging, curative anti-aging, antioxidant, anti-pigmentation, moisturizing, anti-wrinkle, and elasticity-promoting activities.

Description

[Technical field]

[0001] The present invention relates to novel recombinant collagen polypeptides and collagen molecules, polynucleotides encoding them, methods for their preparation, and compositions and uses thereof.The novel recombinant collagen polypeptides or collagen molecules can be used in cosmetics, personal care, dietary supplements, and biomedical applications due to their multiple benefits for the skin, including preventive anti-aging, curative anti-aging, antioxidant, anti-pigmentation, moisturizing, anti-wrinkle, and elasticity-promoting activities. [Background technology]

[0002] For several years, the cosmetic industry has focused on preventing skin aging and skin damage using various chemical and mechanical treatments. Intrinsic skin aging is an inevitable process that is responsible for the thinning, drying, and appearance of wrinkles of the skin. On the other hand, extrinsic damage to the skin is caused by environmental insults such as air pollution or sun exposure. This also leads to the appearance of wrinkles, loss of elasticity, and deterioration of the skin structure.

[0003] Collagen is the main structural protein in human skin. Human skin has an upper layer (epidermis) and a lower layer (dermis). Collagen is specifically present in the extracellular matrix (ECM) and is responsible for providing structure and support to the surrounding cells present in the epidermis and dermis. Structural changes in the human ECM are known to cause skin aging over time. The mechanisms that lead to these changes include both intrinsic and extrinsic factors that affect the molecular and mechanical integrity of the main components of the skin.

[0004] Therefore, strategies to manage skin aging have been to (1) slow down the intrinsic aging process by properly nourishing and moisturizing the skin, (2) protect against extrinsic factors such as UV exposure and skin oxidation by shielding the skin, and (3) compensate for the loss of endogenous stable collagen that accompanies aging.

[0005] Skin care products that aim to use the above strategies often contain collagen. Collagen and gelatin, a denatured and partially degraded collagen, are isolated from animal tissues. Typical collagen products on the cosmetic market can be classified as full-length or hydrolyzed collagen products. Collagen products using extracted full-length collagen tend to have a moisturizing effect, but are generally too large to penetrate the skin barrier. Full-length collagen tends to act as a layer placed on top of the skin. Hydrolyzed collagen, i.e., full-length protein degraded into smaller pieces (<1000 Da), is believed to have antimicrobial properties and higher bioavailability than longer collagen polypeptides, and can be produced by enzymatic digestion or incubation in acid or base. In such a way, many peptides derived from full-length animal collagen proteins are produced, representing a heterogeneous mixture. Some of these peptides are short enough to penetrate the skin more effectively than full-length collagen, but their active concentration is necessarily lower.

[0006] However, concerns related to the cross-species biocompatibility and immunogenicity of animal-derived collagen, the risk of animal-derived contaminating pathogens, and the difficulty of reproducibly isolating collagen preparations with identical chemical composition have created the need for alternative collagen sources for skin care products.In fact, the variability of gelatin preparations creates significant problems in their use.In addition, many consumers prefer collagen preparations that are derived from non-animal (i.e. vegan) sources.

[0007] Cosmetics that use collagen from alternative sources (e.g., bacterial collagen) may use a protein that is similar in structure to the human protein, but typically does not replicate the actual amino acid composition of the human protein and therefore is not as effective as human collagen.

[0008] Thus, there exists a need for alternative sources of collagen that are not only structurally the same or similar to the human protein, but also provide superior anti-aging and skin healing properties for cosmetic, personal care, nutraceutical, and biomedical uses compared to prior art collagen molecules. The novel collagen polypeptides and collagen molecules of the present invention meet these needs. Summary of the Invention

[0009] The present invention provides collagen polypeptides that contain sequences derived from human type I alpha 1 (A1) collagen and can be secreted from cultured yeast cells. These collagen polypeptides can also form higher-order structures, particularly homotrimeric collagen molecules. Notably, the collagen polypeptides and collagen molecules described herein are associated with a variety of beneficial effects on the skin, including preventative anti-aging, curative anti-aging, antioxidant, anti-pigmentation, moisturizing, anti-wrinkle, and elasticity-promoting activities. Thus, the collagen polypeptides and molecules described herein are associated with noteworthy anti-aging effects.

[0010] Thus, one aspect of the present invention provides a collagen polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:2, having a molecular weight of 1 to 80 kDa.

[0011] Another aspect of the present invention provides a collagen molecule comprising a homotrimer of three collagen polypeptides as described herein.

[0012] Another aspect of the invention provides nucleic acids encoding the collagen polypeptides described herein, or polypeptides which are at least 85% identical thereto, and expression vectors containing same.

[0013] Another aspect of the invention provides a host cell comprising an expression vector or nucleic acid described herein.

[0014] Another aspect of the present invention provides compositions comprising a collagen polypeptide or collagen molecule as described herein and one or more excipients, as well as uses of said compositions.

[0015] Another aspect of the present invention is a method for producing a (a) providing a nucleotide sequence encoding a collagen polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:2 and has a molecular weight of 1 to 80 kDa; (b) optionally providing a nucleotide sequence encoding a human prolyl-4-hydroxylase enzyme; (c) inserting the nucleotide sequence of (a) and optionally (b) into at least one vector; (d) expressing the vector of (c) in a cultured yeast cell, preferably Pichia pastoris; (e) isolating the recombinant collagen polypeptide or collagen molecule from the cultured yeast cells. The present invention provides a method for preparing a recombinant collagen polypeptide or collagen molecule, comprising:

[0016] Yet another aspect of the present invention provides a recombinant collagen polypeptide or collagen molecule prepared by the methods described herein. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a diagram of an SDS-PAGE gel showing expression and secretion of various collagen polypeptides by Pichia pastoris (generated using constructs #2 (SEQ ID NO:8), #4 (SEQ ID NO:10), #6 (SEQ ID NO:12), and #8 (SEQ ID NO:14) - see Table 2). Arrows point to compounds of interest. Asterisks indicate the P4H subunit. [Diagram 2]FIG. 11 shows a comparison of SDS-PAGE 4-16% analysis of pepsin digested and non-digested protein suspensions (full-length constructs #5 (SEQ ID NO:11) and #6 (SEQ ID NO:12)). The bands indicated by the arrows are resistant to digestion with pepsin, indicating that homotrimeric collagen molecules are produced and secreted by Pichia pastoris. The same appears to be true for the cleavage products (SEQ ID NO:21 and SEQ ID NO:22), shown as lower weight bands on the gel. [Diagram 3] FIG. 1 shows the cytotoxic activity of the tested collagen polypeptides and buffer solutions against normal human dermal keratinocytes (Panels A-G show the results for Samples 1-7, respectively). [Figure 3-1] Same as above. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above. [Figure 4] Figure 1 shows the biological activity of samples 2, 4, 5, and 7 on the expression of various genes in NHEK cells. The vertical axis indicates the percentage of over- or under-expression compared to cells treated with a blank formulation (buffer solution, see samples 1, 3, and 6). Samples 2, 4, 5, and 7 are compared with samples 1, 3, 3, and 6, respectively. MMP1: matrix metallopeptidase 1, SIRT2 / SIRT6: sirtuins 2 and 6, SOD2: mitochondrial superoxide dismutase 2, GLRX: glutaredoxin, HMOX1: heme oxygenase 1), AZGP1: alpha-2-glycoprotein 1, zinc binding. (-) indicates that under-expression of this gene may be associated with beneficial effects on the skin. (+) indicates that over-expression of this gene may be associated with beneficial activity on the skin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition In order that the invention may be readily understood, definitions of some of the terms used in the course of the invention are set forth below.

[0019] As used herein, the abbreviation "kDa" stands for kilodalton.

[0020] As used herein, the term "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue. Preferably, the term "amino acid" refers to one of the 20 naturally occurring amino acids.

[0021] As used herein, the term "polypeptide" or "protein" refers to a polymer composed of a sequence of amino acids. A protein can be a native protein, i.e., a protein produced by a naturally occurring non-recombinant cell, or it can be produced from a genetically engineered or recombinant cell, and includes molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence.

[0022] The term "sequence identity" refers to a quantitative measure of the degree of homology between two sequences, which can be nucleotide (also called nucleic acid) sequences or amino acid sequences. If the two sequences to be compared are not of equal length, they must be aligned to allow for the insertion of gaps or truncation at the ends of the nucleic acid or amino acid sequences to provide the best possible fit. Those skilled in the art will understand that various means are available for comparing sequence identity (see below).

[0023] As used herein, "conservative amino acid substitution" means that an amino acid may be substituted by another amino acid in its respective group according to the following six groups: [1] alanine (A), serine (S), threonine (T), [2] aspartic acid (D), glutamic acid (E), [3] asparagine (N), glutamine (Q), [4] arginine (R), lysine (K), [5] isoleucine (I), leucine (L), methionine (M), valine (V), and [6] phenylalanine (F), tyrosine (Y), tryptophan (W).

[0024] As used herein, the term "homotrimer" refers to a complex of three collagen polypeptides of the same type, ie, three human type I A1 collagen polypeptides.

[0025] As used herein, "recombinant" refers to a polypeptide or protein molecule that has been produced using recombinant techniques, i.e., does not occur in nature. Methods and techniques for producing recombinant nucleic acids and polypeptides are well known in the art.

[0026] As used herein, "isolated" refers to a protein that has been removed from a cell culture and separated from cell culture components, e.g., it can be separated from at least 90% of the cell culture components.

[0027] As used herein, a molecule that is an "antioxidant" has antioxidant activity, i.e., is capable of stimulating the native antioxidant pathway, for example in skin cells.

[0028] As used herein, a molecule having "anti-aging activity" can slow the intrinsic aging process (relative to baseline expression levels) in skin cells, e.g., normal human dermal keratinocytes, by differential transcriptional regulation of genes involved in the aging process, including genes (determined, e.g., via mRNA sequencing) that have been described to be involved in preventative and curative anti-aging, antioxidant, and anti-pigmentation.

[0029] A "vector" is a nucleic acid that can be used to introduce another nucleic acid (or "construct") linked to it into a cell, for example, by electroporation. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that can introduce additional DNA segments into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and are replicated along with the host genome by culturing under selective pressure. Vectors can be used to direct the expression of a selected nucleic acid in a cell.

[0030] A "host cell" is a cell that can be used to express a nucleic acid, such as a nucleic acid disclosed herein. A host cell according to the invention is a yeast cell, preferably Pichia pastoris.

[0031] As used herein, a "subject" includes all mammals, preferably humans.

[0032] As used herein, "patient" refers to the subject to be treated.

[0033] As used herein, "treatment" is synonymous with alleviating the symptoms of a disease or condition, inhibiting the progression of a disease or condition, causing the relapse of a disease or condition, and / or curing a disease or condition. In the present invention, the term "treatment" is intended to include therapeutic treatment, as well as prophylactic or suppressive measures for a disease or condition.

[0034] As used herein, the term "inventive" or "according to the invention" is intended to refer to all aspects and embodiments of the invention as disclosed and / or claimed herein. Any aspect, feature, or embodiment referred to herein as "disclosed herein" or "described herein" is to be understood to be an aspect, feature, or embodiment "of the invention" or "according to the invention."

[0035] As used herein, the term "comprising" is to be interpreted as embracing both "including" and "consisting of," both meanings being specifically contemplated and therefore individually disclosed embodiments according to the present invention.

[0036] As used herein, the articles "a" and "an" preceding an element or component are intended to be non-restrictive regarding the number of instances (ie, occurrences) of the element or component.

[0037] Thus, "a" or "an" is to be read as including one or at least one, and the singular form of an element or component also includes the plural, unless the number is clearly implied to be singular.

[0038] Collagen Polypeptides In one aspect, the invention provides a collagen polypeptide having a molecular weight of 1-80 kDa and comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 2. The amino acid sequence of human type I A1 collagen is provided herein in SEQ ID NO: 20.

[0039] In some embodiments, the collagen polypeptides have a molecular weight of 2 to 45 kDa. In some embodiments, the collagen polypeptides have a molecular weight of 5 to 20 kDa. It is believed that the size of the collagen polypeptides of the present invention allows them to penetrate the skin more effectively than full-length collagen.

[0040] In certain embodiments, the collagen polypeptide is a fragment of human type I collagen A1.

[0041] In certain embodiments, the collagen polypeptide may contain at least two PGP amino acid sequences.

[0042] In one embodiment, the amino acid sequence of the collagen polypeptide comprises at least 10% proline. Preferably, the amino acid sequence of the collagen polypeptide comprises at least 15% proline. More preferably, the amino acid sequence of the collagen polypeptide comprises at least 20% proline.

[0043] In some embodiments, the amino acid sequence of collagen polypeptide comprises at least 1.5%, at least 2.5%, at least 5%, or at least 7.5% hydroxyproline.Higher hydroxyproline ratio in collagen is expected to correspond to higher moisturizing and anti-aging activity.Higher hydroxyproline ratio is also expected to improve performance as a dietary supplement, since one potential mechanism of action is related to the passage of hydroxyproline-containing dipeptides and tripeptides into the bloodstream.

[0044] In one embodiment, the amino acid sequence of the collagen polypeptide comprises at least 15% glycine. Preferably, the amino acid sequence of the collagen polypeptide comprises at least 20% glycine. More preferably, the amino acid sequence of the collagen polypeptide comprises at least 25% glycine.

[0045] In certain embodiments, the amino acid sequence of the collagen polypeptide comprises a hydroxylated lysine.

[0046] It is believed that the proline / hydroxyproline, glycine, and / or hydroxylated lysine rich amino acid sequence of the collagen polypeptides described herein contributes to their superior moisturizing and anti-aging effects on human skin.

[0047] In certain embodiments, the collagen polypeptide has an isoelectric point >7, preferably >8.

[0048] In certain embodiments, the collagen polypeptide is an antioxidant.

[0049] For example, a collagen polypeptide can preferably comprise an amino acid sequence that is GFSGLDGAKGD (SEQ ID NO: 6) or that differs from GFSGLDGAKGD (SEQ ID NO: 6) by up to three conservative amino acid substitutions.

[0050] In certain embodiments, the collagen polypeptides have anti-aging activity. Thus, in certain embodiments, products comprising the collagen polypeptides of the present invention slow down the intrinsic aging process in skin cells by differential transcriptional regulation of genes involved in the aging process.

[0051] Thus, in one embodiment, the collagen polypeptide downregulates the expression of MMP1 in skin cells by at least 20%, preferably at least 40%, more preferably at least 60% (compared to baseline expression level). In another embodiment, the collagen polypeptide upregulates the expression of SIRT2 / SIRT6 in skin cells by at least 10%, preferably at least 30%, more preferably at least 50% (compared to baseline expression level). In another embodiment, the collagen polypeptide upregulates the expression of SOD2 in skin cells by at least 30%, preferably at least 60%, more preferably at least 90% (compared to baseline expression level). In one embodiment, the collagen polypeptide upregulates the expression of GLRX in skin cells by at least 20%, preferably at least 40%, more preferably at least 60% (compared to baseline expression level). In another embodiment, the collagen polypeptide upregulates the expression of HMOX1 in skin cells by at least 40%, preferably at least 80%, more preferably at least 120%, most preferably at least 150% (compared to baseline expression level). In other embodiments, the collagen polypeptide upregulates expression of AZGP1 in skin cells by at least 30%, preferably at least 60%, more preferably at least 90% (compared to baseline expression levels).

[0052] In a preferred embodiment, collagen polypeptide upregulates the expression of one or more of SIRT2 / SIRT6, SOD2, GLRX, HMOX1 and AZGP1 in skin cells (compared to baseline expression levels).In some particularly preferred embodiments, collagen polypeptide upregulates the expression of SIRT2 / SIRT6, SOD2, GLRX, HMOX1 and AZGP1 in skin cells (compared to baseline expression levels).In some of these embodiments, collagen polypeptide downregulates the expression of MMP1 in skin cells (compared to baseline expression levels).

[0053] In a preferred embodiment, the collagen polypeptide upregulates expression of one or more of SIRT2 / SIRT6, SOD2, GLRX, HMOX1, and AZGP1 in skin cells by at least 50% (compared to baseline expression levels) and downregulates expression of MMP1 in skin cells by at least 50% (compared to baseline expression levels).

[0054] In certain embodiments, the collagen polypeptides are hypoallergenic. Thus, in certain embodiments, a product comprising the collagen polypeptides of the invention, when applied to a subject, does not induce an allergic reaction in said subject.

[0055] In certain embodiments, the collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any one of SEQ ID NOs: 1-4 or 7-17. Preferably, the collagen polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-4 and 7-17.

[0056] In preferred embodiments, the collagen polypeptide comprises an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 98% identical to SEQ ID NO: 2. In particularly preferred embodiments, the collagen polypeptide comprises an amino acid sequence that is SEQ ID NO: 2.

[0057] In other preferred embodiments, the collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any one of SEQ ID NOs: 3, 21, and 22. In some of these embodiments, the collagen polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 3, 21, and 22.

[0058] In certain embodiments, the collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to SEQ ID NO: 8. In particularly preferred embodiments, the collagen polypeptide comprises an amino acid sequence that is SEQ ID NO: 8.

[0059] In particularly preferred embodiments, the collagen polypeptide comprises the amino acid sequence of SEQ ID NO: 2 and has a molecular weight of 5-20 kDa. In some of these embodiments, the collagen polypeptide has an isoelectric point of > 8. In some of these embodiments, the collagen polypeptide comprises at least 2.5% hydroxyproline.

[0060] Protein and / or nucleic acid sequence identity (homology) can be evaluated using any of a variety of sequence comparison algorithms and programs known in the art. In sequence comparison, typically, one sequence serves as a reference sequence (e.g., a sequence disclosed herein) to which test sequences are compared. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0061] The percent identity of two amino acid or two nucleic acid sequences can be determined, for example, by the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol., 215:403-410; Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA, 90:5873-5787. Alternatively, the percent identity of two amino acid or two nucleic acid sequences can be determined, for example, by the DIAMOND algorithm described in Benjamin Buchfink, Chao Xie, and Daniel H. Huson, Fast and Sensitive Protein Alignment using DIAMOND, Nature Methods, 12, 59-60 (2015).

[0062] In some embodiments, the collagen polypeptide is a recombinant collagen polypeptide.

[0063] In certain embodiments, the collagen polypeptide is an isolated collagen polypeptide.

[0064] Nucleic Acids, Vectors, and Host Cells In another aspect, the invention provides a nucleic acid encoding a collagen polypeptide as described herein having all of its embodiments.

[0065] Thus, the nucleic acids of the invention encode a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a polypeptide at least 85% identical thereto, having a molecular weight of 1-80 kDa. In some embodiments, the encoded collagen polypeptide has a molecular weight of 2-45 kDa. In some preferred embodiments, the encoded collagen polypeptide has a molecular weight of 5-20 kDa.

[0066] In certain embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 3, 21, and 22, or a polypeptide at least 85%, at least 90%, at least 95%, or at least 98% identical thereto. Preferably, the nucleic acid encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 3, 21, and 22.

[0067] In other embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 8, 10, 11, 12, 15, and 16, or a polypeptide at least 85%, at least 90%, at least 95%, or at least 98% identical thereto. Preferably, the nucleic acid encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 8, 10, 11, 12, 15, and 16.

[0068] In a preferred embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, or a polypeptide that is at least 85%, at least 90%, at least 95%, or at least 98% identical thereto. Preferably, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

[0069] In another aspect, the invention provides an expression vector comprising a nucleic acid described herein having any of its embodiments.

[0070] In yet another aspect, the invention provides a host cell comprising an expression vector or a nucleic acid described herein having any of its embodiments.

[0071] Collagen molecule In another aspect, the present invention provides a collagen molecule comprising a homotrimer of three collagen polypeptides as described herein having all of its embodiments.

[0072] Thus, as homotrimers, the collagen molecules of the present invention differ from native human type I collagen, which is a heterotrimer consisting of two alpha 1 polypeptides and one alpha 2 polypeptide.

[0073] In certain embodiments, the collagen polypeptide or collagen molecule is glycosylated.

[0074] Preferably, the collagen polypeptide or collagen molecule is secreted from cultured yeast cells, such as Pichia pastoris. In some embodiments, the cultured yeast cells are wild-type strains. Alternatively, the cultured yeast cells may have methanol oxidase knocked out of their genome, which results in them growing slower on methanol. In a preferred embodiment, the cultured yeast cells have a deletion of the AOX1 gene. In some embodiments, the cultured yeast cells stably express prolyl-4-hydroxylase.

[0075] Those skilled in the art will appreciate that collagen polypeptides or collagen molecules recombinantly produced in yeast cells can be classified as vegan, i.e., they are not sourced from animals or animal products. Thus, in certain embodiments, the collagen polypeptides and collagen molecules described herein are vegan.

[0076] Compositions and uses thereof In another aspect, the present invention provides a composition comprising a collagen polypeptide as described herein with all of its embodiments, or a collagen molecule as described herein with all of its embodiments, and one or more excipients.

[0077] The excipient can be any excipient known to those of skill in the art, such as, for example, starch, glucose, lactose, sucrose, silica gel, sodium stearate, glycerol, glycerol monostearate, talc, sodium chloride, propylene, glycol, and ethanol, and combinations thereof.

[0078] The compositions described herein may comprise a continuous aqueous phase comprising an aqueous carrier and a thickener. The aqueous phase may further comprise other hydrophilic materials that exhibit limited solubility in the oil phase, such as, but not limited to, water-soluble ingredients, water-soluble sunscreens, and other water-soluble skin care actives. The compositions described herein comprise from about 60% to about 98%, preferably from about 65% to about 97%, of an aqueous phase.

[0079] The compositions described herein may further comprise components such as carriers, stabilizers, preservatives, thickeners, emulsifiers, humectants, softeners, and / or other components.

[0080] The carrier can be any carrier known to those skilled in the art, such as, for example, aqueous liquids, dextrose solutions, glycerol solutions, microemulsions, nanoparticles, liposomal suspensions; oils such as those of vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil; isopropyl alcohol, gaseous fluorocarbons, ethyl alcohol, polyvinylpyrrolidone, propylene glycol, gel-forming materials, stearyl alcohol, stearic acid, sorbitan monooleate, methylcellulose, and combinations thereof.

[0081] Particularly useful carriers in the present invention include water and aqueous solutions of lower alkyl alcohols. The lower alkyl alcohols useful in the present invention are monohydric alcohols having 1 to 6 carbons, more preferably ethanol and glycerol. Preferably, the aqueous carrier is substantially water. Deionized water is preferably used. Water from natural sources containing mineral cations can also be used, depending on the desired characteristics of the product.

[0082] The pH of the compositions described herein is preferably about 3 to about 8. The pH may be adjusted to a pH that provides optimal collagen efficacy. Buffers and other pH adjusting agents may be included to achieve the desired pH. Suitable pH adjusting agents herein include acetate, phosphate, citrate, sodium hydroxide, triethanolamine, aminomethylpropanol, and carbonate.

[0083] The stabilizer can be any stabilizer known to those of skill in the art, such as, for example, amino acids; ascorbic acid; surfactants, such as poloxamers; polyhydric sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol; and combinations thereof.

[0084] The aqueous phase of the compositions described herein comprises about 0.1% to about 2%, preferably about 0.2% to about 1.5%, of thickening agents such as thickeners, gelling agents, and structurants. The level and type of thickening agent is selected according to compatibility with other components and other desired characteristics of the product. Thickening agents include crosslinked polyacrylate polymers and copolymers, hydrophobically modified polyacrylate polymers and copolymers, polyacrylamide polymers and copolymers, polyacryloyldimethyltaurate, aminomethylpropanol (AMP)-based polymers and copolymers, polysaccharides, and gums. Carboxylic acid / carboxylate copolymers are useful in the present invention. Non-limiting examples of carboxylic acid / carboxylate copolymers useful in the present invention include those having the CTFA name Acrylates / C10-30 Alkyl Acrylate Crosspolymers having the trade names Pemulen TR-1, Pemulen TR-2, Carbopol Ultrez 10, Carbopol Ultrez 20, Carbopol Ultrez 21, Carbopol 1342, Carbopol 1382, and Carbopol ETD 2020 (all from Noveon), xanthan gum.Additional commercially available water-soluble polymers that are highly useful in the present invention include xanthan gum having the trade name KELTROL series available from Kelco; carbomers having the trade names Carbopol 934, Carbopol 940, Carbopol 950, Carbopol 980, and Carbopol 981 (all available from Noveon); acrylates / steareth-20 methacrylate copolymer having the trade name ACRYSOL 22 (available from Rohm and Hass); polyacrylamide having the trade name SEPIGEL 305 (available from Seppic); glyceryl polymethacrylate having the trade name LUBRAGEL NP, and a mixture of glyceryl polymethacrylate, propylene glycol, and PVM / MA copolymer having the trade name LUBRAGEL OIL (all available from ISP); scleroglucan having the trade name Clearogel SC11 (available from Michel Mercier Products Inc); CARBOWAXPEG, POLYOX WASR, and UCON trade names. and ethylene oxide and / or propylene oxide based polymers with FLUIDS (all from Amerchol). Another class of thickening agents includes structuring agents such as stearyl alcohol, cetyl alcohol, and behenyl alcohol.

[0085] The compositions described herein may contain emulsifiers useful for dispersing and suspending the oil phase in the aqueous phase. When the compositions described herein contain an emulsifier, they contain no more than 1% of the emulsifier, preferably no more than 0.5%, more preferably no more than 0.2%. Examples of emulsifiers include polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), steareth-20, ceteareth-20, PPG-2 methyl glucose distearate, ceteth-10, polysorbate 80, cetyl phosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate, polysorbate 60, glyceryl stearate, PEG-100 stearate, polyoxyethylene 20 sorbitan trioleate (polysorbate 85), sorbitan monolaurate, polyoxyethylene 4 lauryl ether sodium stearate, polyglyceryl-4 isostearate, hexyl laurate, PPG-2 methyl glucose distearate, ceteth-10, diethanolamine cetyl phosphate, glyceryl stearate, PEG 40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, glycereth-25 PCA isostearate, and mixtures thereof.

[0086] The preservative can be any preservative known to those of skill in the art, such as, for example, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, diols, such as propanediol, butylene glycol, and pentylene glycol. Examples of emulsifiers include triethyl acetate, glyceryl caprate, benzoic acid, sorbitan caprate, glycerin, sodium levulinate, sodium anisate, pentylene glycol, phenylpropanol, gluconolactone, Lactobacillus ferment, Lactobacillus spp., Cocos Nucifera (coconut) fruit extract, fruit extract, Leuconostoc / radish root ferment filtrate, benzyl alcohol, dehydroacetic acid, potassium sorbate, sorbic acid, and mixtures thereof.

[0087] The compositions described herein may contain one or more additional cosmetic active ingredients that are useful for enhancing the effectiveness of the final cosmetic product.When the compositions described herein contain additional active ingredients, they contain the additional active ingredients at 20% or less, preferably 10% or less, preferably 1% or less, more preferably 0.1% or less.Examples of active ingredients include, but are not limited to, hyaluronic acid, hydrolyzed collagen, animal-derived collagen, marine collagen, copper peptides, peptides and derivatives, retinoids and derivatives, niacinamide, vitamin C, vitamin E, vitamin K, alpha hydroxy acid (AHA), beta hydroxy acid (BHA), ferulic acid, ceramide, and mixtures thereof.

[0088] The compositions described herein may further include moisturizing agents, emollients, exfoliants, non-vitamin antioxidants and radical scavengers, hair growth regulators, minerals, preservatives, plant sterols and / or plant hormones, protease inhibitors, tyrosinase inhibitors, anti-inflammatory agents, and N-acyl amino acid compounds.Suitable moisturizing agents include, but are not limited to, polyhydric alcohols such as polyalkylene glycols and their derivatives.Exemplary are propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerin, propoxylated glycerin, and mixtures thereof.Suitable emollients include, but are not limited to, hydrocarbons, fatty acids, fatty alcohols, and esters.

[0089] Further components in the compositions described herein can be hydrophilic polymers such as polyethylene glycol (PEG), monosaccharides, disaccharides such as mannose and trehalose, oligosaccharides, polysaccharides, other carbohydrates such as dextrins or dextrans, chelating agents such as EDTA, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and fatty acid esters, fatty acid ethers, or sugar esters.

[0090] The compositions described herein can be formulated in a variety of different forms, such as liquid, semi-solid, or solid dosage forms, depending on intended use.In one embodiment, the compositions described herein are formulated in a cream, emulsion, serum, aqueous solution, ointment, paste, lotion, suspension, gel, mask, skin cleanser such as soap, cleaning cream, cleaning lotion, cleaning milk, cleaning pad, face wash, hair shampoo, hair conditioner, or body shampoo.Preferably, the composition is included in a cream.In another embodiment, the compositions described herein are formulated as a solid dosage form, such as tablet, capsule, granule, or powder.

[0091] The compositions described herein are intended to be used externally (i.e., as a cosmetic or personal care product) on a subject. The compositions described herein may also be used internally (i.e., as a dietary supplement or biomedical product). Thus, in some embodiments, the compositions described herein are applied topically, for example, superficially (e.g., via microneedles) or via deep injection. In other embodiments, the compositions described herein are applied internally.

[0092] The compositions described herein compensate for the loss of collagen associated with aging and skin damage.

[0093] Thus, the compositions described herein with all of their embodiments may be used in repairing damaged skin or protecting the skin from oxidative damage, pigmentation, or aging.

[0094] Also provided herein is a method of repairing damaged skin or protecting skin from oxidative damage, pigmentation, or aging, comprising administering to the skin or skin cells of a subject a composition as described herein, including all of its embodiments. Alternatively, the composition may be administered internally.

[0095] Further provided herein is the use of the composition described herein with all of its embodiments as a skin moisturizer.

[0096] The compositions described herein are expected to nourish the skin, improve skin texture, skin balance, skin plumping, skin smoothness and reduce wrinkles.

[0097] In some embodiments, the compositions described herein are for use in treating skin disorders, including acne, rosacea, psoriasis, atopic dermatitis (eczema), contact dermatitis, and vitiligo.

[0098] Methods for preparing recombinant collagen polypeptides or collagen molecules In another aspect, the present invention provides a method for producing a composition comprising: (a) providing a nucleotide sequence encoding a collagen polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:2 and has a molecular weight of 1 to 80 kDa; (b) optionally providing a nucleotide sequence encoding a human prolyl-4-hydroxylase enzyme; (c) inserting the nucleotide sequence of (a) and optionally (b) into at least one vector; (d) expressing the vector of (c) in a cultured yeast cell, preferably Pichia pastoris; (e) isolating the recombinant collagen polypeptide or collagen molecule from the cultured yeast cells. The present invention provides a method for preparing a recombinant collagen polypeptide or collagen molecule, comprising:

[0099] In some embodiments, the encoded collagen polypeptide has a molecular weight of 2-45 kDa, hi some embodiments, the encoded collagen polypeptide has a molecular weight of 5-20 kDa.

[0100] In certain embodiments, the encoded collagen polypeptide may contain at least two PGP amino acid sequences.

[0101] In some embodiments, the amino acid sequence of the encoded collagen polypeptide comprises at least 10% proline. Preferably, the amino acid sequence of the encoded collagen polypeptide comprises at least 15% proline. More preferably, the amino acid sequence of the encoded collagen polypeptide comprises at least 20% proline.

[0102] In certain embodiments, the amino acid sequence of the collagen polypeptide comprises at least 1.5%, at least 2.5%, at least 5%, or at least 7.5% hydroxyproline.

[0103] In some embodiments, the amino acid sequence of the encoded collagen polypeptide comprises at least 15% glycine. Preferably, the amino acid sequence of the encoded collagen polypeptide comprises at least 20% glycine. More preferably, the amino acid sequence of the encoded collagen polypeptide comprises at least 25% glycine.

[0104] In some embodiments, the amino acid sequence of the encoded collagen polypeptide comprises a hydroxylated lysine.

[0105] In certain embodiments, the amino acid sequence of the encoded collagen polypeptide has an isoelectric point >7, preferably >8.

[0106] In some embodiments, the encoded collagen polypeptide is an antioxidant.

[0107] For example, the encoded collagen polypeptide can comprise an amino acid sequence that is GFSGLDGAKGD (SEQ ID NO:6) or that differs from GFSGLDGAKGD (SEQ ID NO:6) by up to three conservative amino acid substitutions.

[0108] In some embodiments, the encoded collagen polypeptide has anti-aging activity.

[0109] In some embodiments, the encoded collagen polypeptide is hypoallergenic.

[0110] In certain embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to one of SEQ ID NOs: 1-4 or 7-17. Preferably, the encoded collagen polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-4 and 7-17.

[0111] In preferred embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is at least 90% identical, at least 95% identical, or at least 98% identical to SEQ ID NO: 2. In particularly preferred embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is SEQ ID NO: 2.

[0112] In other preferred embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to one of SEQ ID NOs: 3, 21, and 22. In some of these embodiments, the encoded collagen polypeptide comprises the amino acid sequence of one of SEQ ID NOs: 3, 21, and 22.

[0113] In certain embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to one of SEQ ID NOs: 8, 10, 11, 12, 15, and 16. Preferably, the encoded collagen polypeptide comprises an amino acid sequence selected from one of SEQ ID NOs: 8, 10, 11, 12, 15, and 16.

[0114] In preferred embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to SEQ ID NO: 8. In some of these embodiments, the encoded collagen polypeptide comprises an amino acid sequence that is SEQ ID NO:8.

[0115] Various means for comparing sequence identity are disclosed hereinabove.

[0116] In certain embodiments, the recombinant collagen molecule produced by the present method comprises a homotrimer of three collagen polypeptides described herein having all of its embodiments.

[0117] In certain embodiments, the recombinant collagen polypeptide or collagen molecule produced by the present method is glycosylated.

[0118] In an embodiment, the nucleotide sequence encoding the collagen polypeptide that is a fragment of human type I collagen A1 is preceded by a nucleotide sequence encoding a secretory signal sequence.For example, the nucleotide sequence encoding the secretory signal sequence can encode the MatAlphaD secretory signal sequence (amino acid sequence of SEQ ID NO:5).Other secretory signal sequences, such as OST1, MatAlphaF, invertase, and native secretory signals from Homo sapiens, can also be used.

[0119] In certain embodiments, the human prolyl-4-hydroxylase enzyme is encoded by the amino acid sequence according to SEQ ID NO:18 and SEQ ID NO:19.

[0120] In one embodiment, the nucleotide sequences of (a) and / or (b) are inserted into a vector that uses a bidirectional promoter.

[0121] In certain embodiments, the nucleotide sequences of (a) and / or (b) are inserted into a vector that uses an inducible and derepressible promoter. For example, the promoter can be a methanol-inducible promoter.

[0122] In one embodiment, the nucleotide sequences of (a) and / or (b) are inserted into a vector that allows post-translational import into the endoplasmic reticulum.

[0123] The vector can be introduced into the yeast cells by conventional means such as transfection, transduction, or electroporation. Preferably, the vector is introduced into the yeast cells by electroporation of a linearized vector.

[0124] Yeast cells can be cultured at about 17 to about 23° C. (e.g., about 20° C.) with sufficient nutrients, such as a carbon source (e.g., glucose), a nitrogen source (e.g., NH4+), salts (e.g., Na+, K+, Mg2+, Ca2+, etc.), trace elements, peptone (a water-soluble mixture of polypeptides and amino acids formed by partial hydrolysis of proteins), and culture at an appropriate pH (e.g., acidic pH). Sufficient nutrients can be provided by yeast growth media known in the art, such as yeast and mold "YM" medium or yeast extract-peptone-dextrose (YPD) medium. Typically, yeast growth media are selective growth media with an acidic pH that permits the growth of yeast while preventing the growth of bacteria and other acid-intolerant organisms.

[0125] In an embodiment, the yeast cells used in the method are wild-type strains. Alternatively, the yeast cells used in the method may have methanol oxidase knocked out of their genome, which results in them growing slower on methanol. In a preferred embodiment, the yeast strain used in the method has a deletion of the AOX1 gene. An example of such a yeast is the MutS strain available from Invitrogen (KM71H), Graz University of Technology (CBS7435MutS), or Biogrammatics (BG11). The MutS strain still expresses Aox2 when methanol is used as the only carbon source, but more slowly than the wild-type strain.

[0126] In some embodiments, the recombinant collagen polypeptide or collagen molecule is isolated from the culture supernatant of cultured yeast cells. In this regard, for example, centrifugation, precipitation, or filtration may be used.

[0127] In some embodiments, the recombinant collagen molecule is an antioxidant.

[0128] In certain embodiments, the recombinant collagen polypeptides or collagen molecules produced by the methods described herein are vegan, i.e., the methods do not use animal products.

[0129] Also provided herein is a recombinant collagen polypeptide or collagen molecule prepared by the methods described herein, including all of its embodiments, wherein the collagen molecule comprises a homotrimer of three collagen polypeptides.

[0130] In certain embodiments, the recombinant collagen polypeptide prepared by the methods described herein contains at least 1.5%, at least 2.5%, at least 5%, or at least 7.5% hydroxyproline.

[0131] The definitions of words or drawing elements described herein are intended to include not only the literal combination of the described elements, but also all equivalent structures, materials, or operations for performing substantially the same function in substantially the same manner to obtain substantially the same results. Thus, in this sense, it is contemplated that for any one of the described elements and its various embodiments, an equivalent substitution of two or more elements may be made, or a single element may be substituted for two or more elements in a claim.

[0132] Variations from the claimed subject matter, now known or later invented, that are anticipated by those skilled in the art are expressly contemplated as equivalents within the intended scope and various embodiments thereof. Thus, obvious substitutions now or later known to those skilled in the art are defined as being within the scope of the defined elements. Thus, the present disclosure is intended to be understood to include those specifically illustrated and described herein, those that are conceptually equivalent, those that can be obviously substituted, and those that incorporate the essential idea.

[0133] matters A collagen polypeptide having an amino acid sequence that is at least 85% identical to SEQ ID NO:2 and having a molecular weight of 1.1 to 80 kDa.

[0134] 2. The collagen polypeptide according to claim 1, having a molecular weight of 2.2 to 45 kDa.

[0135] 2. The collagen polypeptide according to claim 1, having a molecular weight of 3.5 to 20 kDa.

[0136] 4. A collagen polypeptide described in any one of items 1 to 3, which is a human type I collagen A1 fragment.

[0137] 5. A collagen polypeptide described in any one of paragraphs 1 to 4, comprising at least two PGP amino acid sequences.

[0138] 6. A collagen polypeptide described in any one of paragraphs 1 to 5, wherein the amino acid sequence of the collagen polypeptide contains at least 2.5% hydroxyproline.

[0139] 7. A collagen polypeptide described in any one of paragraphs 1 to 6, wherein the amino acid sequence of the collagen polypeptide contains at least 15% glycine.

[0140] 8. A collagen polypeptide according to any one of paragraphs 1 to 7, comprising a hydroxylated lysine.

[0141] 9. A collagen polypeptide according to any one of paragraphs 1 to 8, having an isoelectric point of >7, preferably >8.

[0142] 10. A collagen polypeptide described in any one of paragraphs 1 to 9, which is an antioxidant.

[0143] 11. A collagen polypeptide described in any one of items 1 to 10, having anti-aging activity.

[0144] 12. A collagen polypeptide described in any one of paragraphs 1 to 11, which upregulates expression of one or more of SIRT2 / SIRT6, SOD2, GLRX, HMOX1, and AZGP1 in skin cells by at least 50% compared to baseline expression levels.

[0145] 13. A collagen polypeptide according to any one of clauses 1 to 12, which downregulates expression of MMP1 in skin cells by at least 50% compared to baseline expression levels.

[0146] 14. A collagen polypeptide according to any one of clauses 1 to 13, comprising an amino acid sequence which is GFSGLDGAKGD (SEQ ID NO: 6) or which differs from GFSGLDGAKGD (SEQ ID NO: 6) by up to three conservative amino acid substitutions.

[0147] 15. A collagen polypeptide according to any one of paragraphs 1 to 14, which is hypoallergenic.

[0148] 16. A collagen polypeptide according to any one of paragraphs 1 to 15, comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-4 or 7-17.

[0149] 17. A collagen polypeptide according to any one of paragraphs 1 to 16, comprising an amino acid sequence selected from SEQ ID NOs: 1 to 4 and 7 to 17.

[0150] 18. A collagen polypeptide described in any one of paragraphs 1 to 17, comprising an amino acid sequence which is SEQ ID NO:2.

[0151] 19. A collagen polypeptide described in any one of paragraphs 1 to 18, comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 3, 21, and 22.

[0152] 20. A collagen polypeptide described in any one of paragraphs 1 to 19, comprising an amino acid sequence selected from SEQ ID NOs: 3, 21, and 22.

[0153] 21. A collagen polypeptide according to any one of paragraphs 1 to 20, which is a recombinant collagen polypeptide.

[0154] 22. A collagen polypeptide described in any one of paragraphs 1 to 21, which is an isolated collagen polypeptide.

[0155] 23. A collagen molecule comprising a homotrimer of three collagen polypeptides according to any one of paragraphs 1 to 22.

[0156] 24. A collagen polypeptide according to any one of paragraphs 1 to 22 or a collagen molecule according to paragraph 23, which is glycosylated.

[0157] 25. A collagen polypeptide or collagen molecule according to any one of the preceding claims secreted from cultured yeast cells, preferably Pichia pastoris.

[0158] 26. A nucleic acid encoding a collagen polypeptide according to any one of paragraphs 1 to 22 or paragraphs 24 to 25.

[0159] 27. A nucleic acid encoding a collagen polypeptide comprising the amino acid sequence of one of SEQ ID NOs: 8, 10, 11, 12, 15, and 16, or a polypeptide at least 85% identical thereto.

[0160] 28. The nucleic acid of paragraph 27, wherein the encoded collagen polypeptide comprises the amino acid sequence of one of SEQ ID NOs: 8, 10, 11, 12, 15, and 16.

[0161] 29. An expression vector comprising a nucleic acid according to any one of paragraphs 26 to 28.

[0162] 30. A host cell comprising an expression vector according to paragraph 29 or a nucleic acid according to any one of paragraphs 26 to 28.

[0163] 31. A composition comprising a collagen polypeptide according to any one of paragraphs 1 to 22 or paragraphs 24 to 25, or a collagen molecule according to any one of paragraphs 23 to 25, and one or more excipients.

[0164] 32. The composition according to paragraph 31, which is contained in a cream, emulsion, serum, aqueous solution, ointment, paste, lotion, suspension, gel, mask, skin cleanser, cleansing cream, cleansing lotion, cleansing milk, cleansing pad, facial cleanser, hair shampoo, hair conditioner, or body shampoo.

[0165] 33. The composition according to paragraph 31, which is contained in a tablet, capsule, granule, or powder.

[0166] 34. A composition according to any one of paragraphs 31 to 33, comprising one or more additional active ingredients.

[0167] 35. A composition according to any one of paragraphs 31 to 34, for topical use on a subject.

[0168] 36. A composition according to any one of paragraphs 31 to 35 for use in treating a skin disorder.

[0169] 37. Use of a composition described in any one of paragraphs 31 to 36 in repairing damaged skin or protecting the skin from oxidative damage, pigmentation, or aging.

[0170] 38. A method for repairing damaged skin or protecting the skin from oxidative damage, pigmentation, or aging, comprising administering to a subject a composition described in any one of paragraphs 31 to 36.

[0171] 39. The method according to paragraph 38, wherein the composition is administered to the skin or skin cells of the subject.

[0172] 40. Use of a composition described in any one of paragraphs 31 to 36 as a skin moisturizer.

[0173] 41. (a) providing a nucleotide sequence encoding a collagen polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:2 and has a molecular weight of 1 to 80 kDa; (b) optionally providing a nucleotide sequence encoding a human prolyl-4-hydroxylase enzyme; (c) inserting the nucleotide sequence of (a) and optionally (b) into at least one vector; (d) expressing the vector of (c) in a cultured yeast cell, preferably Pichia pastoris; (e) isolating the recombinant collagen polypeptide or collagen molecule from the cultured yeast cells. 2. A method for preparing a recombinant collagen polypeptide or collagen molecule comprising:

[0174] 42. The method of paragraph 41, wherein the encoded collagen polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0175] 43. The method of paragraph 41 or 42, wherein the encoded collagen polypeptide has a molecular weight of 2 to 45 kDa.

[0176] 44. The method of paragraph 41 or 42, wherein the encoded collagen polypeptide has a molecular weight of 5 to 20 kDa.

[0177] 45. The method of any one of paragraphs 41 to 44, wherein the encoded collagen polypeptide comprises at least two PGP amino acid sequences.

[0178] 46. ​​The method of any one of paragraphs 41 to 45, wherein the amino acid sequence of the encoded collagen polypeptide contains at least 2.5% hydroxyproline.

[0179] 47. The method of any one of paragraphs 41 to 46, wherein the amino acid sequence of the encoded collagen polypeptide contains at least 15% glycines.

[0180] 48. The method of any one of paragraphs 41 to 47, wherein the encoded collagen polypeptide comprises a hydroxylated lysine.

[0181] 49. The method of any one of clauses 41 to 48, wherein the encoded collagen polypeptide has an isoelectric point >7, preferably >8.

[0182] 50. The method of any one of paragraphs 41 to 49, wherein the encoded collagen polypeptide is an antioxidant.

[0183] 51. The method of any one of clauses 41 to 50, wherein the encoded collagen polypeptide has anti-aging activity.

[0184] 52. The method of any one of clauses 41 to 51, wherein the encoded collagen polypeptide comprises an amino acid sequence that is GFSGLDGAKGD (SEQ ID NO: 6) or differs from GFSGLDGAKGD (SEQ ID NO: 6) by up to three conservative amino acid substitutions.

[0185] 53. The method of any one of paragraphs 41 to 52, wherein the encoded collagen polypeptide is hypoallergenic.

[0186] 54. The method of any one of clauses 41 to 53, wherein the encoded collagen polypeptide comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 1-4 or 7-17.

[0187] 55. The method of any one of clauses 41 to 53, wherein the encoded collagen polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-4 and 7-17.

[0188] 56. The method of any one of clauses 41 to 53, wherein the encoded collagen polypeptide comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 3, 21, and 22.

[0189] 57. The method of any one of clauses 41 to 53, wherein the encoded collagen polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 3, 21, and 22.

[0190] 58. The method of any one of paragraphs 41 to 57, wherein the recombinant collagen molecule comprises a homotrimer of three collagen polypeptides described in any one of paragraphs 1 to 22 and 24 to 28.

[0191] 59. The method of any one of clauses 41 to 58, wherein the recombinant collagen polypeptide or collagen molecule is glycosylated.

[0192] 60. The method of any one of paragraphs 41 to 59, wherein a nucleotide sequence encoding a collagen polypeptide comprising an amino acid sequence at least 85% identical to SEQ ID NO:2 is preceded by a nucleotide sequence encoding a secretory signal sequence.

[0193] 61. The method according to paragraph 60, wherein the nucleotide sequence encoding the secretory signal sequence encodes the amino acid sequence of SEQ ID NO:5.

[0194] 62. The method of any one of clauses 41 to 61, wherein the human prolyl-4-hydroxylase enzyme is encoded by the amino acid sequences according to SEQ ID NO:18 and SEQ ID NO:19.

[0195] 63. The method of any one of clauses 41 to 62, wherein the nucleotide sequences of (a) and / or (b) are inserted into a vector that uses a bidirectional promoter.

[0196] 64. The method of any one of clauses 41 to 63, wherein the nucleotide sequences of (a) and / or (b) are inserted into a vector that uses an inducible and derepressible promoter.

[0197] 65. The method of claim 64, wherein the promoter is methanol inducible.

[0198] 66. The method of any one of paragraphs 41 to 65, wherein the nucleotide sequences of (a) and / or (b) are inserted into a vector enabling post-translational import into the endoplasmic reticulum.

[0199] 67. The method of any one of paragraphs 41 to 66, wherein the recombinant collagen polypeptide or collagen molecule is isolated from the culture supernatant of cultured yeast cells.

[0200] 68. A recombinant collagen polypeptide or collagen molecule prepared by the method of any one of paragraphs 41 to 68.

[0201] 69. The recombinant collagen molecule of paragraph 68, comprising a homotrimer of three collagen polypeptides. EXAMPLES

[0202] Example 1: Development of collagen polypeptides and collagen molecules A collagen amino acid sequence (SEQ ID NO:22) corresponding to a fragment of human type I collagen A1 was used in the examples (see, for example, SEQ ID NO:1 and SEQ ID NO:2).

[0203] In order to balance water solubility and skin permeability, an amino acid sequence of human type I collagen A1 having a molecular weight of 1 to 80 kDa, preferably 2 to 45 kDa, was selected.

[0204] Most proteins reach their net charge state in the pH range of 5.5 to 8. This is the lowest solubility state of the protein, called the isoelectric point. To facilitate the preparation of skin care (typically pH 5.5) or personal care formulations, the protein should be in its soluble state at pH 5.5, i.e., charged on its surface.

[0205] The amino acid sequence of human type I collagen A1 is selected to contain proline, preferably to be rich in proline.Proline in the amino acid sequence of collagen is hydroxylated in endoplasmic reticulum during collagen synthesis.Higher hydroxyproline ratio in collagen is expected to correspond to higher moisturizing and anti-aging activity.Higher hydroxyproline ratio is also expected to improve performance as a dietary supplement, since one potential mechanism of action is related to the passage of hydroxyproline-containing dipeptides and tripeptides into the bloodstream.

[0206] The collagen polypeptide of the invention is based on a fragment of human type I collagen A1 comprising the amino acid sequence of SEQ ID NO:2 (see table below).

[0207] To further facilitate formation of higher-order structures of collagen polypeptides, some constructs for expression of collagen polypeptides were prepared with sequences encoding the human type I collagen A1 propeptide region at the N-terminus (SEQ ID NO:3) and / or C-terminus (SEQ ID NO:4). In these constructs, a KEX2 cleavage site ("EKR") was engineered between the collagen sequence and the C- and N-terminal propeptide regions to allow cleavage removal of the C- and / or N-terminal propeptides. KEX2 is a protease native to Pichia pastoris.

[0208] Some constructs were prepared with nucleotide sequences encoding the yeast MatAlphaD secretion signal sequence (SEQ ID NO:5) to facilitate secretion of the encoded collagen polypeptide from Pichia pastoris cells. MatAlphaD refers to a deletion mutant of the mating factor alpha 1 prepropeptide that has been shown to result in increased levels of some secreted proteins compared to the full-length signal sequence (GPLin-Cereghino et al., Gene, 2013, 519, 311-317). MatAlphaD is believed to facilitate post-translational translocation into the endoplasmic reticulum (ER). Without wishing to be bound by theory, it is believed that secretion signal sequences that increase residence time in the ER increase hydroxylation of collagen polypeptides by the co-expressed prolyl-4 hydroxylase in the ER. Controlling the time spent in the ER in this way facilitates a balance between collagen hydroxylation and collagen polypeptide secretion.

[0209] The above-referenced amino acid sequences are shown in Table 1.

[0210] [Table 1]

[0211] As can be seen above, SEQ ID NOs: 1-4 contain a PGP motif.

[0212] Constructs were designed and prepared for expression of collagen polypeptides using combinations of the sequences in Table 1. Some constructs were prepared for expression in yeast, such as Pichia pastoris.

[0213] Examples of these constructs, along with the amino acid sequences they encode, are provided in Table 2. SEQ ID NOs: 7-17 correspond to constructs #1-11, respectively.

[0214] The amino acid sequences of collagen polypeptides produced using constructs #1-11 are shown in bold and italics in Table 2.

[0215] [Table 2] JPEG2025502343000003.jpg252157JPEG2025502343000004.jpg254157JPEG2025502343000005.jpg253157JPEG2025502343000006.jpg64156

[0216] In the above table, underlined sequences = MatAlphaD secretion signal (SEQ ID NO: 5), bold and italicized sequences: sequences of collagen polypeptides produced with the respective constructs.

[0217] The promoter used in the construct is a methanol-inducible promoter that is also a derepressible promoter, meaning that its activity is already initiated at low concentrations of a carbon source (e.g. glucose) and can then be further boosted by the addition of methanol. This promoter therefore allows recombinant protein production under methanol-free conditions, if desired. Since some transcriptional activity is already present before methanol introduction, the cells may already be adapted to this condition before expression is boosted.

[0218] As can be derived from Table 2 above, the collagen polypeptides produced using constructs #2, #4, #5, #6, #9, and #10 (corresponding to SEQ ID NOs: 8, 10, 11, 12, 15, and 16, respectively) contain the "80AA" fragment of human type I collagen A1 (SEQ ID NO: 2).

[0219] The sequences of collagen polypeptides produced using constructs #2, #4, #5, #6, #9, and #10 are set forth in Table 3 below.

[0220] The collagen polypeptides obtained using constructs #2, #4, and #9 correspond to the amino acid sequence of SEQ ID NO: 2. The collagen polypeptides obtained using constructs #5, #6, and #10 correspond to the amino acid sequences of SEQ ID NOs: 21, 22, and 3, respectively, which include SEQ ID NO: 2.

[0221] [Table 3]

[0222] The amino acid sequences of human prolyl-4-hydroxylase (P4HA and P4HB) and human type I collagen A1 are provided in Table 4 below.

[0223] [Table 4]

[0224] Example 2: Protein secretion screening and characterization of fragments of Col1a1 collagen from Pichia pastoris The inventors overexpressed a construct encoding the human type I collagen A1 polypeptide according to the construct defined in Example 1.

[0225] We co-overexpressed the proline hydroxylation machinery (prolyl-4-hydroxylase (P4HA and B heterotetramer A2B2, from Homo sapiens) in the Pichia pastoris system under bidirectional promoters by random integration of the construct in the genome.

[0226] material and method: Transformation of Pichia pastoris P. pastoris P4H platform strain was transformed with 1 μg of the SmiI linear expression construct by electroporation. After 3 h of regeneration at 30 °C and 550 rpm in 1 mL of YPD / sorbitol (1:1), cells were plated onto YPD agar plates containing 100 mg / L Zeocin.

[0227] Cultivation of Pichia pastoris and induction of expression Deep Well Plate Culture Protocol: Methanol-dependent deep-well plate culture protocol (screening and rescreening): · Medium is prepared under sterile conditions in 96-well deep-well plates. A fresh single colony of the target P. pastoris strain is inoculated into 250 μL of BMG1 using a sterile toothpick. Incubate for 60 hours at 28°C, 320 rpm, and 80% humidity. After 60 h of growth, preserve the clones by stamping them onto YPD agar square plates and induce the cultures by adding 250 μL of BMM2. Further induction by addition of 50 μL of BMM10 at 68, 84 and 92 hours after inoculation. Cultures are stopped after a total of 108 hours (= 48 hours of induction). · Centrifuge the cells at 4000 rpm for 10 min at 4°C, harvest the supernatant and apply activity assays or perform SDS-PAGE analysis to detect the target protein.

[0228] Derepressed (no methanol) deep well plate culture protocol (screening): · The medium is prepared under sterile conditions in 96-well deep well plates. Inoculate 250 μL of BMG1 with a fresh single colony of the target P. pastoris strain using a sterile toothpick. Incubate for 60 hours at 28°C, 320 rpm, and 80% humidity. After 60 h of growth, preserve the clones by stamping them onto YPD agar square plates and induce the cultures by adding 250 μL of BMG0.5. Further induction by addition of 50 μL of BMG2.5 at 68, 84 and 92 hours after inoculation. Cultures are stopped after a total of 108 hours (= 48 hours of induction). · Centrifuge the cells at 4000 rpm for 10 min at 4°C, harvest the supernatant and apply activity assays or perform SDS-PAGE or native gel analysis to detect the target protein.

[0229] Reagents, chemicals, and materials composition · Sterile 250mL to 2.5L baffled shake flask. · YPD (1% w / v yeast extract, 2% w / v peptone, 2% w / v glucose). · BMM10 (buffered minimal methanol containing 5% methanol: 1.34% yeast nitrogen-based amino acid free, 4 × 10-5% biotin, 200 mM potassium phosphate buffer, pH 6.0, and 5% methanol). · BMM2 (Buffered Minimal Methanol containing 5% methanol: 1.34% yeast nitrogen-based amino acid free, 4 × 10-5% biotin, 200 mM potassium phosphate buffer, pH 6.0, and 1% methanol). · BMG1 (buffered minimal methanol containing 5% methanol:1.34% yeast nitrogen base free amino acids, 4 × 10-5% biotin, 200 mM potassium phosphate buffer, pH 6.0, and 1% glycerol). · BMG 0.5 (buffered minimal methanol containing 5% methanol:1.34% yeast nitrogen-based amino acid free, 4 x 10-5% biotin, 200 mM potassium phosphate buffer, pH 6.0, and 0.5% glycerol). · BMG2.5 (buffered minimal methanol containing 5% methanol:1.34% yeast nitrogen-based amino acid free, 4 x 10-5% biotin, 200 mM potassium phosphate buffer, pH 6.0, and 2.5% glycerol). Pure methanol.

[0230] SDS PAGE Protocol: Protein analysis by electrophoresis in denaturing gels (SDS-PAGE): Evaluate protein production profiles by separating proteins based on their molecular weight. Standard protocol for running Bolt Bis-Tris Plus Mini Gels: Centrifuge the broth (4000rcf, 10 minutes). Separate the supernatant from the cellular fraction. Measure the protein concentration of each sample. Normalize the protein concentrations in the samples for analysis. Mix the sample with 4x loading buffer containing LDS and 10x reducing agent containing DTT. Complete with distilled water to 30 μL. Load 15-20 μL of sample / well into a Bis-Tris 4-16% gel. Run in MES running buffer (200V, 25 minutes) Resolve the gel by incubating in BlueSafe protein stain for 45 minutes and washing overnight with distilled water.

[0231] SDS PAGE using pepsin digested protein suspension: After lyophilization, the protein solution was diluted in 50 mM potassium phosphate buffer (pH 2.2) and treated with 0.05 mg / mL pepsin at 22°C for 20 hours.

[0232] result: The overexpressed collagen fragments in Pichia pastoris are shown by SDS-PAGE gels in Figure 1. These SDS PAGE gels show the expression of collagen polypeptides of constructs #2, 4, 6, and 8 (constructs #2 (SEQ ID NO:8), 4 (SEQ ID NO:10), 6 (SEQ ID NO:12), and 8 (SEQ ID NO:14) - see Table 2). The stars indicate the two subunits of the P4H machinery.

[0233] Characterization of homotrimerization of the COL1A1 fragment of human collagen in Pichia pastoris According to Vuorela et al., EMBO Journal, 1997, vol. 16, no. 22, there is a complex regulation between overexpression of the P4H machinery and assembled collagen fragments. The P4HA subunit has previously been described as being retained in the ER. In our experiments, it was occasionally secreted into the supernatant (see FIG. 1), which indicates that, for example, when coexpressed with construct #2 (SEQ ID NO: 8), the P4H subunit was secreted into the supernatant.

[0234] Further SDS-PAGE analysis in Figure 2 shows homotrimeric collagen fragments produced and secreted by Pichia pastoris, with full-length constructs migrating at approximately 25 kDa (indicated by red arrows for constructs #5 and 6 (SEQ ID NO:11 and SEQ ID NO:12, respectively)) as well as cleavage products (e.g., SEQ ID NO:21 and 22) shown to be resistant to digestion with pepsin, which is the canonical way to show homotrimeric structures.

[0235] Example 3: Amino acid sequencing method ·Analysis of generated peptides by NanoHPLC-MS / MS. · Identification of proteins found in the secretome of selected strains. Analysis of proline hydroxylation as a post-translational modification (PTM). At least 50% coverage is expected. · Proteolytic enzymes: Trypsin is the recommended standard.

[0236] result The peptide analysis results from the two isolated bands from the SDS gel are shown below: Collagen polypeptide 1 (SEQ ID NO: 2): 7.3 kDa band, produced with construct #2 (see FIG. 2) Collagen polypeptide 2 (SEQ ID NO: 22): 11.2 kDa band, produced with construct #6 (see FIG. 2) Proteolytic in-gel digestion (trypsin) and analysis of the resulting peptides on a tandem mass spectrometer revealed that all selected protein bands were likely collagens. The peptides belonging to the collagen in question were found among the 10 most abundant peptides in the samples analyzed. · Since none of the samples showed a peptide corresponding to the secretion signal MatAlphaD, it is assumed that processing of the secretion peptide was complete for all constructs analyzed.

[0237] The following results are a further exploration of the bands mentioned above.

[0238] Collagen Polypeptide 1 (SEQ ID NO:2) Collagen sequence coverage is 55%. The analysis revealed that at least 28% of the native sites of proline hydroxylation were hydroxylated in the peptide, corresponding to 2.5% hydroxyproline.

[0239] Collagen Polypeptide 2 (SEQ ID NO:22) Collagen sequence coverage is 43%. Collagen fragments as well as N- and C-terminal fragments were found. The analysis revealed that at least 15% of the native sites of proline hydroxylation were hydroxylated in the peptide, corresponding to 1.6% hydroxyproline.

[0240] Example 4: Cytotoxicity assays and gene expression analysis Changes in the expression of various genes described to be associated with skin aging were determined by mRNA sequencing in normal human dermal keratinocytes (NHEKs) after treatment with various collagens.

[0241] method 1. Cytotoxicity Assay Test model: First, the cytotoxicity of various products (samples 1-7, below) at various concentrations on normal human dermal keratinocytes (NHEK) was determined.

[0242] sample: The products tested are labeled as follows: Sample 1 - Buffer solution (PBS) for resuspending the target protein Sample 2 - 2.25 wt% of target protein (SEQ ID NO:2) in a buffer solution (see Sample 1) - Cambrium vegan human collagen Sample 3 - Buffer solution for resuspending bovine collagen and hydrolyzed collagen (20 mM acetic acid) Sample 4 - 2.25 wt% bovine collagen in buffer solution (see Sample 3) - Gibco™ Collagen I, bovine (sold by ThermoFisher, product code A1064401, November 23, 2018) Sample 5 - 2.25 wt% hydrolyzed collagen in buffer solution (see sample 3) - iCONFIT hydrolyzed collagen (obtained from iCONFIT European Foods OU, Tallinn) Sample 6 - Buffer solution for resuspending synthetic collagen (Aqua, composed of 5 w / v% hexanediol and 5 w / v% butylene glycol) Sample 7 - Synthetic collagen solution in buffer solution (see sample 6) - INCI: sh-polypeptide-121

[0243] protocol: All samples were directly diluted in the appropriate culture medium at four different concentrations: 2.5%, 1%, 0.5%, and 0.1% (dilution factors of 40, 100, 200, and 1000). Experiments were performed in triplicate (n=3).

[0244] The products were incubated with NHEKs for 24 hours at 37°C. During the last 3 hours of the 24-hour incubation period, the cell proliferation reagent WST1 (Roche) was introduced into the culture medium. WST1 contains a tetrazolium salt that is cleaved by metabolically active cells into a formazan dye (yellow indicator). The level of yellow staining (determined by absorbance at 450 nm) is proportional to the number of live cells, and values ​​between 80% and 120% of the untreated control indicate the absence of cytotoxicity of the products.

[0245] 2. Gene Expression Analysis Test model: Gene expression changes in normal human dermal keratinocytes (NHEKs) following treatment with samples 1-7 were determined by mRNA sequencing.

[0246] sample: Experiments were performed in triplicate (n=3) using the combinations shown below. Untreated NHEK (NT) NHEK+2.5% sample 1 NHEK+2.5% sample 2 NHEK+2.5% sample 3 NHEK+2.5% sample 4 NHEK+2.5% sample 5 NHEK+2.5% sample 6 NHEK+2.5% sample 7

[0247] In detail, NHEKs were cultured in 96-well plates at a concentration of 10,000 cells / well in the presence of appropriate culture medium (Promocell). After attachment, 2.5% of samples 1-7 (see above) were added to the NHEKs and incubated for 24 h.

[0248] Subsequently, mRNA was extracted from NHEKs, subjected to reverse transcription, and DNA was prepared for sequencing according to the Fluidigm protocol used for real-time PCR (96 genes).

[0249] The samples were tested against a list of genes known to be involved in skin cell pathways relevant to common cosmetic applications.

[0250] result 1. Cytotoxicity Assay For samples 1-7, no cytotoxic activity against NHEKs was detected at concentrations below 2.5% (as indicated by a viable cell percentage of 80-120% of untreated control cells, see Figure 4).

[0251] 2. Gene Expression Analysis Table 5 and Figure 5 show changes in expression of specific genes associated with curative and preventative anti-aging and other beneficial skin effects in NHEKs following treatment with samples 2, 4, 5, and 7 (percentage of up / down regulation compared to basal levels in NHEKs is shown).

[0252] Only changes greater than 50% induction and less than 50% inhibition are shown.

[0253] [Table 5]

[0254] In the above table, MMP1: matrix metallopeptidase 1, SIRT2 / SIRT6: sirtuins 2 and 6, SOD2: mitochondrial superoxide dismutase 2, GLRX: glutaredoxin, HMOX1: heme oxygenase 1, AZGP1: alpha-2-glycoprotein 1, zinc binding. (-) indicates that underexpression of this gene may be associated with beneficial effects on the skin. (+) indicates that overexpression of this gene may be associated with beneficial activities on the skin.

[0255] Consideration Notably, treatment with Sample 2 affected the expression (up- and down-regulation) of a variety of different genes described to be involved in preventive and curative anti-aging, anti-oxidation, and anti-pigmentation in NHEK cells compared to treatment with other collagens (see Table 5: inhibition of expression of MMP1, activation of expression of SIRT2 / SIRT6, SOD2, GLRX, HMOX1, and AZGP1).

[0256] As summarized in Table 6 below, the up- and down-regulation of expression of these genes in NHEKs following treatment with Sample 2 suggests a beneficial effect on the skin.

[0257] [Table 6]

[0258] Thus, the data generated show that treatment with sample 2 was associated with beneficial skin activity in preventing and curing aging by activating the skin's antioxidant defenses as an effective way to prevent aging, as well as by inhibiting collagen degradation and contributing to the regulation of cell proliferation and longevity as an effective way to modulate some of the effects caused by aging.

[0259] conclusion These data suggest that the collagen polypeptides of the present invention exhibit potent anti-aging activity by affecting the transcriptional regulation of a variety of different genes that have been linked to skin aging, thereby slowing the intrinsic aging process in skin cells.

Claims

1. A collagen polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:2, said collagen polypeptide having a molecular weight of 1 to 80 kDa.

2. The collagen polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO:

2.

3. 3. A collagen polypeptide according to claim 1 or claim 2, wherein the amino acid sequence of the collagen polypeptide contains at least 2.5% hydroxyproline.

4. A collagen polypeptide according to any one of claims 1 to 3, having an isoelectric point of >7, preferably >8.

5. A collagen polypeptide according to any one of claims 1 to 4, having a molecular weight of 2 to 45 kDa.

6. The collagen polypeptide of claim 5 having a molecular weight of 5 to 20 kDa.

7. A collagen polypeptide according to any one of claims 1 to 6, having anti-aging activity.

8. A collagen molecule comprising a homotrimer of three collagen polypeptides according to any one of claims 1 to 7.

9. A nucleic acid encoding a collagen polypeptide according to any one of claims 1 to 7.

10. An expression vector comprising the nucleic acid of claim 9.

11. 11. A host cell comprising the expression vector of claim 10 or the nucleic acid of claim 9.

12. A composition comprising a collagen polypeptide according to any one of claims 1 to 7 or a collagen molecule according to claim 8, and one or more excipients.

13. 13. The composition of claim 12 comprised in a cream, emulsion, serum, aqueous solution, ointment, paste, lotion, suspension, gel, mask, skin cleanser, cleansing cream, cleansing lotion, cleansing milk, cleansing pad, facial cleanser, hair shampoo, hair conditioner, or body shampoo.

14. The composition of claim 12 contained in a tablet, capsule, granule, or powder.

15. 15. The composition of any one of claims 12 to 14, comprising one or more additional active ingredients.

16. 16. Use of a composition according to any one of claims 12 to 15 in repairing damaged skin or protecting the skin from oxidative damage, pigmentation or aging.

17. 16. Use of a composition according to any one of claims 12 to 15 as a skin moisturizer.

18. (a) providing a nucleotide sequence encoding a collagen polypeptide comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:2, the collagen polypeptide having a molecular weight of 1-80 kDa; (b) optionally providing a nucleotide sequence encoding a human prolyl-4-hydroxylase enzyme; (c) inserting the nucleotide sequence of (a) and optionally (b) into at least one vector; (d) expressing the vector of (c) in cultured yeast cells; (e) isolating the recombinant collagen polypeptide or collagen molecule from the cultured yeast cells; 2. A method for preparing a recombinant collagen polypeptide or collagen molecule comprising:

19. 20. The method of claim 18, wherein the yeast cell is a Pichia pastoris cell.

20. 20. The method of claim 18 or 19, wherein the nucleotide sequences of (a) and / or (b) are inserted into a vector enabling post-translational import into the endoplasmic reticulum.

21. 21. The method of any one of claims 18 to 20, wherein the recombinant collagen polypeptide or collagen molecule is isolated from the culture supernatant of cultured yeast cells.

22. 22. A recombinant collagen polypeptide or collagen molecule prepared by the method of any one of claims 18 to 21.

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