Topical formulations of recombinant collagen

Abstract

The present disclosure provides methods for improving the firmness, elasticity, brightness, hydration, tactile texture, or visual texture of the skin. The methods include topically applying to the skin a non-naturally occurring truncated collagen.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 827,662, filed on April 1, 2019, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Collagen and similar proteins are the most abundant proteins in the biosphere. Collagen and structured proteins are found in the skin, connective tissue, and bones of animals, as well as in other tissues. The amount of collagen present in the body is approximately one-third of the total protein and accounts for about three-fourths of the dry weight of the skin.

[0004] The structure of native collagen can be a triple helix, where three polypeptide chains come together to form a helical coil. A single polypeptide chain consists of a repeating tripeptide amino acid sequence designated GLY-X-Y. X and Y can be any amino acid, and the first amino acid is glycine. Collagen has a relatively high concentration of the amino acids proline and hydroxyproline. The most common tripeptide is glycine-proline-hydroxyproline (Gly-Pro-Hyp), which accounts for approximately 10.5% of the tripeptides in collagen.

[0005] Gelatin is a product obtained by partial hydrolysis of some (e.g., native) collagen. Typically, gelatin is produced by acid hydrolysis, base hydrolysis, and enzymatic hydrolysis, or by exposing collagen to heat in an aqueous solution (e.g., boiling animal bones and skin, boiling fish scales, etc.).

[0006] Gelatin is used in a variety of products, including cosmetics, food, pharmaceuticals, medical devices, film, adhesives, binders, and so on. The physical and chemical properties of gelatin can be adjusted according to the specific application. These physical / chemical properties include gel strength, melting point temperature, viscosity, color, turbidity, pH, isoelectric point, and so on. SUMMARY OF THE INVENTION

[0007] In certain embodiments, provided herein are various polypeptides, compositions comprising such polypeptides, and methods of using such polypeptides and / or their compositions. In certain embodiments, such polypeptides include non-natural and / or recombinant polypeptides, such as those comprising one or more amino acid sequences that are truncated relative to native collagen, such as the native collagen described herein. In certain instances, such polypeptides are described herein as "truncated collagen." In particular embodiments, the polypeptide comprises one or more (e.g., two or more) truncated amino acid sequences of native human collagen. In particular embodiments, the polypeptide comprises one or more (e.g., two or more) truncated amino acid sequences of native jellyfish collagen. In one aspect, provided is a method of providing a benefit to the skin (e.g., the skin of an individual such as a human), such as increasing the firmness, elasticity, brightness, hydration, tactile texture, or visual texture of the skin. In some embodiments, the method comprises topically applying to the skin a polypeptide described herein (e.g., a non-naturally occurring truncated collagen, such as those described herein) or a formulation (e.g., comprising a polypeptide, such as a non-naturally occurring truncated collagen).

[0008] In a particular aspect, provided is a method of reducing the presence of folds or wrinkles on the skin or reducing skin redness. In some embodiments, the method comprises topically applying to the skin a polypeptide described herein (e.g., a non-naturally occurring truncated collagen, such as those described herein) or a formulation thereof. In some embodiments, provided herein are also formulations comprising a polypeptide described herein, such as a non-naturally occurring truncated collagen.

[0009] In certain instances, the polypeptides described herein (e.g., truncated collagen) can be used to increase the firmness, elasticity, brightness, hydration, tactile texture, and / or visual texture of the skin. In some instances, the polypeptides described herein (e.g., truncated collagen) can be used to reduce the presence of folds or wrinkles on the skin or reduce skin redness. In particular embodiments, the polypeptide (e.g., truncated collagen) is or comprises truncated jellyfish collagen (e.g., a truncated amino acid sequence of native jellyfish collagen). In other particular embodiments, the polypeptide (e.g., truncated collagen) is or comprises truncated human collagen (e.g., a truncated amino acid sequence of native human collagen).

[0010] In some embodiments, the polypeptide (e.g., truncated collagen) (such as can be used in the methods disclosed herein) is or comprises a truncated amino acid sequence relative to native (e.g., human or jellyfish (hydrozoan)) collagen. In some cases, such polypeptides are referred to herein as non-naturally occurring collagens. In certain embodiments, the non-naturally occurring collagen is or comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, and / or SEQ ID NO: 29, or homologs thereof (e.g., having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity thereto). In more specific embodiments, the non-naturally occurring collagen is the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29, or homologs thereof (e.g., having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity thereto).

[0011] In certain embodiments, compositions are provided herein. In some embodiments, such compositions comprise a polypeptide (e.g., truncated or non-native collagen) described herein. In one aspect, a composition is provided that comprises any suitable amount, such as 0.005% to 30% w / w, of any polypeptide provided herein (e.g., truncated or non-naturally occurring collagen). The composition may further comprise at least one additional ingredient, the at least one additional ingredient including an excipient, a topical carrier, or a preservative.

[0012] In certain embodiments, the compositions provided herein are topical compositions, e.g., compositions that are formulated and / or suitable for topical administration or use. In one aspect, provided herein is a method of providing (e.g., as described herein) a benefit to the skin of an individual, the method comprising topically administering the topical composition to the skin. In certain embodiments, the topical composition is used in a method for reducing skin damage, promoting repair of damaged skin, or stimulating skin cells to produce collagen. In certain embodiments, the topical composition is used in a method for increasing, promoting, stimulating, or otherwise increasing elastin production in the skin.

[0013] One aspect provides a method of applying the collagen or a composition comprising collagen to the skin of a subject.

[0014] In some embodiments, as discussed herein, the polypeptides provided herein are or comprise truncated amino acid sequences relative to native (e.g., human or jellyfish (hydrozoan)) collagen. In certain embodiments, such polypeptides are truncated collagen (e.g., comprising one or more truncated amino acid sequences relative to native collagen). In some embodiments, the truncated collagen is jellyfish collagen or human collagen. In some embodiments, the collagen (e.g., relative to native collagen) is truncated at the C-terminus, N-terminus, internally, or at both the C-terminus and N-terminus. In one embodiment, the collagen (e.g., relative to native collagen) is truncated at both the C-terminus and N-terminus. In certain embodiments, the polypeptides provided herein are or comprise truncated collagen having any suitable truncation, such as C-terminal truncation, N-terminal truncation, and / or one or more internal truncations. In some embodiments, the truncation of the collagen is suitable for achieving a beneficial result (e.g., an improved result relative to native collagen and / or an additional benefit relative to native collagen) and / or shortening the length of the collagen while retaining one or more beneficial aspects of collagen. In some embodiments, the polypeptides provided herein are or comprise collagen truncated in a manner that, e.g., retains one or more local benefits of collagen.

[0015] In some embodiments, the truncated collagen (amino acid sequence thereof), such as the polypeptides provided herein, is truncated at the C-terminus by any suitable number of amino acid residues, such as up to 10, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, etc. In certain embodiments, the truncated collagen (amino acid sequence thereof), such as the polypeptides provided herein, is truncated at the N-terminus by any suitable number of amino acid residues, such as up to 10, 10 to 900, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, etc. In some embodiments, the truncated collagen (amino acid sequence thereof), such as the polypeptides provided herein, is truncated internally by any suitable number of amino acid residues, such as up to 10, 10 to 900, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, etc. In certain embodiments, the truncated collagen (amino acid sequence thereof), such as the polypeptides provided herein, is truncated at the C-terminus by 10 to 800 amino acids and / or at the N-terminus by 10 to 800 amino acids. In another embodiment, the truncated collagen (amino acid sequence thereof), such as the polypeptides provided herein, has a length of 10 to 900 amino acids, 10 to 800 amino acids, 10 to 700 amino acids, 10 to 600 amino acids, 10 to 500 amino acids, 10 to 400 amino acids, 10 to 300 amino acids, 10 to 200 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, 50 to 800 amino acids, 50 to 700 amino acids, 50 to 600 amino acids, 50 to 500 amino acids, 50 to 400 amino acids, 50 to 300 amino acids, 50 to 200 amino acids, or 50 to 100 amino acids.

[0016] In certain embodiments, the present disclosure provides polypeptides that are or comprise the amino acid sequence of human (e.g., human type XXI) collagen. In specific embodiments, the truncated human collagen is truncated human type XXI collagen. In various embodiments, the truncation is according to any disclosure provided herein. In a specific embodiment, the disclosed truncated human type XXI collagen is SEQ ID NO: 16 (or a homolog thereof, e.g., having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or other sequence identities provided herein with the amino acid sequence of SEQ ID NO: 16). In various embodiments, such polypeptides are provided in any composition, formulation, or method provided herein.

[0017] In certain embodiments, the present disclosure provides polypeptides that are or comprise the amino acid sequence of jellyfish (hydrozoan) collagen. In various embodiments, the truncation is according to any disclosure provided herein. In a specific embodiment, the disclosed truncated jellyfish collagen is SEQ ID NO: 5 (or a homolog thereof, e.g., having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or other sequence identities provided herein with the amino acid sequence of SEQ ID NO: 5). In various embodiments, such polypeptides are provided in any composition, formulation, or method provided herein.

[0018] In certain embodiments, the present disclosure provides a method comprising administering a polypeptide (e.g., that is or comprises a truncated collagen as described herein) to the skin of an individual, e.g., to provide a benefit to the individual or their skin. In some cases, the benefit provided to the skin is improving the firmness of the skin, improving the elasticity of the skin, improving the hydration of the skin, improving the texture of the skin, improving the brightness of the skin, reducing the wrinkles of the skin, reducing the redness of the skin, improving collagen production in the skin, improving or increasing elastin production in the skin, providing antioxidant protection to the skin, reducing the flushing of the skin, or other benefits or combinations of benefits such as those described herein. In many cases, the improvement or benefit in skin properties provided by the methods provided herein is determined in any suitable manner, e.g., by using an instrument or by clinician evaluation. In one aspect, a method of increasing skin firmness is provided, wherein the firmness of the skin is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 75%. In one embodiment, the firmness of the skin is measured using a cutometer.

[0019] In another aspect, a method for increasing skin elasticity is provided, wherein the elasticity of the skin is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75%. In one embodiment, the elasticity of the skin is measured using a skin elasticity tester.

[0020] In another aspect, a method for increasing skin hydration is provided, wherein the hydration of the skin is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or 75%. In one embodiment, skin hydration is measured on a corneometer.

[0021] In one aspect, a method for increasing skin firmness is provided, wherein the firmness of the skin is increased. In one embodiment, the firmness of the skin is determined by a professional clinical rater.

[0022] In one aspect, a method for increasing skin elasticity is provided, wherein the elasticity of the skin is increased. In one embodiment, the elasticity of the skin is determined by a professional clinical rater.

[0023] In one aspect, a method for increasing skin brightness is provided, wherein the brightness of the skin is increased. In one embodiment, the brightness of the skin is determined by a professional clinical rater.

[0024] In another aspect, a method for increasing the tactile texture of the skin is provided, wherein the tactile texture of the skin is increased. In one embodiment, the tactile texture of the skin is determined by a professional clinical rater.

[0025] In one aspect, a method for increasing the visual texture of the skin is provided, wherein the visual texture of the skin is increased. In one embodiment, the visual texture of the skin is determined by a professional clinical rater.

[0026] In one aspect, a method for reducing the folds or wrinkles present on the skin is provided, wherein the folds or wrinkles present on the skin are reduced. In one embodiment, the amount of folds or wrinkles present on the skin is determined by a professional clinical rater.

[0027] In one aspect, a method for reducing skin rash is provided, wherein the skin rash is reduced. In one embodiment, the skin rash is determined by a professional clinical rater.

[0028] On the other hand, a method for stimulating collagen production in skin cells is provided. In one embodiment, the method comprises applying to the skin a non-naturally occurring truncated collagen or a preparation comprising a non-naturally occurring truncated collagen. In one embodiment, the truncated jellyfish collagen or the truncated human collagen stimulates collagen production. In a particular embodiment, the truncated human collagen is the truncated human type XXI collagen of SEQ ID NO: 16. In another particular embodiment, the truncated jellyfish collagen is the truncated jellyfish collagen of SEQ ID NO: 5.

[0029] In yet another aspect, a method for stimulating collagen production in skin cells is provided, wherein the collagen in the skin is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10%.

[0030] A topical preparation is disclosed that comprises a truncated collagen and one or more additional components selected from water, oil, glycereth-8 ester, glycerin, cocoalkane, hydroxyethyl acrylate / sodium acryloyldimethyltaurate copolymer, pentylene glycol, disodium EDTA, caprylyl glycol, chlorphenesin and phenoxyethanol. In one embodiment, the truncated collagen is a truncated jellyfish collagen or a truncated human collagen. In another embodiment, the truncated collagen is the truncated human type XXI collagen. Another embodiment disclosed herein is a topical preparation that comprises collagen and also comprises a vegetable oil. In one embodiment, the vegetable oil is olive oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which:

[0032] Figure 1 Shows the effect of an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence) on the secretion of type I collagen in fibroblasts.

[0033] Figure 2A Shows the expression of type I collagen mRNA in fibroblasts treated with an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence).

[0034] Figure 2B Shows the expression of elastin mRNA in fibroblasts treated with an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence).

[0035] Figure 2C Shows the expression of fibronectin mRNA in fibroblasts treated with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0036] Figure 3 Shows the expression of IL-1α in primary human keratinocytes treated with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0037] Figure 4 Shows the antioxidant capacity of the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0038] Figure 5 Shows the viability of UVB-irradiated keratinocytes treated with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0039] Figure 6 Shows the skin elasticity after treatment with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0040] Figure 7 Shows the skin collagen content after treatment with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0041] Figure 8 Shows the quantification of skin redness after treatment with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0042] Figure 9 Shows the quantification of skin wrinkles after treatment with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

[0043] Figure 10 Shows the secretion of type I collagen in a human skin tissue model treated with the exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

[0044] Figure 11 Shows the UVB-induced TT dimers in keratinocytes treated with the exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

[0045] Figure 12 Shows the viability of UVB-irradiated keratinocytes treated with the exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

[0046] Figure 13 Shows the cell viability after treatment with urban dust and the exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

[0047] Figure 14 Shows the relative expression of IL-1a induced by UVB after treatment with the exemplary polypeptides provided herein (comprising a truncated jellyfish collagen amino acid sequence).

[0048] Figure 15 Shows the antioxidant capacity of the exemplary polypeptides provided herein (comprising a truncated jellyfish collagen amino acid sequence).

[0049] Figure 16 Shows the skin hydration after treatment with the exemplary polypeptides provided herein (comprising a truncated jellyfish collagen amino acid sequence).

[0050] Figure 17 Shows the skin elasticity after treatment with the exemplary polypeptides provided herein (comprising a truncated jellyfish collagen amino acid sequence). Detailed Description

[0051] In the following description, certain specific details are set forth in order to provide a thorough understanding of the various embodiments of the present disclosure. However, those skilled in the art will understand that the present disclosure may be practiced without these details.

[0052] As used herein, the term "about" generally refers to ±10%.

[0053] The term "consisting of" means "including and limited to". Generally, the disclosure of "including" includes the disclosure of "consisting of".

[0054] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, provided that the additional ingredients, steps and / or parts do not materially alter the basic and novel features of the claimed composition, method or structure. Generally, the disclosure of "including" includes the disclosure of "consisting essentially of".

[0055] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include multiple compounds, including mixtures thereof.

[0056] Throughout this document, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description of a range format is merely for convenience and brevity and should not be construed as a strict limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a description of a range from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of how broad the range is.

[0057] Whenever a numerical range is indicated herein, it is intended to include any recited number (fractional or integral) within the indicated range. The phrases "range between" a first recited number and a second recited number and "range from" a first recited number "to" a second recited number are used interchangeably herein and are intended to include the first and second recited numbers and all fractional and integral numbers therebetween.

[0058] As used herein, the term "collagen" or "collagen-like" in some cases refers to a polypeptide (e.g., a monomer) that can associate with one or more collagens or collagen-like polypeptides to form a quaternary structure. Non-limiting examples of collagen include human type XXI α1 collagen (e.g., SEQ ID NO: 31), human type I α2 collagen (e.g., SEQ ID NO: 32), and jellyfish (hydrozoan) collagen (e.g., SEQ ID NO: 33). In some cases, collagen can be treated with acid, base, or heat to prepare gelatin. The quaternary structure of native collagen is a triple helix, typically composed of three polypeptides, but it should be noted that the "truncated collagen" provided herein or polypeptides containing "truncated collagen" may or may not have such a quaternary structure and thus do not necessarily have such a quaternary structure. In some cases, of the three polypeptides that form native collagen, two are usually the same and are referred to as α-chains. The third polypeptide is referred to as the β-chain. In certain cases, a typical native collagen can be referred to as AAB, where the collagen is composed of two α ("A") chains and one β ("B") chain. In some cases, the polypeptides provided herein that contain "truncated collagen" may or may not have such structural elements. The term "collagen" or "collagen-like" can refer to an α-chain polypeptide, a β-chain polypeptide, or both an α-chain polypeptide and a β-chain polypeptide. As used herein, the term "procollagen" generally refers to a polypeptide produced by a cell that can be processed into a naturally occurring collagen.

[0059] As used herein, the term "expression vector" or "vector" generally refers to a nucleic acid assembly capable of directing the expression of a foreign gene. An expression vector may include a promoter operably linked to a foreign gene, restriction endonuclease sites, nucleic acids encoding one or more selectable markers, and other nucleic acids useful in the practice of recombinant techniques.

[0060] As used herein, the term "fibroblast" generally refers to cells that synthesize procollagen and other structural proteins. Fibroblasts are widely distributed in the body and are present in the skin, connective tissue, and other tissues.

[0061] The term "fluorescent protein" generally refers to a protein that can be used as a reporter molecule for the expression of exogenous polynucleotides in genetic engineering techniques. The protein fluoresces and emits bright visible light when exposed to ultraviolet or blue light. Proteins that emit green light include green fluorescent protein (GFP), and proteins that emit red light include red fluorescent protein (RFP).

[0062] As used herein, the term "gelatin" generally refers to collagen that has been further processed by exposure to acid, base, or heat. In some cases, gelatin solutions form reversible gels and are used in food, cosmetics, pharmaceuticals, industrial products, medical products, laboratory culture growth media, and many other applications.

[0063] As used herein, the term "gene" generally refers to a polynucleotide that encodes a specific protein, and it can refer to the coding region alone or can include regulatory sequences (5' non-coding sequences) before and regulatory sequences (3' non-coding sequences) after the coding sequence.

[0064] The term "histidine tag" generally refers to a series of 2 - 30 consecutive histidine residues on a recombinant polypeptide.

[0065] The term "host cell" generally refers to a cell that has been engineered to express an introduced exogenous polynucleotide.

[0066] The term "keratinocyte" generally refers to cells that produce keratin present in the epidermal layer of the skin.

[0067] As used herein, the term "lactamase" generally refers to an enzyme that hydrolyzes antibiotics containing a lactam (cyclic amide) moiety. "Beta-lactamase" or "β-lactamase" is an enzyme that hydrolyzes antibiotics containing a β-lactam moiety.

[0068] As used herein, the term "non-naturally occurring" refers to a gene, polypeptide, or protein that is not normally found in nature, such as collagen. Non-naturally occurring collagen can be prepared recombinantly. Non-naturally occurring collagen can be recombinant collagen. In one embodiment, the non-naturally occurring collagen is truncated collagen. Other non-naturally occurring collagen polypeptides include chimeric collagen. Chimeric collagen is a polypeptide in which a portion of one collagen polypeptide is adjacent to a portion of a second collagen polypeptide. For example, a collagen molecule that includes a portion of jellyfish collagen adjacent to a portion of human collagen is a chimeric collagen. In another embodiment, the non-naturally occurring collagen includes a fusion polypeptide that comprises additional amino acids, such as a secretion tag, a histidine tag, green fluorescent protein, a protease cleavage site, a GEK repeat, a GDK repeat, and / or a β-lactamase.

[0069] Generally, the disclosure of a collagen or truncated collagen (e.g., having a specific amino acid sequence) provided herein includes polypeptides having or comprising that exact amino acid sequence and homologs thereof. In some cases, homologs of the amino acid sequences provided herein can have longer or shorter sequences and can have substitutions of one or more amino acid residues of that amino acid sequence. Such homologs have a specific sequence identity to the sequence (e.g., in the amounts of sequence identity provided herein). Sequence identity (e.g., for purposes of assessing percent identity) can be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (e.g., see the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally using default settings), the BLAST algorithm (e.g., see the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally using default settings), or the Smith-Waterman algorithm (e.g., see the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally using default settings). Any suitable parameters of the selected algorithm (including default parameters) can be used to evaluate the optimal alignment. In some cases, non-naturally occurring collagen can have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the sequences disclosed herein.

[0070] The term "protease cleavage site" generally refers to an amino acid sequence that is cleaved by a specific protease.

[0071] The term "secretion tag" or "signal peptide" generally refers to an amino acid sequence that recruits the cellular machinery of a host cell to transport an expressed protein to a specific location or organelle of the host cell.

[0072] The term "truncated collagen" generally refers to a monomeric polypeptide that is less than full-length collagen, where one or more portions of the full-length collagen are absent. The collagen polypeptide is truncated at the C-terminus, truncated at the N-terminus, truncated by removing an internal portion of the full-length collagen polypeptide (e.g., internal truncation), or truncated at both the C-terminus and the N-terminus. In non-limiting embodiments, the truncated human collagen may comprise the amino acid sequence according to SEQ ID NO: 16 or a homolog thereof. In another non-limiting example, the truncated jellyfish collagen may comprise the amino acid sequence according to SEQ ID NO: 5 or a homolog thereof. Generally, the truncated collagen provided herein may have similar or substantially similar functions and / or provide similar or substantially similar benefits as native or full-length collagen (e.g., as provided herein). In some cases, compared to native or full-length collagen, the truncated collagen provided herein may have improved or increased functions and / or benefits (e.g., as provided herein).

[0073] When used in reference to an amino acid position, "truncated" includes that amino acid position. For example, an N-terminal truncation at amino acid position 100 of a full-length protein means that 100 amino acids are truncated from the N-terminus of the full-length protein (i.e., the truncated protein lacks amino acids 1 to 100 of the full-length protein). Similarly, a C-terminal truncation of a full-length protein (assuming a full-length protein of 1000 amino acids) at amino acid position 901 means that 100 amino acids are truncated from the C-terminus (i.e., the truncated protein lacks amino acids 901 to 1000 of the full-length protein). Similarly, an internal truncation at amino acid positions 101 and 200 refers to an internal truncation of 100 amino acids of the full-length protein (i.e., the truncated protein lacks amino acids 101 to 200 of the full-length protein).

[0074] In some embodiments, the cell culture may further comprise one or more of the following: ammonium chloride, ammonium sulfate, calcium chloride, amino acids, ferrous(II) sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate, and yeast extract.

[0075] The host bacterial cells can be cultured continuously or discontinuously; batch processing, fed-batch processing, or repeated fed-batch processing.

[0076] Typically, a signal sequence can be part of an expression vector or can be part of an exogenous gene inserted into the vector. The signal sequence selected can be a signal sequence that is recognized and processed by the host cell (e.g., cleaved by a signal peptidase). For bacterial host cells that do not recognize and process the native signal sequence of an exogenous gene, the signal sequence can be replaced by any commonly known bacterial signal sequence. In some embodiments, the DsbA signal sequence can be used to target recombinantly produced polypeptides to the periplasmic space. Dinh and Bernhardt, J Bacteriol, September 2011, 4984-4987.

[0077] In one aspect, provided is a non-naturally occurring collagen produced by a host cell. The non-naturally occurring collagen can be jellyfish collagen or human collagen. The non-naturally occurring collagen can be a truncated collagen. The truncation can be an internal truncation (e.g., truncation of an internal portion), truncation at the N-terminal portion of the collagen, truncation at the C-terminal portion of the collagen, or truncation at both the C-terminal and N-terminal ends. The collagen can be truncated by 50 amino acids to 1000 amino acids, 50 amino acids to 950 amino acids, 50 amino acids to 900 amino acids, 50 amino acids to 850 amino acids, 50 amino acids to 800 amino acids, 50 amino acids to 850 amino acids, 50 amino acids to 800 amino acids, 50 amino acids to 750 amino acids, 50 amino acids to 700 amino acids, 50 amino acids to 650 amino acids, 50 amino acids to 600 amino acids, 50 amino acids to 550 amino acids, 50 amino acids to 500 amino acids, 50 amino acids to 450 amino acids, 50 amino acids to 400 amino acids, 50 amino acids to 350 amino acids, 50 amino acids to 300 amino acids, 50 amino acids to 250 amino acids, 50 amino acids to 200 amino acids, 50 amino acids to 150 amino acids, or 50 amino acids to 100 amino acids. In another embodiment, the collagen can be truncated by about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acids. The non-naturally occurring collagen can be encoded by a portion of the polynucleotide sequence or the entire polynucleotide sequence disclosed herein.

[0078] The truncated collagen disclosed herein can include a truncation relative to the full-length collagen. In some embodiments, the truncated collagen disclosed herein can include a truncation relative to full-length human type XXI alpha 1 collagen. In some embodiments, the truncated collagen disclosed herein can include a truncation relative to full-length human type I alpha 2 collagen. In some embodiments, the truncated collagen disclosed herein includes a truncation relative to full-length jellyfish (hydrozoan) collagen. Non-limiting examples of full-length collagen are provided in Table 1 below.

[0079] Table 1. Amino Acid Sequences of Full-Length Collagen

[0080]

[0081] In some cases, the truncated collagen as described herein can include an N-terminal truncation at any amino acid position between amino acid positions 1 to 548; 1 to 553; 1 to 558; 1 to 563; 1 to 568; or 1 to 573 of SEQ ID NO: 31. In some cases, the truncated collagen as described herein can include a C-terminal truncation at any amino acid position between amino acid positions 726 to 957; 731 to 957; 736 to 957; 741 to 957; 746 to 957; 751 to 957; or 756 to 957 of SEQ ID NO: 31. In some cases, the truncated collagen as described herein can include both an N-terminal truncation and a C-terminal truncation. For example, the truncated collagen as described herein can include an N-terminal truncation at any amino acid position between amino acid positions 1 to 548; 1 to 553; 1 to 558; 1 to 563; 1 to 568; or 1 to 573 of SEQ ID NO: 31; and a C-terminal truncation at any amino acid position between amino acid positions 726 to 957; 731 to 957; 736 to 957; 741 to 957; 746 to 957; 751 to 957; or 756 to 957 of SEQ ID NO: 31. In a particular embodiment, the truncated collagen disclosed herein can include an N-terminal truncation at amino acid position 558 of SEQ ID NO: 31; and a C-terminal truncation at amino acid position 746 of SEQ ID NO: 31.

[0082] In some cases, a truncated collagen as described herein can include an N-terminal truncation at any amino acid position between amino acid positions 1 to 401; 1 to 406; 1 to 411; 1 to 416; 1 to 421; 1 to 426; or 1 to 431 of SEQ ID NO: 32. In some cases, a truncated collagen as described herein can include a C-terminal truncation at any amino acid position between amino acid positions 585 to 1366; 590 to 1366; 595 to 1366; 600 to 1366; 605 to 1366; 610 to 1366; 615 to 1366; or 620 to 1366 of SEQ ID NO: 32. In some cases, a truncated collagen as described herein can include both an N-terminal truncation and a C-terminal truncation. For example, a truncated collagen as described herein can include an N-terminal truncation at any amino acid position between amino acid positions 1 to 401; 1 to 406; 1 to 411; 1 to 416; 1 to 421; 1 to 426; or 1 to 431 of SEQ ID NO: 32; and a C-terminal truncation at any amino acid position between amino acid positions 585 to 1366; 590 to 1366; 595 to 1366; 600 to 1366; 605 to 1366; 610 to 1366; 615 to 1366; or 620 to 1366 of SEQ ID NO: 32. In a particular embodiment, a truncated collagen as provided herein can include an N-terminal truncation at amino acid position 416 of SEQ ID NO: 32; and a C-terminal truncation at amino acid position 605 of SEQ ID NO: 32.

[0083] In some cases, a truncated collagen as described herein can include an N-terminal truncation at any amino acid position between amino acid positions 1 and 101 of SEQ ID NO: 32; between amino acid positions 1 and 106; between amino acid positions 1 and 111; between amino acid positions 1 and 116; between amino acid positions 1 and 121; or between amino acid positions 1 and 126. In some cases, a truncated collagen as described herein can include a C-terminal truncation at any amino acid position between amino acid positions 276 and 1366 of SEQ ID NO: 32; between amino acid positions 281 and 1366; between amino acid positions 286 and 1366; between amino acid positions 291 and 1366; between amino acid positions 296 and 1366; between amino acid positions 301 and 1366; or between amino acid positions 306 and 1366. In some cases, a truncated collagen as described herein can include both an N-terminal truncation and a C-terminal truncation. For example, a truncated collagen as described herein can include an N-terminal truncation at any amino acid position between amino acid positions 1 and 101 of SEQ ID NO: 32; between amino acid positions 1 and 106; between amino acid positions 1 and 111; between amino acid positions 1 and 116; between amino acid positions 1 and 121; or between amino acid positions 1 and 126; and a C-terminal truncation at any amino acid position between amino acid positions 276 and 1366 of SEQ ID NO: 32; between amino acid positions 281 and 1366; between amino acid positions 286 and 1366; between amino acid positions 291 and 1366; between amino acid positions 296 and 1366; between amino acid positions 301 and 1366; or between amino acid positions 306 and 1366. In a particular embodiment, a truncated collagen as provided herein can include an N-terminal truncation at amino acid position 111 of SEQ ID NO: 32; and a C-terminal truncation at amino acid position 291 of SEQ ID NO: 32.

[0084] In some cases, a truncated collagen as described herein can comprise between amino acid positions 16 and 240 of SEQ ID NO: 33; between amino acid positions 16 and 245; between amino acid positions 16 and 250; between amino acid positions 16 and 255; between amino acid positions 16 and 260; between amino acid positions 16 and 265; between amino acid positions 6 and 255; between amino acid positions 11 and 255; between amino acid positions 21 and 255; between amino acid positions 26 and 255; between amino acid positions 31 and 255; between amino acid positions 21 and 250; between amino acid positions 21 and 245; between amino acid positions 26 and 250; between amino acid positions 26 and 245; between amino acid positions 31 and 250; or between amino acid positions 31 and 245 of SEQ ID NO: 33. In certain embodiments, a truncated collagen as described herein can comprise an internal truncation at any amino acid position between amino acid positions 16 and 255 of SEQ ID NO: 33.

[0085] In some cases, a truncated collagen can comprise any of the amino acid sequences provided in Table 2 below. In some cases, a truncated collagen can consist of any of the amino acid sequences provided in Table 2 below. In some cases, a truncated collagen can consist essentially of any of the amino acid sequences provided in Table 2 below. In certain embodiments, a non-naturally occurring collagen is or comprises the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29. In some embodiments, a truncated collagen comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29.

[0086] Table 2. Non-limiting examples of truncated collagens

[0087]

[0088]

[0089]

[0090] In some cases, the truncated collagen can be between 100 and 300 amino acids, between 150 and 250 amino acids, between 160 and 250 amino acids, between 160 and 220 amino acids, between 170 and 200 amino acids, between 180 and 190 amino acids, or between 185 and 190 amino acids in length.

[0091] In some embodiments, the non-naturally occurring collagen can further include an amino acid sequence comprising a secretion tag. The secretion tag can direct the collagen to the periplasmic space of a host cell. In a specific embodiment, the signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, Hy1A, DegP, or a hybrid secretion tag comprising a portion of one secretion tag fused to a portion of a second secretion tag. In one aspect, the secretion tag can be attached to the non-naturally occurring collagen. In another aspect, the secretion tag can be cleaved from the non-naturally occurring collagen.

[0092] In some embodiments, the non-naturally occurring collagen comprises a histidine (or polyhistidine) tag. In a particular embodiment, the histidine tag or polyhistidine tag is or comprises a sequence of 2 to 20 histidine residues attached to the collagen. In various embodiments, the histidine tag comprises 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues, or 7 to 10 histidine residues. The histidine tag can be used for protein purification by a chromatographic method employing a nickel-based chromatographic medium. Exemplary fluorescent proteins include green fluorescent protein (GFP) or red fluorescent protein (RFP). Fluorescent proteins are well known in the art. In one embodiment, the non-naturally occurring collagen comprises GFP and / or RFP. In one embodiment, superfolder GFP is fused to the non-naturally occurring collagen. Superfolder GFP can be a GFP that can fold correctly even when fused to a misfolded polypeptide. In one aspect, the histidine tag can be attached to the non-naturally occurring collagen. In another aspect, the histidine tag can be cleaved from the non-naturally occurring collagen.

[0093] In some embodiments, the non-naturally occurring collagen further comprises protease cleavage sites. The protease cleavage sites can be used to cleave recombinantly produced collagen to remove one or more portions of the polypeptide. Polypeptide portions that can be removed include secretion tags, histidine tags, fluorescent protein tags, and / or beta-lactamase. Proteases can include endoproteases, exoproteases, serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and metalloproteases. Exemplary protease cleavage sites include amino acids cleaved by thrombin, TEV protease, factor Xa, enteropeptidase, and rhinovirus 3C protease. In one aspect, a cleavage tag is attached to the non-naturally occurring collagen. In another aspect, the cleavage tag is removed from the non-naturally occurring collagen by an appropriate protease.

[0094] In some embodiments, the non-naturally occurring collagen further comprises an enzyme that is beta-lactamase. The beta-lactamase can be used as a selectable marker. In one aspect, the beta-lactamase is attached to the non-naturally occurring collagen. In another aspect, the beta-lactamase is cleaved from the non-naturally occurring collagen.

[0095] Provided in certain embodiments herein are (e.g., topical) compositions or formulations comprising one or more of the polypeptides provided herein. In some embodiments, the composition provides any suitable amount of the polypeptides provided herein in any suitable amount (e.g., an amount suitable to provide a benefit when administered or applied to an individual or cell). In some specific embodiments, the composition comprises an amount suitable to provide a beneficial effect to the skin of an individual when (e.g., topically) applied to the skin of the individual. In a particular embodiment, the composition comprises from 0.001% to 30% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein. In a more particular embodiment, the composition comprises from 0.001% to 20% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein, from 0.001% to 10% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein, from 0.001% to 5% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein, from 0.001% to 2% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein, from 0.001% to 1% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein, from 0.001% to 0.5% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein, and from 0.001% to 0.2% w / w of a polypeptide (or non-naturally occurring collagen) such as provided herein.

[0096] In one aspect, a composition comprising non-naturally occurring collagen can be a personal care product (e.g., a cosmetic). In some embodiments, the composition is formulated for topical application. The composition can include other cosmetic ingredients suitable for human use. The personal care product can be used to prevent or treat damage to human skin or hair from ultraviolet radiation. The personal care product can be used to increase skin firmness, elasticity, brightness, hydration, tactile texture, or visual texture, and / or stimulate collagen production. The personal care product can be used to reduce skin redness. The personal care product can be applied to the skin or hair. The composition includes, for example, facial masks, skin cleansers (such as soaps, cleansing creams, cleansing lotions, facial washes, cleansing milks, cleansing pads, facial cleansers), facial and body creams and moisturizers, facial serums, facial and body masks, toners and facial sprays, eye creams and eye care products, exfoliating formulations, lip balms and lipsticks, shampoos, conditioners and body washes, hair and scalp serums, hair mists and sprays, eyeshadows, concealers, mascaras, and other color cosmetics.

[0097] The composition comprising non-naturally occurring collagen can further comprise at least one additional ingredient, including a topical carrier or a preservative. The topical carrier can include a topical carrier selected from liposomes, biodegradable microcapsules, lotions, sprays, aerosols, powdered powders, biodegradable polymers, mineral oil, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, alcohols, emulsifiers, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, waxes, sorbitan monostearate, polysorbate, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and water. The preservative can include a preservative selected from tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride cis isomer, glutaraldehyde, 4,4-dimethyl oxazolidine, 7-ethyl bicyclo oxazolidine, phenoxyethanol, butylene glycol, 1,2-hexanediol, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA.

[0098] In certain embodiments of the present disclosure, methods are also provided for reducing skin damage, promoting the repair of damaged skin, protecting the skin from ultraviolet (UV) damage, and / or protecting skin cells from exposure to urban dust. In another embodiment, methods are provided for improving skin firmness, elasticity, brightness, hydration, tactile texture, or visual texture and / or stimulating collagen production. The method may include the step of applying a composition comprising non-naturally occurring collagen to the skin of a subject. Without being bound by a particular theory or mechanism, the collagen in the composition may reduce skin damage by preventing UV damage. In some cases, the collagen in the composition may promote the repair of damaged skin by increasing cell viability. In some cases, when the collagen in the composition is applied to the skin, skin damage and / or cell repair may be reduced by increasing procollagen synthesis and / or promoting the viability of skin cells. In some cases, collagen reduces the formation of thymine-thymine (TT) dimers.

[0099] The methods provided herein include using the composition for the treatment indicated in the method (e.g., by the steps provided herein). In an embodiment, the present disclosure provides the use of the compositions provided herein (e.g., truncated collagen or a formulation comprising truncated collagen) in a method for reducing skin damage, promoting the repair of damaged skin, protecting the skin from UV damage, and / or protecting skin cells from exposure to urban dust (e.g., by administering the composition provided herein to the skin of a subject). In an embodiment, the present disclosure provides the use of the compositions provided herein (e.g., truncated collagen or a formulation comprising truncated collagen) in a method for improving skin firmness, elasticity, brightness, hydration, tactile texture, or visual texture and / or stimulating collagen production.

[0100] In some embodiments, the truncated collagen provided herein may stimulate fibroblasts and / or keratinocytes to produce type I collagen (see, e.g., Example 4 and Example 6). In some cases, the level of type I procollagen C-peptide (a readout of collagen production) may be measured. In some cases, an in vitro MatTek full-thickness human skin tissue model (see, e.g., Example 6) may be used to evaluate type I procollagen C-peptide levels. In some cases, type I collagen levels may be measured or determined by enzyme-linked immunosorbent assay (ELISA). In some cases, the truncated collagen provided herein may stimulate the production of type I collagen at a higher level compared to untreated cells, cells treated with retinol, and / or cells treated with vitamin B3.

[0101] In some embodiments, the truncated collagen provided herein can stimulate fibroblast overexpression of extracellular matrix genes (see, e.g., Example 4). In some cases, the levels of extracellular matrix genes can be measured by RNA sequencing. In some cases, the truncated collagen provided herein can stimulate fibroblast overexpression of one or more of the type I collagen gene (COL1A), elastin gene (ELN), and fibronectin gene (FN1). In some cases, the levels of extracellular matrix genes produced by fibroblasts treated with the truncated collagen provided herein can be higher than those of untreated fibroblasts or fibroblasts treated with retinol. In some cases, the levels of extracellular matrix genes produced by fibroblasts treated with the truncated collagen provided herein can be similar to or higher than those of fibroblasts treated with vitamin C.

[0102] In some embodiments, the truncated collagen provided herein can reduce the inflammation of keratinocytes irradiated with UVB light (see, e.g., Example 4 and Example 6). In some cases, keratinocytes can be irradiated with UVB light and then treated with the truncated collagen provided herein. In some cases, the inflammation can be measured by measuring the level of IL-1α produced by UVB-irradiated keratinocytes (e.g., by ELISA). In some cases, for UVB-irradiated keratinocytes, when treated with the truncated collagen provided herein, a lower level of IL-1α can be produced compared to untreated keratinocytes.

[0103] In some embodiments, the truncated collagen provided herein can increase the viability of keratinocytes irradiated with UVB light (see, e.g., Example 4). In some cases, keratinocytes can be pretreated (before UVB irradiation) and post-treated (after UVB irradiation) with the truncated collagen provided herein. In some cases, the cell viability can be measured using the MTT metabolic colorimetric assay. In some cases, keratinocytes treated with the truncated collagen provided herein can exhibit greater cell viability after UVB irradiation compared to untreated keratinocytes.

[0104] In some embodiments, the truncated collagen provided herein can reduce DNA damage in keratinocytes after exposure to UVB light (see, e.g., Example 6). In some cases, the DNA damage can be evaluated by measuring the level of thymine dimers (TT-dimers). In a non-limiting example, the OxiSelect UV-induced DNA damage ELISA kit can be used to measure the level of TT-dimers. In some cases, UVB-irradiated keratinocytes treated with the truncated collagen provided herein can show lower TT-dimer levels compared to untreated keratinocytes.

[0105] In some embodiments, the truncated collagen provided herein can have antioxidant capacity (see, e.g., Example 4 and Example 6). In some cases, the oxygen radical absorbance capacity (ORAC) assay can be used to measure the antioxidant capacity of the truncated collagen. In a non-limiting example, the truncated collagen in the form of a 0.1% solution can have antioxidant properties of at least 10 μM Trolox (vitamin E) equivalents (TE), at least 50 μM TE, at least 100 μM TE, at least 150 μM TE, at least 160 μM TE, at least 170 μM TE, at least 180 μM TE, at least 190 μM TE, or at least 200 μM TE.

[0106] In some embodiments, compared to untreated cells, the truncated collagen provided herein can increase the cell viability of keratinocytes exposed to urban dust pollution (see, e.g., Example 6). In some cases, cell viability can be measured by the MTT metabolic colorimetric assay.

[0107] In some embodiments, topical application of the truncated collagen provided herein to a subject's face can result in an increase in the elasticity of the facial skin compared to baseline at 1 week, 2 weeks, 4 weeks, 8 weeks, or longer after treatment (see, e.g., Example 5 and Example 7). In some cases, the elasticity of the facial skin can be measured by a skin elasticity tester.

[0108] In some embodiments, topical application of the truncated collagen provided herein to a subject's face can result in an increase in the collagen content of the facial skin compared to baseline at 1 week, 2 weeks, 4 weeks, 8 weeks, or longer after treatment (see, e.g., Example 5). In some cases, the collagen content of the facial skin can be measured by SIAscope.

[0109] In some embodiments, topical application of the truncated collagen provided herein can result in a reduction in facial skin redness (rash) compared to baseline at 1 week, 2 weeks, 4 weeks, 8 weeks, or longer after treatment (e.g., see Example 5). In some cases, the facial skin redness (rash) can be scored by a blinded clinical rater (e.g., using the 5-point ordinal scale provided in Table 4).

[0110] In some embodiments, topical application of the truncated collagen provided herein can result in a reduction in facial wrinkles compared to baseline at 1 week, 2 weeks, 4 weeks, 8 weeks, or longer after treatment (see, e.g., Example 5). In some cases, the facial wrinkles can be scored by a blinded clinical rater.

[0111] In some embodiments, topical administration of the truncated collagen provided herein can result in an increase in the moisture content of facial skin compared to baseline at 1 week, 2 weeks, 4 weeks, 8 weeks, or longer after treatment (see, e.g., Example 7). In some cases, topical administration of the truncated collagen provided herein can result in an increase in the moisture content of facial skin compared to topical administration of marine collagen. In some cases, the hydration of the skin can be measured by a skin moisture tester.

[0112] One aspect of the present disclosure provides a polynucleotide encoding a non-naturally occurring collagen. The polynucleotide can encode a collagen from jellyfish or human. The polynucleotide can encode a full-length or truncated collagen. In various embodiments, the polynucleotide can include a polynucleotide according to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30 or homologs thereof (e.g., having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity thereto). In some cases, the polynucleotide can be codon-optimized (e.g., for expression in a host cell).

[0113] In another aspect, the present disclosure provides a polynucleotide encoding a collagen fusion protein. The collagen fusion protein can comprise a secretion tag, a histidine tag, a fluorescent protein tag, a protease cleavage site, a β-lactamase, and / or a GEK amino acid trimer repeat sequence and / or a GDK amino acid trimer repeat sequence, as well as collagen.

[0114] In one aspect, a vector comprising a polynucleotide encoding a collagen can be used to transform a host cell and express the polynucleotide. The polynucleotide can further comprise a nucleic acid encoding an enzyme that allows the host organism to grow in the presence of a selection agent. The selection agent can include certain sugars, including galactose-containing sugars, or antibiotics, including ampicillin, hygromycin, G418, etc. Enzymes that can be used to confer resistance to the selection agent include β-galactosidase or β-lactamase.

[0115] In one aspect, a host cell expressing the polynucleotide of the present invention is provided. The host cell can be any host cell, including Gram-negative bacterial cells, Gram-positive bacterial cells, yeast cells, insect cells, mammalian cells, plant cells, or any other cell used for expressing exogenous polynucleotides. An exemplary Gram-negative host cell is Escherichia coli.

[0116] In addition to a carbon, nitrogen, and inorganic phosphate source, any desired or necessary supplements may be included at appropriate concentrations, introduced separately or as a mixture with another supplement or medium (such as a complex nitrogen source). In certain embodiments, the culture medium further comprises one or more components selected from the group consisting of ammonium chloride, ammonium sulfate, calcium chloride, casein amino acids, ferrous sulfate (II), magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate, and yeast extract.

[0117] β-lactamase is an enzyme that confers resistance to β-lactam antibiotics in prokaryotic cells. Typically, when β-lactamase is expressed in a bacterial host cell, the expressed β-lactamase protein also contains a targeting sequence (secretion tag) that directs the β-lactamase protein to the periplasmic space. β-lactamase only functions when transported to the periplasmic space. A β-lactamase that targets the periplasmic space is provided without the use of a separate secretion tag that targets the enzyme to the periplasmic space. By creating a fusion protein in which a periplasmic secretion tag is added to the N-terminus of a protein (such as GFP, collagen, or a GFP / collagen chimera), the function of β-lactamase lacking a native secretion tag can be used to select for the complete translation and secretion of the N-terminal fusion protein. Using this method, a DsbA-GFP-collagen-β-lactamase fusion can be used to select for truncated products of the target collagen that are favorable for translation and secretion.

[0118] Another embodiment provides a method for producing a polypeptide (or non-naturally occurring collagen) such as provided herein. In some embodiments, the method comprises the steps of inoculating a culture medium with a recombinant host cell that comprises a polynucleotide encoding the polypeptide or "collagen"; culturing the host cell; and isolating the polypeptide (or non-naturally occurring collagen) from the host cell.

[0119] A method for fermentatively producing a polypeptide (or protein) is provided. The method comprises the steps of:

[0120] (a) culturing recombinant Gram-negative bacterial cells in a culture medium comprising a magnesium salt, wherein the concentration of magnesium ions in the culture medium is at least about 6 mM, and wherein the bacterial cells comprise an exogenous gene encoding the protein; and

[0121] (b) harvesting the protein from the culture medium.

[0122] The bacteria can be cultured in any suitable manner, such as continuous culture (as described, for example, in WO 05 / 021772) or discontinuous culture in a batch process (batch culture) or in a fed-batch or repeated fed-batch process, for the production of the target protein. In some embodiments, protein production is carried out on a large scale. Various large-scale fermentation procedures can be used to produce recombinant proteins. Large-scale fermentation has a capacity of at least 1,000 liters, preferably a capacity of about 1,000 to 100,000 liters. In some cases, the fermenter uses agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon source / energy source). Small-scale fermentation generally refers to fermentation in a fermenter with a capacity of no more than about 20 liters.

[0123] To accumulate the target protein, the host cells can be cultured under conditions sufficient to accumulate the target protein. Such conditions include, for example, temperature, nutrients, and cell density conditions that allow the cells to express and accumulate the protein. In addition, as is known to those skilled in the art, such conditions can refer to conditions under which the cells can perform the following basic cellular functions for secreted proteins: transcription, translation, and transfer of the protein from one cellular compartment to another.

[0124] Any suitable bacterial cells can be optionally used in the methods provided herein. The bacterial cells can be cultured at any suitable temperature. In a particular embodiment, the bacterial cells are Escherichia coli cells. For E. coli growth, for example, the typical temperature range is from about 20°C to about 39°C. In one embodiment, the temperature is from about 20°C to about 37°C. In another embodiment, the temperature is about 30°C. In one embodiment, the host cells can be cultured at one temperature in a non-switching state or a switching state and switched to a different temperature to induce protein production. The host cells can be first cultured at one temperature to propagate the cells, and then the cells can be cultured at a lower temperature to induce protein production. The first temperature can be about 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. The second temperature can be about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C. The culturing at the second temperature can be carried out for 1 hour to 100 hours, 5 hours to 90 hours, 5 hours to 80 hours, 5 hours to 75 hours, 5 hours to 70 hours, 10 hours to 70 hours, 15 hours to 70 hours, 15 hours to 65 hours, 15 hours to 60 hours, 20 hours to 60 hours, 20 hours to 55 hours, 20 hours to 50 hours, 24 hours to 50 hours, 24 hours to 48 hours, 30 hours to 50 hours, 30 hours to 45 hours, or 30 hours to 40 hours.

[0125] The pH of the culture medium can be any pH from about 5 - 9, mainly depending on the host organism. For Escherichia coli, the pH can be from about 6.0 to about 7.4, about 6.2 to about 7.2, about 6.2 to about 7.0, about 6.2 to about 6.8, about 6.2 to about 6.6, about 6.4 or about 6.5.

[0126] To induce gene expression, cells can generally be cultured until a certain optical density is reached, e.g., OD 600 of about 1.1, at which point induction (e.g., by adding an inducer, by depleting a repressor, inhibitor or culture medium component, etc.) is started to induce the expression of the foreign gene encoding the target protein. In some embodiments, the expression of the foreign gene can be induced by an inducer selected from, for example, isopropyl - β - D - 1 - thiogalactopyranoside, lactose, arabinose, maltose, tetracycline, anhydrotetracycline, vavlycin, xylose, copper, zinc, etc. The induction of gene expression can also be achieved by reducing the dissolved oxygen level during the fermentation process. The dissolved oxygen level during cell propagation in the fermentation can be between 10% and 30%. To induce gene expression, the dissolved oxygen level can be reduced to below 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%. In host cells, whether in a physiological state or in a transition state, protein production can be induced by reducing the fermentation temperature as disclosed herein.

[0127] Examples

[0128] Example 1. Production of Truncated Collagen

[0129] A codon - optimized DNA sequence was synthesized and expressed, which was optimized for expression in Escherichia coli and encoded a truncated (relative to full - length jellyfish collagen (SEQ ID NO: 33)) jellyfish collagen with 240 internal amino acids. This DNA sequence is shown in SEQ ID NO: 1 below. In SEQ ID NO: 1, the DsbA secretion tag is encoded by nucleotides 1 - 72 and encodes amino acids 1 - 24 of SEQ ID NO: 2. The histidine tag containing 9 histidine residues is encoded by nucleotides 73 - 99 of SEQ ID NO: 1 and encodes amino acids 25 - 33 of SEQ ID NO: 2. The linker is encoded by nucleotides 100 - 111 of SEQ ID NO: 1 and encodes amino acids 34 - 37 of SEQ ID NO: 2. The thrombin cleavage site is encoded by nucleotides 112 - 135 of SEQ ID NO: 1 and encodes amino acids 38 - 45 of SEQ ID NO: 2. The truncated collagen is encoded by nucleotides 136 - 822 of SEQ ID NO: 1 and encodes amino acids 46 - 274 of SEQ ID NO: 2.

[0130] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATCACCATCACCACCACCACCATCACCACTCTGGCTCGAGCCTGGTGCCGCGCGGCAGCCATATGGGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTGAAAAAGGAGAACAGGGACGTACAGGTGCAGCAGGTAAACAGGGCAGCCCGGGTGCCGATGGTGCCCGTGGCCCGCTGGGTAGCATTGGTCAGCAGGGTGCAAGAGGCGAACCGGGCGATCCGGGTAGTCCGGGCCTGCGTGGTGATACGGGTCTGGCCGGTGTTAAAGGCGTTGCAGGTCCTTCAGGTCGTCCAGGTCAACCGGGTGCAAATGGTCTGCCGGGTGTTAATGGTCGTGGCGGTCTGGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:1)

[0131] The truncated collagen is approximately 54% of the full-length jellyfish collagen (SEQ ID NO: 33) and is disclosed in SEQ ID NO: 2 below.

[0132] MKKIWLALAGLVLAFSASAAQYEDHHHHHHHHHSGSSLVPRGSHMGPQGVVGADGKDGTPGEKGEQGRTGAAGKQGSPGADGARGPLGSIGQQGARGEPGDPGSPGLRGDTGLAGVKGVAGPSGRPGQPGANGLPGVNGRGGLERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:2)

[0133] A polynucleotide encoding a truncated jellyfish collagen without a DsbA secretion tag, a histidine tag, a linker, and a thrombin cleavage site is disclosed in SEQ ID NO:3.

[0134] GTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTGAAAAAGGAGAACAGGGACGTACAGGTGCAGCAGGTAAACAGGGCAGCCCGGGTGCCGATGGTGCCCGTGGCCCGCTGGGTAGCATTGGTCAGCAGGGTGCAAGAGGCGAACCGGGCGATCCGGGTAGTCCGGGCCTGCGTGGTGATACGGGTCTGGCCGGTGTTAAAGGCGTTGCAGGTCCTTCAGGTCGTCCAGGTCAACCGGGTGCAAATGGTCTGCCGGGTGTTAATGGTCGTGGCGGTCTGGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:3)

[0135] The truncated jellyfish collagen amino acid sequence without the DsbA secretion tag, histidine tag, linker, and thrombin cleavage site is disclosed in SEQ ID NO: 4.

[0136] GPQGVVGADGKDGTPGEKGEQGRTGAAGKQGSPGADGARGPLGSIGQQGARGEPGDPGSPGLRGDTGLAGVKGVAGPSGRPGQPGANGLPGVNGRGGLERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:4)

[0137] The polynucleotide of SEQ ID NO:1 was codon-optimized and synthesized by in-house synthesis at Gen9DNA (now Ginkgo Bioworks). The overlapping sequence between the pET28 vector and SEQ ID NO:1 was designed to be 30 to 40 bp long and added using PCR with the enzyme GXL polymerase (www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase? sitex=10020:22372:US). Then the opened pET28a vector and the insert DNA (SEQ ID NO:1) were assembled together into the final plasmid using SGIGibson (us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc). The plasmid sequence was then verified by Sanger sequencing at Eurofins Genomics (www.eurofinsgenomics.com).

[0138] The transformed cells were cultured in minimal medium and frozen in 1.5 ml aliquots with glycerol at a 50:50 cell:glycerol ratio. A vial of this frozen culture was thawed overnight at 37 °C, 200 rpm in 50 ml of minimal medium. The cells were transferred to 300 ml of minimal medium and grown for 6 - 9 hours to reach an OD600 of 5 - 10.

[0139] Prepare a bioreactor with 2.7 L of basal medium + glucose and add 300 ml of a culture with an OD600 of 5 - 10 to bring the starting volume to 3 L. Grow the cells at 28 °C and pH 7, where the dissolved oxygen is maintained at 20% saturation using a cascade of stirring, air, and oxygen. Control the pH with 28% w / w ammonium hydroxide solution. Once the initial bolus of 40 g / L is depleted in about 13 hours, a DO-stat based feeding algorithm is used to perform fermentation in fed-batch mode. After an initial growth of 24 - 26 hours, the OD600 reaches above 100. At this point, 300 mL of 500 g / L sucrose is added and the temperature is reduced to 25 °C. Induce high-density culture with 1 mM IPTG to produce protein. Continue fermentation for an additional 20 - 24 hours and harvest the cells by centrifugation at 9000 rcf and 15 °C for 60 minutes using a tabletop centrifuge. Resuspend the centrifuged cell pellet in a buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at pH 8 at a weight ratio of 2x buffer to 1x cell.

[0140] Break the harvested cells twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contains collagen as well as other proteins.

[0141] Perform fermentations at different temperatures ranging from 25 °C to 28 °C. For some fermentations, maintain the fermentation temperature at a constant temperature and immediately purify the collagen at the end of fermentation (OD 600 is 5 - 10). For other fermentations, maintain the fermentation temperature for a desired period of time and when the OD 600 cell density reaches 5 - 10, reduce the temperature to induce protein production. Typically, reduce the temperature from 28 °C to 25 °C. After continuing fermentation at 25 °C for 40 - 60 hours, isolate the collagen.

[0142] Purify the collagen by acid treatment of the homogenized cell broth. Additionally, after centrifugation and resuspension in the above buffer, acid treatment was also performed on the unhomogenized intact cells recovered from the bioreactor. Use 6 M hydrochloric acid to lower the pH of the homogenized slurry or resuspended intact cells to pH 3. Incubate the acidified cell slurry overnight at 4 °C with mixing and then centrifuge. Test the supernatant of the acidified slurry on a polyacrylamide gel and find that the abundance of collagen contained is relatively high compared to the starting precipitate. The collagen slurry thus obtained has a high salt content. To reduce the volume and salt content, perform concentration and diafiltration steps using an EMD Millipore tangential flow filtration system (each using a 0.1 m2 ultrafiltration cassette). The total filtration area using two parallel cassettes is 0.2 m2. During the TFF phase, the volume is reduced by 5x and the salt content is reduced by 19x. Electrophorese the final collagen slurry on an SDS-PAGE gel to confirm the presence of collagen. Dry the slurry for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0143] The purified truncated collagen obtained from the homogenized cell broth or the non-homogenized cells was analyzed on an SDS-PAGE gel, and a clear thick band was observed at the expected size of 27 kDa. The purified collagen was also analyzed by mass spectrometry, confirming that the 27 kDa protein is jellyfish collagen.

[0144] Alternative purification methods for full-length collagen and truncated collagen are provided below.

[0145] The fermentation broth was mixed with 0.3 - 0.5% w / v of polyethylenimine (PEI). After incubating with PEI for 15 minutes, the fermentation broth was centrifuged at 9000 rcf for 15 minutes to recover the supernatant containing collagen. The pellet containing cells was discarded, and the PEI-treated supernatant containing collagen was mixed with sodium bentonite (final 0.2% w / v) ( Wyoming bentonite) and centrifuged. The pellet containing bentonite was discarded, and the supernatant was recovered.

[0146] The bentonite-treated supernatant was concentrated 3 - 6-fold using a 5 kDa cassette in a tangential flow filtration system (TFF) (EMD Millipore). Collagen was retained with little loss of permeate flow. To remove salts, the retentate from the concentration step was diafiltered using the same TFF device. The final conductivity of the protein solution < 10 millisiemens. Typical conductivities are between 400 microsiemens and 1.5 millisiemens. Higher conductivities for more concentrated collagen solutions are close to 4 millisiemens. Those skilled in the art will understand that conductivities above 10 millisiemens can be observed depending on the concentration of collagen. Subsequently, the desalted concentrated protein was treated with activated carbon using a W-L 9000 10×40 granular resin (Carbon Activated Corporation). 5% w / v of the carbon resin was mixed with the collagen-containing feed and gently stirred at 45 - 50 °C. The carbon-treated slurry was filtered using a Buchner funnel lined with an Ertel filter pad M-953 (Ertel Alsop) in the presence or absence of a filter aid such as diatomaceous earth (Sigma Aldrich). After filtration, the collagen solution was filtered through a 0.2 micron filter and then treated with sodium bentonite (final 0.2% w / v) ( Wyoming bentonite) for one to several hours and centrifuged at 9000 rcf for 15 - 30 minutes to obtain a high-purity, clear, and particle-free collagen solution. When endotoxin proteins need to be removed, the protein was specifically removed of endotoxin proteins through a chromatographic filter such as Sartobind-Q (Sartorius-Stedim).

[0147] The purified collagen was analyzed on an SDS-PAGE gel, and a clear thick band was observed at 30 kDa. The upward shift in size is due to the structure of the collagen molecule and the high glycine / proline amino acid content. The purified collagen was also analyzed by mass spectrometry, and it was confirmed that the 30 kDa protein is truncated collagen.

[0148] The truncated collagen was further analyzed by HPLC using an Agilent 1100 series HPLC. The column was a 50 mm AgilentPLRP-S reversed-phase chromatography column with an inner diameter of 4.6 mm, a particle size of μM, and a pore size of 1000 Å.

[0149] Samples were prepared by diluting 1:1 in a 0.04% sodium azide solution in HPLC-grade water. After dilution, the resulting mixture was filtered through a 0.45 μm filter to remove any large particles that could clog the HPLC column. For analysis, the samples were appropriately diluted with 20 mM ammonium acetate buffer in HPLC-grade water at approximately pH 4.5. After mixing the samples, they were transferred to 300 μL vials and then placed in an autosampler. Using the software ChemStation that operates the HPLC, the analysis parameters such as sample flow rate, column temperature, mobile phase flow rate, mobile phase composition, etc. can be changed. In an exemplary but non-limiting analysis, the parameters were: sample flow rate of 1 mL / min, column temperature of 80 °C, column pressure of 60 - 70 bar, mobile phase composition of 97.9% water / 1.9% acetonitrile and 0.2% trifluoroacetic acid; UV wavelength for analysis was 214.4 nm, injection volume was 10 μL, and sample run time was 10 minutes.

[0150] Under these conditions, the truncated jellyfish collagen of SEQ ID NO: 5 had an elution time of approximately 5.4 minutes. ChemStation quantified the peak area of the elution peak and calculated the protein concentration using a calibration curve that directly relates the peak area to the protein concentration. This calibration curve was generated using a known collagen solution that was serially diluted to contain collagen concentrations from 0.06 mg / mL to 1.00 mg / mL.

[0151] Truncated collagen without His-tag-linker-thrombin cleavage site

[0152] Truncated jellyfish collagen without His-tag, linker, and thrombin cleavage site is disclosed below. A codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO: 6. The amino acid sequence is disclosed in SEQ ID NO: 7. The DsbA secretion tag is encoded by nucleotides 1-72 of SEQ ID NO: 6 and encodes amino acids 1-24 of SEQ ID NO: 7. The truncated collagen sequence is encoded by nucleotides 73-639 of SEQ ID NO: 6 and encodes amino acids 25-213 of SEQ ID NO: 7.

[0153] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTAATGCAGGTCAGAAAGGTCCGTCAGGTGAACCTGGCAGCCCTGGTAAAGCAGGTAGTGCCGGTGAGCAGGGTCCGCCGGGCAAAGATGGTAGTAATGGTGAGCCGGGTAGCCCTGGCAAAGAAGGTGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO: 6)

[0154] MKKIWLALAGLVLAFSASAAQYEDGPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:7)

[0155] A polynucleotide encoding truncated jellyfish collagen without a His-tag, linker, and thrombin cleavage site is disclosed in SEQ ID NO: 8.

[0156] GGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTAATGCAGGTCAGAAAGGTCCGTCAGGTGAACCTGGCAGCCCTGGTAAAGCAGGTAGTGCCGGTGAGCAGGGTCCGCCGGGCAAAGATGGTAGTAATGGTGAGCCGGGTAGCCCTGGCAAAGAAGGTGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:8)

[0157] Truncated jellyfish collagen without a His-tag, linker, and thrombin cleavage site is disclosed in SEQ ID NO: 5.

[0158] GPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:5)

[0159] Truncated collagen with DsbA secretion tag - His tag - linker - thrombin cleavage site and GFPβ - lactamase fusion protein (version 1):

[0160] The following discloses jellyfish collagen with DsbA secretion tag - His tag - linker - thrombin cleavage site and GFPβ - lactamase fusion protein. A codon - optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO: 9. The amino acid sequence is disclosed in SEQ ID NO: 10. The DsbA secretion tag is encoded by nucleotides 1 - 72 of SEQ ID NO: 9 and encodes amino acids 1 - 24 of SEQ ID NO: 10. The His tag is encoded by nucleotides 73 - 99 of SEQ ID NO: 9 and encodes a 9 - histidine tag of amino acids 25 - 33 of SEQ ID NO: 10. The linker is encoded by nucleotides 100 - 111 of SEQ ID NO: 9 and encodes amino acids 34 - 37 of SEQ ID NO: 10. The thrombin cleavage site is encoded by nucleotides 112 - 135 of SEQ ID NO: 9 and encodes amino acids 38 - 45 of SEQ ID NO: 10. Green fluorescent protein (GFP) with linker is encoded by nucleotides 136 - 873 of SEQ ID NO: 9 and encodes amino acids 46 - 291 of SEQ ID NO: 10. The truncated collagen sequence is encoded by nucleotides 874 - 1440 of SEQ ID NO: 9 and encodes amino acids 292 - 480 of SEQ ID NO: 10. β - lactamase with linker is encoded by nucleotides 1441 - 2232 of SEQ ID NO: 9 and encodes amino acids 481 - 744 of SEQ ID NO: 10. Even though the polypeptide does not have an independent secretion tag, β - lactamase is correctly targeted to the periplasmic space. The DsbA secretion tag directs the entire transcript (truncated collagen with DsbA secretion tag - His tag - linker - thrombin cleavage site and GFPβ - lactamase fusion protein) to the periplasmic space and β - lactamase functions normally.

[0161]

[0162] MKKIWLALAGLVLAFSASAAQYEDHHHHHHHHHSGSSLVPRGSHMSGSSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSGAPGGPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLRHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW(SEQ ID NO: 10)

[0163] The polynucleotide of SEQ ID NO: 9 was constructed by assembling multiple DNA fragments. The sequence containing collagen was codon-optimized and synthesized by internal synthesis at Gen9DNA (now Ginkgo Bioworks). GFP was also synthesized by Gen9. Using PCR with enzymes The GXL polymerase (www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase? sitex=10020:22372:US) was used to clone the β-lactamase from the plasmid pKD46 (cgsc2.biology.yale.edu / Strain.php? ID=68099). The overlapping sequences between the pET28 vector, GFP, collagen, and β-lactamase were designed to be 30 to 40 bp long and added using PCR with the GXL polymerase. Then the SGI Gibson (us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc) was used to assemble the opened pET28a vector and the insert into the final plasmid. The plasmid sequence was then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0164] The transformed cells were cultured in minimal medium and frozen in 1.5 ml aliquots with glycerol at a 50:50 cell:glycerol ratio. A vial of this frozen culture was thawed overnight at 37 °C, 200 rpm in 50 ml of minimal medium. The cells were transferred to 300 ml of minimal medium and grown for 6 - 9 hours to reach an OD600 of 5 - 10.

[0165] A bioreactor was prepared with 2.7 L of minimal medium + glucose and 300 ml of the culture with an OD600 of 5 - 10 was added to bring the starting volume to 3 L. The cells were grown at 28 °C, pH 7, where the dissolved oxygen was maintained at 20% saturation using a cascade of agitation, air, and oxygen. The pH was controlled with 28% w / w ammonium hydroxide solution. Once the initial bolus of 40 g / L was depleted in about 13 hours, a DO-stat based feeding algorithm was used to perform fermentation in fed-batch mode. After an initial growth of 24 - 26 hours, the OD600 reached above 100. At this point, 300 mL of 500 g / L sucrose was added and the temperature was reduced to 25 °C. High cell density cultivation was induced using 1 mM IPTG to produce the protein. Fermentation was continued for another 20 - 24 hours and the cells were harvested by centrifugation at 9000 rcf, 15 °C for 60 minutes using a benchtop centrifuge. The cell pellet recovered by centrifugation was resuspended in a buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at pH 8 at a weight ratio of 2x buffer to 1x cell.

[0166] The harvested cells were disrupted twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen as well as other proteins.

[0167] After centrifugation and resuspension in the above buffer, the unhomogenized intact cells recovered from the bioreactor were acid-treated to purify collagen. The pH of the resuspended suspension was lowered to pH 3 using 6M hydrochloric acid. The acidified cell slurry was incubated overnight at 4 °C with mixing and then centrifuged. Then the pH was raised to 9 using 10N NaOH, and the supernatant of the slurry was tested on a polyacrylamide gel and found to have a relatively high abundance of collagen compared to the starting precipitate. The collagen slurry thus obtained had a high salt content. To reduce the volume and salt content, concentration and diafiltration steps were performed using an EMD Millipore tangential flow filtration system (each using a 0.1 m2 ultrafiltration cassette). The total filtration area using two parallel cassettes was 0.2 m2. During the TFF stage, the volume was reduced 5x and the salt content was reduced 19x. The final collagen slurry was electrophoresed on an SDS-PAGE gel to confirm the presence of collagen. The slurry was dried for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0168] The purified collagen-GFP-β-lactamase fusion protein was analyzed on an SDS-PAGE gel, and electrophoresis was observed at an apparent molecular weight of 90 kDa. The expected size of this fusion protein was 85 kDa. The 90 kDa band was confirmed by mass spectrometry to be the correct collagen fusion protein.

[0169] Truncated collagen (version 2) with DsbA secretion tag - His tag - linker - thrombin cleavage site and GFPβ-lactamase fusion protein:

[0170] The following discloses jellyfish collagen with a DsbA secretion tag-His tag-linker-thrombin cleavage site and a GFPβ-lactamase fusion protein. A codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO: 11. The amino acid sequence is disclosed in SEQ ID NO: 12. The DsbA secretion tag is encoded by nucleotides 1-72 of SEQ ID NO: 11 and encodes amino acids 1-24 of SEQ ID NO: 12. The His tag is encoded by nucleotides 73-99 of SEQ ID NO: 11 and encodes a 9-histidine tag of amino acids 25-33 of SEQ ID NO: 12. The linker is encoded by nucleotides 100-111 of SEQ ID NO: 11 and encodes amino acids 34-37 of SEQ ID NO: 12. The thrombin cleavage site is encoded by nucleotides 112-135 of SEQ ID NO: 11 and encodes amino acids 38-45 of SEQ ID NO: 12. Green fluorescent protein (GFP) with a linker is encoded by nucleotides 136-873 of SEQ ID NO: 11 and encodes amino acids 46-291 of SEQ ID NO: 12. The truncated collagen sequence is encoded by nucleotides 874-1440 of SEQ ID NO: 11 and encodes amino acids 292-480 of SEQ ID NO: 12. β-lactamase with a linker is encoded by nucleotides 1441-2232 of SEQ ID NO: 11 and encodes amino acids 481-744 of SEQ ID NO: 12.

[0171]

[0172] MKKIWLALAGLVLAFSASAAQYEDHHHHHHHHHSGSSLVPRGSHMSGSSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSGAPGGPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLRHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW(SEQ ID NO:12)

[0173] Example 2. Human Collagen

[0174] Truncated human type XXI alpha1 collagen

[0175] The following discloses a truncated human type XXI α1 collagen without a His-tag, linker, and thrombin cleavage site (truncated relative to full-length human type XXI α1 collagen (SEQ ID NO: 31)). The codon-optimized nucleotide sequence and amino acid sequence encoding this collagen are disclosed below. The DsbA secretion tag is encoded by nucleotides 1-72 of SEQ ID NO: 13 and encodes amino acids 1-24 of SEQ ID NO: 14. The truncated collagen sequence is encoded by nucleotides 73-633 of SEQ ID NO: 13 and encodes amino acids 25-211 of SEQ ID NO: 14.

[0176] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO: 13.

[0177] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGCAGGTTTTCCGGGTCTGCCTGGTCCGGCAGGCGAACCGGGTCGTCATGGTAAAGATGGTCTGATGGGTAGTCCGGGTTTTAAAGGTGAAGCAGGTTCACCGGGTGCACCTGGTCAGGATGGCACCCGTGGTGAACCGGGTATTCCGGGATTTCCGGGTAATCGTGGCCTGATGGGTCAGAAAGGTGAAATTGGTCCGCCTGGTCAGCAGGGTAAAAAAGGCGCACCGGGTATGCCAGGACTGATGGGTTCAAATGGCAGTCCGGGTCAGCCAGGCACACCGGGTTCAAAAGGTAGCAAAGGCGAACCTGGTATTCAGGGTATGCCTGGTGCAAGCGGTCTGAAAGGCGAGCCAGGTGCCACCGGTTCTCCGGGTGAACCAGGTTATATGGGTCTGCCAGGTATCCAAGGCAAAAAAGGTGATAAAGGTAATCAGGGCGAAAAAGGCATTCAGGGCCAGAAAGGCGAAAATGGCCGTCAGGGTATTCCAGGCCAGCAGGGCATCCAGGGTCATCATGGTGCAAAAGGTGAACGTGGTGAAAAGGGCGAACCAGGTGTTCGTTTA(SEQ ID NO: 13)

[0178] The amino acid sequence is disclosed in SEQ ID NO: 14.

[0179] MKKIWLALAGLVLAFSASAAQYEDAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATGSPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVR(SEQ ID NO:14)

[0180] The codon-optimized nucleotide sequence encoding truncated human type XXI α1 collagen without the DsbA secretion tag is provided in SEQ ID NO:15.

[0181] TGCAGGTTTTCCGGGTCTGCCTGGTCCGGCAGGCGAACCGGGTCGTCATGGTAAAGATGGTCTGATGGGTAGTCCGGGTTTTAAAGGTGAAGCAGGTTCACCGGGTGCACCTGGTCAGGATGGCACCCGTGGTGAACCGGGTATTCCGGGATTTCCGGGTAATCGTGGCCTGATGGGTCAGAAAGGTGAAATTGGTCCGCCTGGTCAGCAGGGTAAAAAAGGCGCACCGGGTATGCCAGGACTGATGGGTTCAAATGGCAGTCCGGGTCAGCCAGGCACACCGGGTTCAAAAGGTAGCAAAGGCGAACCTGGTATTCAGGGTATGCCTGGTGCAAGCGGTCTGAAAGGCGAGCCAGGTGCCACCGGTTCTCCGGGTGAACCAGGTTATATGGGTCTGCCAGGTATCCAAGGCAAAAAAGGTGATAAAGGTAATCAGGGCGAAAAAGGCATTCAGGGCCAGAAAGGCGAAAATGGCCGTCAGGGTATTCCAGGCCAGCAGGGCATCCAGGGTCATCATGGTGCAAAAGGTGAACGTGGTGAAAAGGGCGAACCAGGTGTTCGTtaa(SEQ ID NO:15)

[0182] The amino acid sequence of truncated human type 21 α1 collagen without the DsbA secretion tag is provided in SEQ ID NO: 16.

[0183] AGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATGSPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVR(SEQ ID NO: 16)

[0184] The polynucleotide of SEQ ID NO: 13 was synthesized by Twist Bioscience. The overlapping sequence between the pET28 vector and SEQ ID NO: 15 and SEQ ID NO: 16 was designed to be 20 to 40 bp long and added using PCR with the enzyme GXL polymerase (www.takarabio.com / products / pcr / gc-rich-pcr / primestar-gxl-dna-polymerase). Then the opened pET28a vector and the insert DNA (SEQ ID NO: 13) were assembled together into the final plasmid using In-Fusion Cloning (www.takarabio.com / products / cloning / in-fusion-cloning). The plasmid sequence was then verified by Sanger sequencing by Genewiz (www.genewiz.com / en).

[0185] The transformed cells were cultured in minimal medium and frozen in 1.5 ml aliquots with plant glycerol at a 50:50 cell:glycerol ratio. A vial of this frozen culture was thawed overnight at 37 °C and 200 rpm in 50 ml of minimal medium. The cells were transferred to 300 ml of minimal medium and grown for 6 - 9 hours to reach an OD600 of 5 - 10.

[0186] The minimal medium used in this example and throughout the application was prepared as follows:

[0187] 1) 5 L of a 550 g / kg sucrose slurry (VWR, product number 97061 - 170) in DI water was autoclaved.

[0188] 2) The following were autoclaved in 3946 mL of DI water:

[0189] 20 g of (NH4)2HPO4. (VWR, product number 97061 - 932);

[0190] 66.5 g of KH2PO4. (VWR, product number 97062 - 348);

[0191] 22.5 g of H3C6H5O7. (VWR, product number BDH9228 - 2.5KG);

[0192] 8.85 g of MgSO4·7H2O. (VWR, product number 97062 - 134);

[0193] 10 mL of 1000x trace metal preparation (Table 3).

[0194] After autoclaving, add:

[0195] 118 g of (1) to (2);

[0196] 5 mL of 25 mg / mL kanamycin sulfate (VWR - V0408);

[0197] Adjust the pH to 6.1 using 28% NH4OH (VWR, product number BDH3022).

[0198] Table 3. Trace metal preparation

[0199] Ferrous sulfate heptahydrate, 27.8 g / L (Spectrum, 7782-63-0) Zinc sulfate heptahydrate, 2.88 g / L (Spectrum, 7446-20-0) Calcium chloride dihydrate, 2.94 g / L (Spectrum, 2971347) Sodium molybdate dihydrate, 0.48 g / L (Spectrum, 10102-40-6) Manganese chloride tetrahydrate, 1.26 g / L (Spectrum, 13446-34-9) Sodium selenite, 0.35 g / L (Spectrum, 10102-18-8) Boric acid, 0.12 g / L (Spectrum, 10043-35-3

[0200] Fermentation is carried out at different temperatures from 25 °C to 28 °C. For some fermentations, the fermentation temperature is maintained at a constant temperature, and collagen is purified immediately upon completion of fermentation. For other fermentations, the fermentation temperature is maintained for a desired period of time, and when the OD 600 cell density reaches 10 - 20, the temperature is reduced to induce protein production. Typically, the temperature is reduced from 28 °C to 25 °C. After continuing fermentation at 25 °C for 40 - 60 hours.

[0201] Purify collagen as follows: Lower the pH of the fermentation broth to 3 - 3.5 using 5 - 50% sulfuric acid. Then separate the cells using centrifugation. The supernatant of the acidified broth was tested on a polyacrylamide gel and found to have a relatively high abundance of collagen compared to the starting precipitate. The collagen slurry thus obtained has a high salt content. To reduce the volume and salt content, concentration and diafiltration steps are performed using an EMD Millipore tangential flow filtration system (each using a 0.1 m2 ultrafiltration cassette). The total filtration area using two parallel cassettes is 0.2 m2. During the TFF stage, the volume is reduced by 5x and the salt content is reduced by 19x. Electrophorese the final collagen slurry on an SDS - PAGE gel to confirm the presence of collagen.

[0202] The purified collagen was analyzed on an SDS-PAGE gel, and a clear thick band was observed at the expected size of 25 kDa. The titer and purity of the collagen were quantified using reverse-phase and size-exclusion HPLC chromatography. The titer was generally between 3 and 8 grams per liter. The purified collagen was also further analyzed by mass spectrometry, confirming a match to the publicly available sequence of human type 21 collagen.

[0203] Truncated human type 1 α2(1) collagen

[0204] Truncated human type 1 α2 collagen without His tag, linker, and thrombin cleavage site is disclosed below (truncated relative to full-length human type 1 α2 collagen (SEQ ID NO: 32)). The codon-optimized nucleotide sequence and amino acid sequence are disclosed below. The DsbA secretion tag is encoded by nucleotides 1-72 of SEQ ID NO: 17 and encodes amino acids 1-24 of SEQ ID NO: 18. The truncated collagen sequence is encoded by nucleotides 73-636 of SEQ ID NO: 17 and encodes amino acids 25-212 of SEQ ID NO: 18.

[0205] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO: 17.

[0206] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATATGGGTCCGCCTGGTAGCCGTGGTGCAAGTGGTCCGGCAGGCGTTCGTGGTCCGAATGGTGATGCAGGTCGTCCGGGTGAACCGGGTCTGATGGGTCCTCGTGGTCTGCCTGGTTCACCGGGTAATATTGGTCCTGCAGGTAAAGAAGGTCCGGTTGGTCTGCCAGGTATTGATGGCCGTCCGGGTCCGATTGGTCCAGCCGGTGCACGTGGTGAACCTGGCAATATTGGTTTTCCGGGTCCTAAAGGTCCGACCGGTGATCCGGGTAAAAATGGTGATAAAGGTCATGCAGGTCTGGCAGGCGCACGCGGTGCACCTGGTCCGGATGGTAATAATGGTGCACAGGGTCCACCGGGTCCGCAGGGTGTTCAAGGTGGTAAAGGCGAACAGGGTCCTGCCGGTCCTCCGGGTTTTCAGGGACTGCCTGGTCCGAGCGGTCCTGCGGGTGAAGTTGGTAAACCTGGTGAACGCGGTCTGCATGGTGAATTTGGCCTGCCTGGGCCTGCAGGTCCGCGTGGCGAACGTGGTCCGCCAGGTGAAAGCGGTGCAGCAGGTCCGACAGGTTAA(SEQ ID NO: 17)

[0207] The amino acid sequence is provided in SEQ ID NO: 18.

[0208] MKKIWLALAGLVLAFSASAAQYEDMGPPGSRGASGPAGVRGPNGDAGRPGEPGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPAGPPGFQGLPGPSGPAGEVGKPGERGLHGEFGLPGPAGPRGERGPPGESGAAGPTG(SEQ ID NO: 18)

[0209] The nucleic acid sequence of truncated human type 1 α2(1) collagen without the DsbA secretion tag is provided in SEQ ID NO: 19.

[0210] ATGGGTCCGCCTGGTAGCCGTGGTGCAAGTGGTCCGGCAGGCGTTCGTGGTCCGAATGGTGATGCAGGTCGTCCGGGTGAACCGGGTCTGATGGGTCCTCGTGGTCTGCCTGGTTCACCGGGTAATATTGGTCCTGCAGGTAAAGAAGGTCCGGTTGGTCTGCCAGGTATTGATGGCCGTCCGGGTCCGATTGGTCCAGCCGGTGCACGTGGTGAACCTGGCAATATTGGTTTTCCGGGTCCTAAAGGTCCGACCGGTGATCCGGGTAAAAATGGTGATAAAGGTCATGCAGGTCTGGCAGGCGCACGCGGTGCACCTGGTCCGGATGGTAATAATGGTGCACAGGGTCCACCGGGTCCGCAGGGTGTTCAAGGTGGTAAAGGCGAACAGGGTCCTGCCGGTCCTCCGGGTTTTCAGGGACTGCCTGGTCCGAGCGGTCCTGCGGGTGAAGTTGGTAAACCTGGTGAACGCGGTCTGCATGGTGAATTTGGCCTGCCTGGGCCTGCAGGTCCGCGTGGCGAACGTGGTCCGCCAGGTGAAAGCGGTGCAGCAGGTCCGACAGGTTAA(SEQ ID NO: 19)

[0211] The amino acid sequence of truncated human type 1 α2(1) collagen without the DsbA secretion tag is provided in SEQ ID NO: 20.

[0212] MGPPGSRGASGPAGVRGPNGDAGRPGEPGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPAGPPGFQGLPGPSGPAGEVGKPGERGLHGEFGLPGPAGPRGERGPPGESGAAGPTG(SEQ ID NO: 20)

[0213] Truncated human type 1 α2(2) collagen

[0214] Truncated human type 1 α2 collagen without His-tag, linker, and thrombin cleavage site (truncated relative to full-length human type 1 α2 collagen (SEQ ID NO: 32)) is disclosed below. The codon-optimized nucleotide sequence and amino acid sequence are disclosed below. The DsbA secretion tag is encoded by nucleotides 1-72 of SEQ ID NO: 21 and encodes amino acids 1-24 of SEQ ID NO: 22. The truncated collagen sequence is encoded by nucleotides 73-609 of SEQ ID NO: 21 and encodes amino acids 25-203 of SEQ ID NO: 22.

[0215] The codon-optimized nucleotide sequence encoding the collagen is provided in SEQ ID NO: 21.

[0216] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGGTTTTCAGGGTCCTGCCGGTGAACCGGGTGAACCTGGTCAGACAGGTCCGGCAGGCGCACGTGGTCCTGCAGGTCCTCCTGGTAAAGCCGGTGAAGATGGTCATCCGGGTAAACCGGGTCGTCCTGGTGAACGTGGTGTTGTTGGTCCGCAGGGTGCCCGTGGTTTTCCGGGTACTCCGGGTCTGCCAGGTTTTAAAGGTATTCGTGGTCATAATGGTCTGGATGGTCTGAAAGGTCAGCCTGGTGCACCGGGTGTTAAAGGTGAACCAGGTGCTCCGGGTGAAAATGGCACACCGGGTCAGACCGGTGCGCGTGGTCTGCCTGGCGAACGCGGTCGTGTTGGTGCACCTGGTCCAGCCGGTGCACGCGGTAGTGATGGTAGCGTTGGTCCGGTTGGTCCAGCGGGTCCGATTGGTAGCGCAGGTCCACCGGGTTTTCCAGGCGCACCGGGTCCGAAAGGTGAAATTGGTGCAGTTGGTAATGCAGGCCCTGCCGGTCCAGCAGGACCGCGTGGTGAAGTTGGCCTGCCTGGTCTGTAA(SEQ ID NO: 21)

[0217] The amino acid sequence is provided in SEQ ID NO: 22.

[0218] MKKIWLALAGLVLAFSASAAQYEDGFQGPAGEPGEPGQTGPAGARGPAGPPGKAGEDGHPGKPGRPGERGVVGPQGARGFPGTPGLPGFKGIRGHNGLDGLKGQPGAPGVKGEPGAPGENGTPGQTGARGLPGERGRVGAPGPAGARGSDGSVGPVGPAGPIGSAGPPGFPGAPGPKGEIGAVGNAGPAGPAGPRGEVGLPGL(SEQ ID NO:22)

[0219] The nucleic acid sequence of truncated human type 1 α2(2) collagen without the DsbA secretion tag is provided in SEQ ID NO: 23.

[0220] GGTTTTCAGGGTCCTGCCGGTGAACCGGGTGAACCTGGTCAGACAGGTCCGGCAGGCGCACGTGGTCCTGCAGGTCCTCCTGGTAAAGCCGGTGAAGATGGTCATCCGGGTAAACCGGGTCGTCCTGGTGAACGTGGTGTTGTTGGTCCGCAGGGTGCCCGTGGTTTTCCGGGTACTCCGGGTCTGCCAGGTTTTAAAGGTATTCGTGGTCATAATGGTCTGGATGGTCTGAAAGGTCAGCCTGGTGCACCGGGTGTTAAAGGTGAACCAGGTGCTCCGGGTGAAAATGGCACACCGGGTCAGACCGGTGCGCGTGGTCTGCCTGGCGAACGCGGTCGTGTTGGTGCACCTGGTCCAGCCGGTGCACGCGGTAGTGATGGTAGCGTTGGTCCGGTTGGTCCAGCGGGTCCGATTGGTAGCGCAGGTCCACCGGGTTTTCCAGGCGCACCGGGTCCGAAAGGTGAAATTGGTGCAGTTGGTAATGCAGGCCCTGCCGGTCCAGCAGGACCGCGTGGTGAAGTTGGCCTGCCTGGTCTGTAA(SEQ ID NO:23)

[0221] The amino acid sequence of truncated human type 1 α2(2) collagen without the DsbA secretion tag is provided in SEQ ID NO: 24.

[0222] GFQGPAGEPGEPGQTGPAGARGPAGPPGKAGEDGHPGKPGRPGERGVVGPQGARGFPGTPGLPGFKGIRGHNGLDGLKGQPGAPGVKGEPGAPGENGTPGQTGARGLPGERGRVGAPGPAGARGSDGSVGPVGPAGPIGSAGPPGFPGAPGPKGEIGAVGNAGPAGPAGPRGEVGLPGL(SEQ ID NO:24)

[0223] As described herein, the polynucleotide of SEQ ID NO: 13, 17 or 21 was subcloned into the vector pET28a to prepare a transformation vector. The host cell was transformed with the vector and the polynucleotide was expressed as described in Example 1.

[0224] After fermentation was completed, the truncated human collagen was purified from the fermentation broth using the procedure disclosed in Example 2. As disclosed in Example 2, the purified truncated human collagen was analyzed using SDS-PAGE and HPLC.

[0225] In the SDS-PAGE analysis, all three truncated human collagens electrophoresed at the expected molecular weights. When analyzing the truncated human collagen using HPLC, a standard curve using the jellyfish collagen of Example 1 was utilized. The retention times of the human collagens were slightly different from those of the jellyfish collagen. The retention time of SEQ ID NO: 16 was 5.645 minutes, the retention time of SEQ ID NO: 20 was 5.631 minutes, and SEQ ID NO: 24 electrophoresed at two peaks and had retention times of 5.531 minutes and 5.7 minutes.

[0226] Truncated human type I α2 collagen truncated 5 with DsbA secretion tag and FLAG tag

[0227] The amino acid sequence of truncated human type I α2 collagen truncated 5 with DsbA secretion tag and FLAG tag is disclosed in SEQ ID NO: 25. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO: 26 and the amino acid sequence is amino acids 1-19 of SEQ ID NO: 25. The nucleotide sequence of the collagen is nucleotides 58-657 of SEQ ID NO: 26 and the amino acid sequence is amino acids 20-219 of SEQ ID NO: 25. The FLAG nucleotide sequence is nucleotides 658-684 of SEQ ID NO: 26 and the amino acid sequence is amino acids 220-228 of SEQ ID NO: 25.

[0228] MKKIWLALAGLVLAFSASAGDQGPVGRTGEVGAVGPPGFAGEKGPSGEAGTAGPPGTPGPQGLLGAPGILGLPGSRGERGLPGVAGAVGEPGPLGIAGPPGARGPPGAVGSPGVNGAPGEAGRDGNPGNDGPPGRDGQPGHKGERGYPGNIGPVGAAGAPGPHGPVGPAGKHGNRGETGPSGPVGPAGAVGPRGPSGPQGIRGDKGEPGEKGPRGLPGLGDYKDDDDK(SEQ ID NO:25)

[0229] The nucleic acid sequence of truncated human type 1 α2 collagen truncated 5 with DsbA secretion tag and FLAG tag is disclosed in SEQ ID NO: 26.

[0230] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGGTGATCAGGGTCCGGTTGGTCGTACCGGTGAAGTTGGTGCAGTCGGGCCGCCGGGTTTTGCGGGTGAAAAAGGCCCGTCAGGTGAAGCAGGCACCGCTGGCCCTCCTGGCACGCCTGGCCCACAGGGTTTACTGGGCGCACCTGGAATTCTGGGACTGCCGGGCAGCCGTGGAGAACGCGGTTTACCAGGTGTTGCCGGTGCCGTTGGTGAACCTGGTCCACTGGGCATTGCAGGGCCGCCTGGCGCACGGGGACCGCCTGGTGCTGTTGGTAGTCCGGGTGTGAATGGTGCTCCGGGTGAAGCCGGTCGTGACGGTAATCCGGGAAATGACGGCCCGCCAGGCCGCGATGGTCAGCCGGGTCATAAAGGTGAGCGTGGTTACCCAGGTAATATTGGTCCAGTCGGTGCCGCCGGTGCGCCGGGTCCTCATGGCCCTGTCGGTCCAGCCGGTAAACATGGTAATCGCGGTGAGACAGGTCCGTCAGGACCAGTGGGCCCTGCTGGCGCAGTCGGTCCGCGCGGGCCGAGTGGCCCTCAGGGTATTCGTGGCGATAAAGGGGAACCGGGCGAAAAAGGGCCGCGGGGTCTGCCAGGCCTGGGTGACTACAAAGACGACGACGACAAATAA(SEQ ID NO:26)

[0231] The polynucleotide of SEQ ID NO: 26 was subcloned into the vector pET28a, expressed in the host Escherichia coli cells, and the truncated collagen was purified as described herein. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG Western was observed at approximately 100 kilodaltons. In the absence of expression of this protein, no band appeared at this position on the gel.

[0232] Truncated human type I α2 collagen truncated 6 with DsbA secretion tag and FLAG tag

[0233] The amino acid sequence of truncated human type 1 α2 collagen truncated 6 with DsbA secretion tag and FLAG tag is disclosed in SEQ ID NO: 27. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO: 28 and the amino acid sequence is amino acids 1-19 of SEQ ID NO: 27. The nucleotide sequence of collagen is nucleotides 58-657 of SEQ ID NO: 28 and the amino acid sequence is amino acids 20-219 of SEQ ID NO: 27. The FLAG nucleotide sequence is nucleotides 658-684 of SEQ ID NO: 28 and the amino acid sequence is amino acids 220-228 of SEQ ID NO: 27.

[0234] MKKIWLALAGLVLAFSASAKGHNGLQGLPGIAGHHGDQGAPGSVGPAGPRGPAGPSGPAGKDGRTGHPGTVGPAGIRGPQGHQGPAGPPGPPGPPGPPGVSGGGYDFGYDGDFYRADQPRSAPSLRPKDYEVDATLKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFSTGETCIRAQPENIPAKNWYRSSKDGDYKDDDDK(SEQ ID NO: 27)

[0235] The nucleic acid sequence of truncated human type 1 α2 collagen truncated 6 with DsbA secretion tag and FLAG tag is disclosed in SEQ ID NO: 28.

[0236] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGAAAGGTCACAATGGACTGCAAGGCCTGCCAGGTATTGCAGGTCATCATGGTGATCAAGGTGCCCCGGGAAGCGTTGGTCCGGCGGGGCCGAGAGGCCCTGCGGGACCTTCAGGTCCGGCAGGCAAAGATGGTCGGACAGGCCATCCGGGCACCGTTGGCCCTGCAGGAATTCGTGGACCGCAGGGTCATCAGGGACCTGCTGGTCCGCCAGGTCCCCCGGGCCCTCCGGGACCACCGGGTGTTAGTGGTGGTGGTTATGATTTTGGCTATGATGGTGATTTTTATCGTGCAGATCAGCCGCGTAGCGCACCGAGCCTGCGTCCTAAAGATTATGAAGTTGATGCAACCCTGAAAAGCCTGAATAATCAGATTGAAACACTGCTGACACCGGAAGGTAGCCGTAAAAATCCGGCCCGTACCTGTCGTGATCTGCGTCTGAGCCACCCGGAATGGAGCAGCGGTTATTATTGGATTGATCCGAATCAAGGTTGTACCATGGATGCAATTAAAGTTTATTGTGATTTTAGCACAGGTGAAACATGTATCCGTGCACAGCCGGAAAATATTCCGGCCAAAAATTGGTATCGTAGTAGCAAAGATGGTGACTACAAAGACGACGACGACAAATAA(SEQ ID NO:28)

[0237] The polynucleotide of SEQ ID NO: 28 was subcloned into the vector pET28a, expressed in the host Escherichia coli cells, and the truncated collagen was purified as described herein. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG Western was observed at approximately 25 kilodaltons. In the absence of expression of this protein, no band appeared at this position on the gel.

[0238] Truncated human type 1 α2 collagen truncated 7 with DsbA secretion tag and FLAG tag

[0239] The amino acid sequence of truncated human type I α2 collagen truncated 7 with DsbA secretion tag and FLAG tag is disclosed in SEQ ID NO: 29. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO: 30 and the amino acid sequence is amino acids 1-19 of SEQ ID NO: 29. The nucleotide sequence of collagen is nucleotides 58-759 of SEQ ID NO: 30 and the amino acid sequence is amino acids 20-253 of SEQ ID NO: 29. The FLAG nucleotide sequence is nucleotides 760-786 of SEQ ID NO: 30 and the amino acid sequence is amino acids 254-262 of SEQ ID NO: 29.

[0240] MKKIWLALAGLVLAFSASAYEVDATLKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFSTGETCIRAQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVEGVTSKEMATQLAFMRLLANYASQNITYHCKNSIAYMDEETGNLKKAVILQGSNDVELVAEGNSRFTYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFKGDYKDDDDK(SEQ ID NO: 29)

[0241] The nucleic acid sequence of truncated human type I α2 collagen truncated 7 with DsbA secretion tag and FLAG tag is disclosed in SEQ ID NO: 30.

[0242] TGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGTATGAAGTTGATGCAACCCTGAAAAGCCTGAATAATCAGATTGAAACACTGCTGACACCGGAAGGTAGCCGTAAAAATCCGGCCCGTACCTGTCGTGATCTGCGTCTGAGCCACCCGGAATGGAGCAGCGGTTATTATTGGATTGATCCGAATCAAGGTTGTACCATGGATGCAATTAAAGTTTATTGTGATTTTAGCACAGGTGAAACATGTATCCGTGCACAGCCGGAAAATATTCCGGCCAAAAATTGGTATCGTAGTAGCAAAGATAAAAAACATGTGTGGCTGGGTGAAACCATTAATGCAGGTAGCCAGTTTGAATACAATGTTGAAGGTGTTACCAGCAAAGAAATGGCAACACAGCTGGCATTTATGCGTCTGCTGGCAAATTATGCAAGCCAGAATATTACATATCATTGTAAAAATAGCATTGCATATATGGATGAAGAAACCGGTAATCTGAAAAAAGCAGTTATTCTGCAGGGTAGCAATGATGTTGAACTGGTTGCCGAAGGTAATAGCCGTTTTACATATACCGTTCTGGTTGATGGTTGTAGCAAAAAAACCAATGAATGGGGTAAAACCATCATTGAATATAAAACCAACAAACCGAGCCGTCTGCCGTTTCTGGATATCGCTCCGCTGGATATTGGTGGTGCCGATCAGGAATTTTTTGTCGATATCGGTCCTGTGTGTTTTAAAGGTGACTACAAAGACGACGACGACAAATAA(SEQ ID NO:30)

[0243] The polynucleotide of SEQ ID NO: 30 was subcloned into the vector pET28a, expressed in the host Escherichia coli cells, and the truncated collagen was purified as described herein. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG Western was observed at approximately 30 kilodaltons. In the absence of expression of this protein, no band appeared at this position on the gel.

[0244] Example 3. Human Clinical Study of Truncated Human Collagen Type 21

[0245] A clinical study was conducted using human subjects to determine the effect of a topical skin care product containing truncated human collagen type 21 (SEQ ID NO: 16). This study was conducted in accordance with US and international Good Clinical Practice (FDA and ICH guidelines) and applicable government regulations.

[0246] A base preparation (control preparation) was prepared from water, olive oil glycereth-8 esters, glycerin, cocoalkane, acryloyldimethyltaurate / VP copolymer, pentylene glycol, disodium EDTA, caprylyl glycol, chlorphenesin, phenoxyethanol. A preparation containing truncated human collagen type 21 was prepared by adding sufficient collagen, thereby preparing a topical preparation containing 0.1% w / w collagen.

[0247] Professional Graded Assessment of Eligibility and Efficacy

[0248] Visual Analog Scales (VAS) are commonly used in clinical studies to measure the intensity or frequency of various symptoms, subjective characteristics, or attitudes that cannot be directly measured. VAS is a reliable scale and is more sensitive to small changes than simple ordinal scales (A. Paul-Dauphin, F. Guillemin, J. Virion, and S. Briancon, "Bias and precision in visual analog scales: A randomized controlled trial," American Journal of Epidemiology, Vol. 150, No. 10, pp. 1117-27, 1999). When answering a VAS item, a professional rater indicates their degree of agreement with a statement by indicating the position along a line (10 cm) between two end points or anchors. Simple VAS was used to evaluate efficacy parameters, where the two ends of the 10 cm horizontal line were defined as limits oriented from left (best) to right (worst). Signs of photoaging can be classified as follows: mild = 1 - 3.9 cm, moderate = 4 - 6.9 cm, severe = 7 - 10 cm.

[0249] The following VAS was used:

[0250]

[0251]

[0252] Ordinal scales allow numbers to relate directly and objectively to the quality of a given attribute. When answering ordinal scale items, professional raters specify their degree of agreement with a statement by selecting a set rank or level.

[0253] The appearance of facial skin flushing (erythema) for each subject was evaluated by a professional rater using the following five-point ordinal scale (for eligibility at baseline) (Table 4). If eligible, each subject will receive further erythema assessments at Week 2, Week 4, Week 6, and Week 8.

[0254] Table 4. Five-point ordinal scale for erythema assessment

[0255]

[0256] The skin moisture tester CM 820 (Courage+Khazaka, Germany) measures the relative degree of skin hydration by applying an alternating current to the skin using a pair of closely spaced electrodes and measuring the capacitance. Changes in skin water content alter the conductivity of the capacitance circuit.

[0257] The skin moisture tester can reproducibly detect minute changes in the hydration level with a measurement time of only about one second. The measurement depth is small (the stratum corneum at about 10 - 20 μm), which ensures that the assessment is not affected by deeper skin layers.

[0258] All subjects received skin moisture tester measurements on their faces at baseline, immediately after the initial application, and at Weeks 2, 4, and 8. The measurements were taken in triplicate and averaged at each time point. The measurement locations were recorded on a facial map to ensure consistency of assessment at each time point.

[0259] The skin elasticity tester MPA 580 (Courage+Khazaka, Germany) measures the viscoelastic properties of the skin as follows: a suction force is applied to the skin surface, the skin is drawn into the hole of the probe, and an optical measurement system is used to determine the penetration depth.

[0260] The resistance (tightness) of the skin sucked up by negative pressure and its ability to return to the original position (elasticity) are calculated and presented in the form of a curve. The output of the skin elasticity tester includes many parameters of different parts of the measurement curve, including R0 (Uf, tightness), R2 (Ua / Uf, total elasticity), R5 (Ur / Ue, net elasticity), R7 (Ur / Uf, elastic part), and R9 (R3[last maxamp] - R0[Uf], fatigue).

[0261] All subjects had skin elasticity measured using a skin elasticity tester on the left or right cheek (following the prepared random code) at baseline, immediately after the first application, and at weeks 2, 4, and 8. Skin elasticity was reported using the R5 (Ur / Ue) and R2 (Ua / Uf) parameters. As the skin becomes more elastic, this value will increase. Skin firmness was reported using the R0 (Uf) parameter. As the skin becomes tighter, this value will decrease. The evaluation location was recorded on a facial map for each subject to ensure measurement consistency between visits.

[0262] COSMETRICS TM The SIAScope (Astron Clinica, Toft, UK) is a non-invasive optical skin imaging instrument that uses spectrophotometric intracutaneous analysis (SIA) or chromophore mapping. This technique is based on a unique combination of dermoscopy and contact remittance spectrophotometry. The hardware consists of a handheld imaging probe connected to a laptop computer. The device is placed in contact with the skin surface, and high-intensity LEDs illuminate the skin at discrete wavelengths from 400 to 1000 nm (covering the visible spectrum and a small range of the near-infrared spectrum). Digital images are captured for each wavelength. Three parameter chromophore maps (up to 2 mm in depth and 11 mm in perimeter) are retrieved, each corresponding to one of the following parameters: epidermal melanin, dermal hemoglobin, and dermal collagen.

[0263] For the purposes of this study, dermal collagen will be measured on the left or right cheek at baseline and at weeks 2, 4, and 8 following the prepared random code. The evaluation location will be recorded on a facial map for each subject to ensure measurement consistency between visits.

[0264] The DermaScan C USB (Cortex Technology ApS, Hadsund, Denmark) is a compact high-resolution ultrasound scanner. It uses a 20 MHz, high-resolution 60×150 μm, 13 mm penetration probe, which provides linear scanning, high-precision operation, and true position detection for image clarity and resolution.

[0265] This instrument was provided by cyberDERM, Inc. (Broomall, PA, USA). All subjects had an ultrasound evaluation on the face at baseline and at weeks 2, 4, and 8. The location of the evaluation was the same at each visit and was recorded on a facial map. After obtaining the ultrasound scan, it was sent to cyberDERM, Inc. for analysis of dermal thickness (density).

[0266] All clinical photography was conducted according to the SOP of IRSI to ensure the reproducibility of high-quality images throughout the study. Imaging was performed in a designated photography suite with matte black walls, and all natural light was blocked. To prepare the subjects for clinical photography, the subjects were required to remove all jewelry, including earrings, necklaces, and any facial jewelry. A trained technician examined the subjects under a light amplification circuit to ensure that no residual colored cosmetics or skin care products were visible on the face, eyes, or lips. The subjects were provided with a black cloak and a black headband and instructed to place them to ensure that all hair was neatly pulled back and covered.

[0267] Clarity TM The 2D research system Ti (Clarity) (BrighTex Bio-Photonics (BTBP), San Jose CA, USA) captured high-quality full-face frontal, left, and right images. The three cameras in the system used real-time feed displays and automatic facial alignment checks for baseline images (for reproducibility) to simultaneously capture 16-bit 18-megapixel SLR images.

[0268] Multispectral lighting (diffuse white light, cross-polarization, blue light, and parallel polarization) revealed skin conditions above and below the skin surface. The system used skin feature recognition to apply automatic skin segmentation and area mapping for subsequent skin analysis. Attributes related to pigmentation, subsurface pigmentation, gloss, skin color, redness, wrinkles, skin texture, pores, acne, and / or lips were analyzed in the images.

[0269] All subjects had their facial images taken in the frontal, left, and right views under standard light and parallel polarization light at baseline, week 2, week 4, and week 8.

[0270] The subjective questionnaire allowed the sponsor to evaluate the subjects' perceptions of their skin, the test product, and the product's effects. Questions were asked using a five-point scale and open-ended responses to inquire about the subjects' degree of agreement with statements.

[0271] Fourteen female subjects were enrolled. The inclusion criteria were Caucasian female subjects with Fitzpatrick type III skin in good health and aged between 35 and 65 years (inclusive at the time of enrollment). The inclusion criteria also included the following signs of facial aging determined by a professional rater at baseline: a) The score of folds / wrinkles on a 10-cm scale was ≥2 cm and ≤6 cm; and b) The score of facial redness (rash) on a five-point ordinal scale was ≥1 and ≤3. Table 5 discloses the demographic data of the study participants.

[0272] Table 5. Demographic Data

[0273]

[0274] After two weeks of treatment, the evaluation results of the professional clinical assessors for wrinkles / folds, firmness (visual), elasticity (tactile), brightness, texture / softness (tactile), texture / smoothness (visual), and erythema are shown in Table 6. All of the tested characteristics were improved. The scores for brightness, texture / softness (tactile), texture / smoothness (visual), and erythema showed statistically significant improvements.

[0275] Table 6. Professional Clinical Assessor Evaluation - Monadic, Compared to Baseline

[0276]

[0277] * Indicates a statistically significant improvement compared to the baseline, p ≤ 0.05

[0278] Table 7 shows the instrumental evaluation of hydration, firmness, and elasticity using a skin moisture tester and a skin elasticity tester. The improvements in skin hydration, firmness, and elasticity were statistically significant. Additionally, as indicated by spectrophotometric intracutaneous analysis (SIA), Table 7 shows the stimulation of collagen production in skin cells.

[0279] Table 7. Instrumental Evaluation - Monadic, Compared to Baseline

[0280]

[0281]

[0282] NI = No Improvement

[0283] * Indicates a statistically significant improvement compared to the baseline, p ≤ 0.05

[0284] ** Indicates a statistically significant deterioration compared to the baseline, p ≤ 0.05

[0285] The results indicate that truncated human type XXI collagen showed statistically significant improvements in skin elasticity, brightness, hydration, tactile texture, or visual texture. In addition, the results showed that truncated human type XXI collagen showed a statistically significant reduction in visible wrinkles / folds and a significant reduction in redness.

[0286] Example 4. In Vitro Study of Truncated Human Type 21 Collagen on Skin Cells Truncated human type XXI α1 collagen stimulates fibroblasts to produce type I collagen

[0287] A series of in vitro experiments were conducted to evaluate the effects of truncated human type XXI collagen on human skin fibroblasts and keratinocytes. In the first experiment, the secretion of type I collagen by human primary fibroblasts was evaluated. Fibroblasts were cultured with 0.03% of the polypeptide according to SEQ ID NO: 16 for 48 hours. The procollagen type I C-peptide in the culture supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA), which is a reading of the total amount of type I collagen secretion. As Figure 1 shown, the level of type I collagen secreted by the cells treated with the polypeptide of SEQ ID NO: 16 ( Figure 1 ; "B") was higher than that of the untreated cells ( Figure 1 ; "A") or the cells treated with retinol ( Figure 1 ; "C").

[0288] Truncated human type XXI α1 collagen stimulates fibroblasts to produce genes of extracellular matrix proteins

[0289] RNA sequencing was performed to analyze the overall gene expression. After 48 hours of exposure, fibroblasts were incubated with 0.03% of the polypeptide according to SEQ ID NO: 16. These fibroblasts expressed higher levels of various extracellular matrix genes compared to the cells incubated only in the medium. As Figure 2A shown, relative to the untreated cells ( Figure 2A ; "A") or the fibroblasts treated with retinol ( Figure 2A ; "B"), the fibroblasts treated with the polypeptide of SEQ ID NO: 16 ( Figure 2A ; "C") upregulated the type I collagen gene (COL1A). This response was similar to that of the fibroblasts treated with vitamin C ( Figure 2A ; "D"). As Figure 2B shown, the fibroblasts treated with the polypeptide of SEQ ID NO: 16 ( Figure 2B ; "B") upregulated the elastin gene (ELN) relative to the untreated cells ( Figure 2B ; "A") and various marine collagens ( Figure 2B ; "C", "D", "E" and "F"). As Figure 2C shown, the fibroblasts treated with the polypeptide of SEQ ID NO: 16 ( Figure 2C ; "B") upregulated the fibronectin gene (FN1) relative to the untreated cells ( Figure 2C ; "A"), retinol ( Figure 2C ; "C") and vitamin C ( Figure 2C ; "D").

[0290] Truncated human type XXI α1 collagen reduces inflammation in UVB-irradiated keratinocytes.

[0291] Irradiate human primary keratinocytes with 40 mJ / cm 2 UVB light, and then treat with 0.1% of the polypeptide of SEQ ID NO: 16 for 24 hours. Determine the level of the pro-inflammatory cytokine IL-1α by ELISA. As Figure 3 shown, compared with the untreated UVB-irradiated keratinocytes ( Figure 3 ; "A"), the UVB-irradiated keratinocytes treated with the polypeptide of SEQ ID NO: 16 ( Figure 3 ; "B") express lower levels of IL-1α.

[0292] Truncated human type XXI α1 collagen has antioxidant capacity.

[0293] Evaluate the antioxidant potential of the polypeptide of SEQ ID NO: 16 using the oxygen radical absorbance capacity (ORAC) assay. The ORAC assay is a cell-free assay that uses a fluorescence readout to measure the antioxidant capacity of a product. The data are reported as Trolox (vitamin E) equivalents. As Figure 4 shown, a 0.1% solution of the polypeptide of SEQ ID NO: 16 has antioxidant properties equivalent to 190 μM Trolox.

[0294] Truncated human type XXI α1 collagen increases the cell viability of UVB-irradiated keratinocytes.

[0295] To further evaluate the effect of treatment with the polypeptide of SEQ ID NO: 16 on UVB-irradiated keratinocytes, experiments with pre-irradiation and post-irradiation treatments were conducted. Pre-treat human primary keratinocytes with 0.1% of the polypeptide of SEQ ID NO: 16 for 24 hours, irradiate with 40 mJ / cm 2 UVB light, and then treat again with 0.1% of the polypeptide of SEQ ID NO: 16 for an additional 24 hours. Evaluate cell viability using the MTT metabolic colorimetric assay. As Figure 5 shown, compared with the UVB-irradiated keratinocytes not treated in this way ( Figure 5 ; "A"), the UVB-irradiated keratinocytes treated with the polypeptide of SEQ ID NO: 16 ( Figure 5 ; "B") show higher cell viability.

[0296] Example 5. Human Clinical Study of Truncated Human Type 21 Collagen

[0297] Topical application of truncated human type XXI α1 collagen is associated with increased facial skin elasticity

[0298] In a clinical study (n = 15 subjects), after a 1-week washout period using a protein-free basic facial essence, the subjects used a topical facial essence containing 0.1% of the polypeptide of SEQ ID NO: 16 for 8 weeks. The topical application of the polypeptide of SEQ ID NO: 16 was associated with an increase in skin elasticity, as measured by a skin elasticity tester. As Figure 6 shown, compared to the baseline ( Figure 6 ; "A"), 100% of the subjects showed improvement, with an increase in skin elasticity at 2 weeks ( Figure 6 ; "B") and 4 weeks ( Figure 6 ; "C").

[0299] The topical application of human type 21 α1 collagen was associated with an increase in facial skin collagen content

[0300] In a clinical study (n = 15 subjects), after a 1-week washout period using a protein-free basic facial essence, the subjects used a topical facial essence containing 0.1% of the polypeptide of SEQ ID NO: 16 for 8 weeks. The topical application of the polypeptide of SEQ ID NO: 16 was associated with an increase in skin collagen content, as measured by SIAscope. As Figure 7 shown, compared to the baseline ( Figure 7 ; "A"), the skin collagen content increased at 2 weeks ( Figure 7 ; "B") and 8 weeks ( Figure 7 ; "C").

[0301] The topical application of human type 21 α1 collagen was associated with a reduction in facial skin redness

[0302] In a clinical study (n = 15 subjects), after a 1-week washout period using a protein-free basic facial essence, the subjects used a topical facial essence containing 0.1% of the polypeptide of SEQ ID NO: 16 for 8 weeks. As Figure 8 shown, compared to the baseline ( Figure 8 ; "A"), the topical application of the polypeptide of SEQ ID NO: 16 was associated with a reduction in skin redness at 4 weeks ( Figure 8 ; "B") and 8 weeks ( Figure 8 ; "C").

[0303] The topical application of human type 21 α1 collagen was associated with a reduction in facial wrinkles

[0304] In a clinical study (n = 15 subjects), after a 1-week washout period using a protein-free basic facial essence, the subjects used a topical facial essence containing 0.1% of the polypeptide of SEQ ID NO: 16 for 8 weeks. As Figure 9 shown, compared to the baseline ( Figure 9; "A"), the topical application of the polypeptide of SEQ ID NO: 16 was associated with a reduction in skin wrinkles at 4 weeks ( Figure 9 ; "B") and 8 weeks ( Figure 9 ; "C").

[0305] Example 6. In Vitro Study of Truncated Jellyfish Collagen on Skin Cells

[0306] Truncated jellyfish collagen stimulates skin cells to produce type I collagen

[0307] A series of in vitro experiments were conducted to evaluate the effects of truncated jellyfish collagen on human skin fibroblasts and keratinocytes. After treatment with the polypeptide of SEQ ID NO: 5 for 48 hours, the secretion of type I collagen in an in vitro full-thickness human skin tissue model (MatTek) containing fibroblasts and keratinocytes was evaluated. The tissue model was then rinsed and re-incubated with fresh medium for 48 hours (96-hour time point). The procollagen type I C-peptide (a readout of the total amount of type I collagen secreted) in the culture supernatant was analyzed by ELISA. As Figure 10 shown, the tissue model treated with the polypeptide of SEQ ID NO: 5 ( Figure 10 ; "A") secreted higher levels of type I collagen than the untreated tissue model ( Figure 10 ; "B") or the tissue model treated with the positive control vitamin B3 ( Figure 10 ; "C").

[0308] Truncated jellyfish collagen reduces DNA damage in keratinocytes after exposure to UVB light

[0309] In further studies, human primary keratinocytes were irradiated with 25 mJ / cm 2 UVB light and then incubated overnight in medium containing 0.03% of the polypeptide of SEQ ID NO: 5. DNA was extracted from the cells and the level of thymine dimers (an indicator of DNA damage) was analyzed using an OxiSelect UV-induced DNA damage ELISA kit. As Figure 11 shown, compared with the untreated cells ( Figure 11 ; "A"), the cells treated with the polypeptide of SEQ ID NO: 5 ( Figure 11 ; "B") showed lower levels of adenine dimers and thus less DNA damage.

[0310] Truncated jellyfish collagen increases the cell viability of keratinocytes irradiated with UVB light.

[0311] Human primary keratinocytes were treated with 40 mJ / cm 2UVB light irradiation, followed by incubation in a culture medium containing 0.03% of the polypeptide of SEQ ID NO: 5 for 48 hours. Cell viability was evaluated using the MTT metabolic colorimetric assay. As Figure 12 shown, compared with untreated UVB-irradiated keratinocytes ( Figure 12 ; "A"), UVB-irradiated keratinocytes treated with the polypeptide of SEQ ID NO: 5 ( Figure 12 ; "B") showed higher cell viability.

[0312] Truncated jellyfish collagen increases the cell viability of keratinocytes exposed to urban dust pollution.

[0313] To test the protection against urban dust, human primary keratinocytes were pretreated with 0.03% of the polypeptide of SEQ ID NO: 5 for 24 hours and then exposed to 2 mg / ml urban dust (NIST 1649B) for 24 hours. Cell viability was evaluated using the MTT metabolic colorimetric assay. As Figure 13 shown, compared with untreated keratinocytes exposed to urban dust ( Figure 13 ; "A"), keratinocytes pretreated with the polypeptide of SEQ ID NO: 5 ( Figure 13 ; "B") showed higher cell viability after exposure to urban dust. Truncated jellyfish collagen reduces the inflammation of UVB-irradiated keratinocytes

[0314] In a further study using an in vitro full-thickness human skin tissue model (MatTek), the MatTek tissue model was irradiated with 300 mJ / cm 2 UVB light and then treated with 0.01% of the polypeptide of SEQ ID NO: 5 for 24 hours. The level of the pro-inflammatory cytokine IL-1α was determined by ELISA. As Figure 14 shown, compared with the untreated UVB-irradiated control tissue model ( Figure 14 ; "B"), the tissue model treated with the polypeptide of SEQ ID NO: 5 ( Figure 14 ; "A") expressed a lower level of IL-1α.

[0315] Truncated jellyfish collagen has antioxidant capacity

[0316] The polypeptide of SEQ ID NO: 5 was also evaluated by the ORAC assay. As Figure 15 shown, a 0.1% solution of the polypeptide of SEQ ID NO: 5 had antioxidant properties equivalent to 193 μM Trolox.

[0317] Example 7. Human Clinical Study of Truncated Jellyfish Collagen

[0318] Topical application of truncated jellyfish collagen is associated with increased facial skin moisture

[0319] In a clinical study (n = 18 subjects), subjects used a topical cream containing 0.05% of the polypeptide of SEQ ID NO: 5 for 2 weeks. As Figure 16 shown, compared with the baseline ( Figure 16 ; "A1"), topical application of the polypeptide of SEQ ID NO: 5 was associated with increased skin hydration at 1 week ( Figure 16 ; "A2") and 2 weeks ( Figure 16 ; "A3"). Compared with topical application of marine collagen at the baseline ( Figure 16 ; "B1"), topical application of the polypeptide of SEQ ID NO: 5 also showed increased skin hydration at 1 week ( Figure 16 ; "B2") and 2 weeks ( Figure 16 ; "B3").

[0320] Topical application of truncated jellyfish collagen is associated with increased facial skin elasticity. In a clinical study (n = 18 subjects), subjects used a topical cream containing 0.05% of the polypeptide of SEQ ID NO: 5 for 2 weeks. As Figure 17 shown, compared with the baseline ( Figure 17 ; "A"), topical application of the polypeptide of SEQ ID NO: 5 was associated with increased skin elasticity at 1 week ( Figure 17 ; "B") and 2 weeks ( Figure 17 ; "C"), and skin elasticity was measured using a skin elasticity tester.

[0321] The embodiments disclosed herein may be implemented in other specific forms without departing from the structures, methods, or other features broadly described herein and claimed below. The described embodiments are to be considered in all respects only as illustrative and not restrictive. All changes that fall within the meaning and range of equivalence of the claims are to be included within the scope of the claims.

Claims

1. A non-naturally occurring recombinant polypeptide, said non-naturally occurring recombinant polypeptide consisting of the amino acid sequence of SEQ ID NO:5, and wherein said non-naturally occurring recombinant polypeptide provides a benefit selected from the following: (i) increasing skin firmness, elasticity, brightness, hydration, tactile texture, visual texture, collagen content, and / or elastin content; (ii) reducing skin damage, folds, and / or wrinkles present on the skin, and / or skin rash or redness; (iii) preventing or treating damage to the skin caused by ultraviolet radiation; (iv) promoting the repair of damaged skin; (v) protecting skin cells from exposure to urban dust; (vi) stimulating collagen production and / or elastin production in the skin; or (vii) any combination of two or more of (i)-(vi).

2. The non-naturally occurring recombinant polypeptide according to claim 1, wherein said non-naturally occurring recombinant polypeptide is produced in a microbial host cell.

3. The non-naturally occurring recombinant polypeptide according to claim 2, wherein said microbial host cell is a bacterial cell or a fungal cell.

4. The non-naturally occurring recombinant polypeptide according to claim 2, wherein said microbial host cell is a yeast cell.

5. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is that the firmness of the skin is increased by at least 5%, and the firmness of the skin is measured by a skin elasticity tester.

6. The non-naturally occurring recombinant polypeptide according to claim 5, wherein the benefit is that the firmness of the skin is increased by at least 10%.

7. The non-naturally occurring recombinant polypeptide according to claim 5, wherein the benefit is that the firmness of the skin is increased by at least 25%.

8. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is that the elasticity of the skin is increased by at least 5%, and the elasticity of the skin is measured by a skin elasticity tester.

9. The non-naturally occurring recombinant polypeptide according to claim 8, wherein the benefit is that the elasticity of the skin is increased by at least 10%.

10. The non-naturally occurring recombinant polypeptide according to claim 8, wherein the benefit is that the elasticity of the skin is increased by at least 25%.

11. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is that the hydration of the skin is increased by at least 5%, and the hydration of the skin is measured by a skin moisture tester.

12. The non-naturally occurring recombinant polypeptide according to claim 11, wherein the benefit is that the hydration of the skin is increased by at least 25%.

13. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is that the brightness of the skin is improved, and the benefit is determined by a professional clinical rater.

14. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is that the collagen content of the skin is increased by at least 5%, and the collagen content of the skin is measured by SIAscope.

15. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is an improvement in the tactile texture of the skin, and the benefit is determined by a professional clinical assessor.

16. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is an improvement in the visual texture of the skin, and the benefit is determined by a professional clinical assessor.

17. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is a reduction in the folds or wrinkles present on the skin, and the benefit is determined by a professional clinical assessor.

18. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the benefit is a reduction of at least 10% in the red bumps present on the skin after 2 weeks of topical application, and the benefit is determined by a professional clinical assessor.

19. The non-naturally occurring recombinant polypeptide according to claim 18, wherein the benefit is a reduction of at least 20% in the red bumps present on the skin.

20. The non-naturally occurring recombinant polypeptide according to claim 1, wherein the non-naturally occurring recombinant polypeptide is formulated as a topical composition comprising 0.001% to 10% w / w of the non-naturally occurring recombinant polypeptide.

21. The non-naturally occurring recombinant polypeptide according to claim 20, wherein the topical composition is a cream, facial essence, facial mask or toner.

22. A topical preparation comprising the non-naturally occurring recombinant polypeptide according to claim 1 and one or more additional ingredients selected from water, oil, glycereth-8 esters, glycerol, caprylyl / capryl wax, sodium acryloyldimethyltaurate / VP copolymer, pentylene glycol, disodium edetate (EDTA), octyldecanol, chlorphenesin and phenoxyethanol.

23. The topical preparation according to claim 22, wherein the oil is a vegetable oil.

24. The topical preparation according to claim 23, wherein the vegetable oil is olive oil.

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