CN116669696A

CN116669696A - Compositions comprising a combination of a collagen or elastin polypeptide and an active ingredient and methods of use thereof

Info

Publication number: CN116669696A
Application number: CN202180080343.1A
Authority: CN
Inventors: 朱莉娅·科; 道恩·辛普森; 尼古拉·奥佐诺夫
Original assignee: Geltor Inc
Current assignee: Geltor Inc
Priority date: 2020-09-30
Filing date: 2021-09-30
Publication date: 2023-08-29

Abstract

The present application provides compositions comprising a collagen polypeptide (e.g., a human type 21 collagen polypeptide or a truncate thereof) or an elastin polypeptide (e.g., a human elastin polypeptide or a truncate thereof) and one or more active ingredients. The application also provides compositions comprising an elastin polypeptide (e.g., a human elastin polypeptide or a truncate thereof) in combination with a collagen polypeptide or a truncate thereof. The application also provides formulations, such as personal care products, comprising the composition. The application also provides methods of using the compositions and formulations provided herein (e.g., for application to the skin and/or hair of a subject).

Description

Compositions comprising a combination of a collagen or elastin polypeptide and an active ingredient and methods of use thereof

Cross reference

The present application claims the benefit of U.S. provisional application No. 63/085,829, U.S. provisional application No. 63/085,857, U.S. provisional application No. 63/091,791, and U.S. provisional application No. 63/091,799, both filed on 30 months of 2020, 9, and 30 months of 2020, 10 and 14, respectively, which are both incorporated herein by reference in their entirety.

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on 9 months 29 of 2021, named 57607-714_601_sl. Txt, of size 123,215 bytes.

Background

Collagen and similar proteins are the most abundant proteins in the biosphere. Collagen is a structural protein found in the skin, connective tissue and bone, and other tissues of animals. In humans, the amount of collagen present in the body is about one third of the total protein and is about three quarters of the dry weight of the skin.

The structure of native collagen may be a triple helix, wherein three polypeptide chains together form a helical coil. A single polypeptide chain consists of a repeating triplet amino acid sequence designated GLY-X-Y. X and Y can be any amino acid, and the first amino acid is glycine. The amino acids proline and hydroxyproline are present in collagen in high concentrations. The most common triplet is glycine-proline-hydroxyproline (Gly-Pro-Hyp), which accounts for about 10.5% of the triplets in collagen.

Gelatin is a product obtained by partial hydrolysis of certain (e.g., natural) collagens. Typically, gelatin is produced by acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis or by exposing collagen to heat in aqueous solutions (e.g., boiling bones and skin of animals, boiling fish scales, etc.).

Gelatin is used in many products including cosmetics, foods, pharmaceuticals, medical devices, films, adhesives, binders, and many others. The physical and chemical properties of gelatin are tailored to the specific application. These physical/chemical properties include gel strength, melting point temperature, viscosity, color, turbidity, pH, isoelectric point, and the like.

Elastin is an elastin that is critical to the normal function of arteries, lungs, tendons, ligaments, skin and other tissues. Elastin provides the ability for tissue to stretch and recover as such. The proteinogenic elastin is the building block for elastin. In contrast to other structural proteins including gene families (e.g., collagen), a tropoelastin gene is present in humans. When the gene encoding tropoelastin is expressed, a single tropoelastin gene is spliced to produce different forms of tropoelastin. Many tropoelastin molecules associate together to form elastin.

The structure of natural elastin is similar to collagen with glycine and proline rich amino acid sequences. A single polypeptide chain consists of a repeating triplet amino acid sequence designated GLY-X-Y interspersed with alanine strings that provide flexibility. X and Y can be any amino acid, and the first amino acid is glycine. The elastin has high concentrations of the amino acids proline and hydroxyproline. Elastin is typically covalently crosslinked to other elastin peptides by the action of lysyl oxidase. Due to the extensive cross-linking of elastin peptides, it is difficult to extract elastin from animal tissue.

Certain domains of elastin can be used as signaling domains for intracellular communications. In addition, certain elastin domains can be used as binding domains for polypeptides and other biomolecules. Degradation products of elastin have been shown to activate cellular processes, such as phagocytosis. These cell interaction domains are spaced apart from the alanine fragments that produce the flexible elastic structure characteristic of elastin. Alanine fragments are also rich in lysine, which is converted to aldehyde lysine by lysyl oxidase in tissue and cross-linked together to form a fishing net-like structure, making it very difficult to extract elastin from animal tissue, requiring the breakdown of elastin molecules for release. Due to this difficult extraction process, the size of commercially available elastin is only 3-5kDa.

In human natural elastin, the molecule has two parts, each of which contains three consecutive repeats of VGVAPG (SEQ ID NO: 34), i.e., VGVAPGVGVAPGVGVAPG (SEQ ID NO: 36). The amino acid sequence VGVAPG (SEQ ID NO: 34) has been reported to promote extracellular matrix degradation.

Both collagen and elastin are typically prepared from animal sources for manufacturing purposes. What is needed in the art are sources of these two useful proteins that are not prepared from animal sources.

Disclosure of Invention

In certain embodiments, herein are various polypeptides, compositions comprising such polypeptides, and methods of using such polypeptides and/or compositions thereof. In certain embodiments, such polypeptides comprise non-native and/or recombinant polypeptides, e.g., comprising one or more amino acid sequences that are truncated relative to native (e.g., full length) collagen or elastin.

In one aspect, a composition is provided comprising a non-naturally occurring recombinant polypeptide, such as human collagen or a truncate thereof, jellyfish collagen or a truncate thereof, or elastin or a truncate thereof, the composition further comprising one or more active ingredients, wherein the polypeptide and the one or more active ingredients provide a benefit to the skin and/or hair. The one or more active ingredients may be, for example, retinoids or vitamin a compounds, vitamin C compounds, alpha hydroxy acids, hyaluronic acid, coenzyme Q10, tea extract, grape seed extract, niacinamide, antibacterial agents, or acne-preventing agents, or any combination of two or more of these. Benefits to the skin may be, for example, (i) increasing the firmness, elasticity, brightness, hydration, tactile texture, visual texture, collagen content, or elastin content of the skin; (ii) Reducing skin damage, lines or wrinkles present on the skin, or erythema or redness of the skin; (iii) preventing or treating ultraviolet radiation damage to the skin; (iv) promoting repair of damaged skin; (v) Protecting skin cells from exposure to urban dust; (vi) Stimulating collagen production or elastin production in the skin; or (vii) any combination of two or more of (i) - (vi). The non-naturally occurring recombinant polypeptide can be a truncated collagen polypeptide or a truncated elastin polypeptide that is truncated at the C-terminus, the N-terminus, the internal truncation, or both the C-terminus and the N-terminus relative to a naturally occurring collagen or elastin. The non-naturally occurring recombinant polypeptide may be truncated by 50 to 800 amino acids at the C-terminus, 50 to 800 amino acids at the N-terminus, or both. Non-naturally occurring recombinant polypeptides may be internally truncated by 50 to 800 amino acids. The non-naturally occurring recombinant truncated collagen polypeptide may be human collagen or jellyfish collagen. The non-naturally occurring recombinant truncated collagen polypeptide can be 50 to 900 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 in length. The non-naturally occurring recombinant truncated collagen polypeptide may be a polypeptide having an N-terminal truncation of 50 to 650 amino acids and a C-terminal truncation of 50 to 250 amino acids.

In one aspect, the non-naturally occurring recombinant truncated collagen polypeptide can be human type 21 collagen. The non-naturally occurring recombinant truncated collagen polypeptide may have at least 80% sequence identity relative to SEQ ID No. 31. The non-naturally occurring recombinant truncated collagen polypeptide may have at least 80% sequence identity to SEQ ID NO. 14 or SEQ ID NO. 16. The non-naturally occurring recombinant truncated collagen polypeptide may comprise SEQ ID NO. 14 or SEQ ID NO. 16. The non-naturally occurring recombinant truncated collagen polypeptide may be human type 1 collagen.

The non-naturally occurring recombinant truncated collagen polypeptide may have at least 80% sequence identity relative to SEQ ID No. 32. The non-naturally occurring recombinant truncated collagen polypeptide can have at least about 80% sequence identity to 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. The non-naturally occurring recombinant truncated collagen polypeptide may comprise the amino acid sequence of 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.

The non-naturally occurring recombinant truncated collagen polypeptide may be jellyfish collagen. The non-naturally occurring recombinant truncated collagen polypeptide may be a polypeptide comprising an amino acid sequence having at least 80% sequence identity relative to SEQ ID No. 33. The non-naturally occurring recombinant truncated collagen polypeptide can be an amino acid sequence having at least 80% sequence identity relative to SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 10 or SEQ ID NO. 12. The non-naturally occurring recombinant truncated collagen polypeptide may comprise SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 10 or SEQ ID NO. 12. The non-naturally occurring recombinant truncated collagen polypeptide may have at least 80% sequence identity to SEQ ID No. 5.

The non-naturally occurring recombinant truncated elastin polypeptide may be human elastin. The elastin may be 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 in length. The elastin may have a C-terminal truncation, N-terminal truncation, internal truncation, or any combination thereof, relative to the full-length elastin polypeptide. Elastin may have an N-terminal truncation of 50 to 200 amino acids and a C-terminal truncation of 50 to 600 amino acids. The elastin may be a polypeptide having at least 80% sequence identity relative to SEQ ID NO. 53. The elastin may have at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50 or SEQ ID NO. 52. The elastin may have at least 80% sequence identity to SEQ ID NO. 52. The elastin may have an amino acid sequence comprising SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50 or SEQ ID NO. 52. One or more additional ingredients or carriers may be provided, such as water, oil glyceryl polyether-8 esters, glycerin, coconut alkane, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, pentanediol, disodium EDTA, octanoyl glycol, chlorobenzeneglycolether, phenoxyethanol, or vegetable oils.

In one aspect, a formulation is provided. The formulation may be a personal care product formulated for application to the skin and/or hair of a subject, and may have one or more carriers. The carrier may be a topical carrier such as liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerol monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, or water. The formulation may also contain a preservative. The non-naturally occurring recombinant polypeptide or truncate thereof may be present in the topical formulation in an amount of about 0.001% (w/w) to about 30% (w/w). The non-naturally occurring recombinant polypeptide or truncate thereof may be present in the topical formulation in an amount of about 0.001% (w/w) to about 10% (w/w). The hyaluronic acid, when present, may be present in an amount of about 0.001% (w/w) to about 10% (w/w). The retinol, when present, may be present in an amount of from about 0.001% (w/w) to about 5% (w/w). The ascorbic acid, when present, may be present in an amount of about 0.01% (w/w) to about 50% (w/w). The salicylic acid, when present, may be present in an amount of about 0.1% (w/w) to about 50% (w/w). The benzoyl peroxide, when present, may be present in an amount of about 0.1% (w/w) to about 20% (w/w). The nicotinamide, when present, may be present in an amount of about 0.1% (w/w) to about 50% (w/w). The alpha hydroxy acid, when present, may be present in an amount of about 0.1% (w/w) to about 50% (w/w).

In one aspect, the formulation may be a personal care product. The personal care product may be, for example, a cosmetic product. The personal care product may be a mask, skin cleanser, cleansing cream, cleansing lotion, facial cleanser, cleansing cream, cleansing pad, cleansing cream, body cream, facial moisturizer, body moisturizer, facial essence, facial mask, body film, toner, facial spray, eye cream, eye care, exfoliating formulation, lipstick, shampoo, hair conditioner, body wash, hair care essence, scalp essence, hair spray, eye shadow, concealer, mascara, or make-up. The personal care product may be configured to form a film.

In yet another aspect, a method of providing a benefit to a subject's skin by applying one of the personal care products described above to the subject's skin is provided. Benefits to the skin may be, for example: (i) Improving skin firmness, elasticity, brightness, hydration, tactile texture, visual texture, collagen content or elastin content; (ii) Reducing skin damage, lines or wrinkles present on the skin, or erythema or redness of the skin; (iii) preventing or treating ultraviolet radiation damage to the skin; (iv) promoting repair of damaged skin; (v) Protecting skin cells from exposure to urban dust; (vi) Stimulating collagen production or elastin production in the skin; or (vii) any combination of two or more of (i) - (vi).

In another aspect, a method of providing benefits to subject hair by applying one of the above personal care products to subject hair is provided. Benefits to the hair may be, for example: (i) improving the appearance and/or quality of hair; (ii) improving hair growth or thickness; (iii) reducing hair loss; (iv) Improving hair tensile strength, hair moisture, hydrophobicity, static control, fiber alignment, gloss, wet comb, dry comb, hair breakage resistance; (v) reduced bifurcation and curling of the hair tips; or (vi) any combination of two or more of (i) - (v).

In yet another aspect, a composition is provided comprising a non-naturally occurring recombinant elastin polypeptide or a truncate thereof and a non-naturally occurring recombinant collagen polypeptide or a truncate thereof. The composition may also contain one or more active ingredients. The active ingredient may be, for example, a retinoid or vitamin a compound, a vitamin C compound, an alpha hydroxy acid, hyaluronic acid, coenzyme Q10, a tea extract, a grape seed extract, niacinamide, an antibacterial agent, or an acne-preventing agent, or any combination of two or more thereof.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of this invention will be better understood by reference to the following detailed description and accompanying drawings in which illustrative embodiments of the principles of the invention are utilized, and in which:

FIG. 1 shows the effect of exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences) on type 1 collagen secretion in fibroblasts.

Fig. 2A shows the expression of type 1 collagen mRNA in fibroblasts treated with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

FIG. 2B shows the expression of elastin mRNA in fibroblasts treated with an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence).

Fig. 2C shows the expression of fibronectin mRNA in fibroblasts treated with the exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

FIG. 3 shows the expression of IL-1a in human primary keratinocytes treated with an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence).

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

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

Figure 6 shows skin elasticity after treatment with an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence).

Figure 7 shows skin collagen content after treatment with an exemplary polypeptide provided herein (comprising a truncated human collagen amino acid sequence).

Figure 8 shows quantification of skin redness after treatment with exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

Figure 9 shows quantification of skin wrinkles after treatment with exemplary polypeptides provided herein (comprising truncated human collagen amino acid sequences).

Figure 10 shows type 1 collagen secretion from a human skin tissue model treated with an exemplary polypeptide provided herein (comprising a truncated jellyfish collagen amino acid sequence).

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

Figure 12 shows keratinocyte viability following UVB irradiation treated with exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

Figure 13 shows cell viability when treated with municipal dust and exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

FIG. 14 shows the relative expression of IL-1a induced by UVB following treatment with the exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

Figure 15 illustrates the antioxidant capacity of exemplary polypeptides (comprising truncated jellyfish collagen amino acid sequences) provided herein.

Figure 16 shows skin hydration after treatment with the exemplary polypeptides provided herein (comprising truncated jellyfish collagen amino acid sequences).

Figure 17 shows skin elasticity after treatment with an exemplary polypeptide provided herein (comprising a truncated jellyfish collagen amino acid sequence).

Fig. 18 shows the expression of type 1 collagen (ng/mL) in human full-thickness skin equivalents (containing human keratinocytes and human fibroblasts) treated with exemplary polypeptides provided herein (comprising truncated human type 21 collagen amino acid sequences) in combination with retinol, ascorbic acid, or hyaluronic acid.

Figure 19 shows the expression of fibronectin (ng/mL) in human whole skin equivalent (containing human fibroblasts and human keratinocytes) treated with an exemplary polypeptide provided herein (comprising truncated human type 21 collagen amino acid sequence) in combination with retinol, ascorbic acid, or hyaluronic acid.

FIG. 20 shows the expression of elastin (ng/mL) in human whole skin equivalents (containing human fibroblasts and human keratinocytes) treated with exemplary polypeptides provided herein (comprising truncated human type 21 collagen amino acid sequences) in combination with retinol, ascorbic acid, or hyaluronic acid.

Figure 21 shows the expression of type III collagen (ng/mL) in human full skin equivalents (containing human keratinocytes and human fibroblasts) treated with exemplary polypeptides provided herein (comprising truncated human type 21 collagen amino acid sequences) in combination with retinol, ascorbic acid, or hyaluronic acid.

Fig. 22 shows a photograph of a protein gel. Lane 1 is a size marker. Lanes 2 and 3 show running of the 23kDa truncated human elastin of example 21 at the expected molecular weight. Lane 4 is a blank without protein loading. Lanes 5 and 6 show running of the 13kDa truncated human elastin of example 22 at the expected molecular weight.

FIG. 23 depicts a SDS-PAGE protein gel showing high purity and homogeneity of recombinantly produced polypeptides (comprising non-naturally occurring truncated elastin amino acid sequences) as described herein.

FIG. 24 depicts secretion levels of procollagen type I C-peptide (pro-collagen type I C-peptide) following treatment of fibroblasts with exemplary polypeptides provided herein, including non-naturally occurring truncated elastin amino acid sequences.

Figure 25 depicts the antioxidant capacity of exemplary polypeptides provided herein (comprising a non-naturally occurring truncated elastin amino acid sequence).

FIG. 26 depicts the expression levels of mRNA of various antioxidant genes after treatment of keratinocytes with exemplary polypeptides provided herein (comprising non-naturally occurring truncated elastin amino acid sequences).

FIG. 27 depicts the mRNA expression levels of various pro-apoptotic genes following treatment of keratinocytes with exemplary polypeptides (comprising non-naturally occurring truncated elastin amino acid sequences) provided herein.

Figure 28 depicts increased elastin production following treatment of keratinocytes with an exemplary polypeptide provided herein (comprising a non-naturally occurring truncated elastin amino acid sequence).

Detailed Description

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details.

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

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

Throughout this document, various embodiments of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Accordingly, the description of a range should be considered to have all possible subranges as specifically disclosed, as well as individual values within the range. For example, a description of a range (e.g., 1 to 6) should be considered to have the specifically disclosed subranges (e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.), as well as individual numbers within the range (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the width of the range.

Whenever a range of values is referred to herein, it is intended to include any reference number (fractional or integer) within the range shown. The phrase "a range between a first indicated number and a second indicated number" and "a range of first indicated numbers" to "second indicated numbers" are used interchangeably herein and are meant to include both the first and second indicated numbers and all fractional and integer numbers therebetween.

In some cases, the term "collagen polypeptide" or "collagen-like polypeptide" as used herein refers to a (e.g., monomeric) polypeptide that can associate with one or more collagens or collagen-like polypeptides to form a quaternary structure. Non-limiting examples of collagens include human type 21 collagen α1 (e.g., SEQ ID NO: 31), human type 1 collagen α2 (e.g., SEQ ID NO: 32), and jellyfish (hydroid) collagen (e.g., SEQ ID NO: 33). In some cases, the collagen may be treated with an acid, base, or heat to produce gelatin. The quaternary structure of native collagen is a triple helix, typically consisting of three polypeptides, but it should be noted that the "collagen polypeptides", "collagen-like polypeptides", "truncated collagens" or "truncated collagens" provided herein may or may not have such quaternary structure, and do not necessarily have such quaternary structure. In some cases, of the three polypeptides that form native collagen, two are typically identical and are referred to as the alpha chain. The third polypeptide is called the beta chain. In some cases, a typical native collagen may be referred to as AAB, wherein the collagen consists of two alpha ("a") chains and one beta ("B") chain. In certain instances, a polypeptide provided herein comprising "truncated collagen" may or may not have such structural elements. The term "collagen polypeptide" or "collagen-like polypeptide" may refer to an alpha chain polypeptide, a beta chain polypeptide, or both an alpha and a beta chain polypeptide. As used herein, the term "procollagen" generally refers to a polypeptide produced by a cell that can be processed into naturally occurring collagen.

In some cases, the term "elastin polypeptide" or "elastin-like polypeptide" as used herein refers to a (e.g., monomeric) polypeptide that can be associated with one or more elastin or elastin-like polypeptides and/or can be bound to another polypeptide, nucleic acid, polysaccharide, lipid, or other molecule. In general, the term "elastin polypeptide" or "elastin-like polypeptide" refers to a polypeptide that has one or more functions associated with a naturally occurring elastin. Non-naturally occurring polypeptides (e.g., truncated elastin) provided herein may be referred to as "elastin polypeptides" or "elastin-like polypeptides" when they have one or more functions associated with naturally occurring elastin. As used herein, the term "tropoelastin" generally refers to polypeptides that may be further processed (e.g., by cleavage, splicing, etc.) to produce elastin polypeptides. A non-limiting example of an elastin is a naturally occurring human elastin having an amino acid sequence according to SEQ ID NO. 53.

As used herein, the term "expression vector" or "vector" generally refers to a nucleic acid component capable of directing expression of a foreign gene. Expression vectors can include promoters, restriction endonuclease sites, nucleic acids encoding one or more selectable markers, and other nucleic acids useful in recombinant technology practice, operably linked to a foreign gene.

As used herein, the term "extracellular matrix" generally refers to a network of extracellular macromolecules (e.g., elastin, collagen, enzymes, and glycoproteins) that provide scaffolds for cells in a multicellular organism. The extracellular matrix may provide structural components that mediate cell adhesion, intercellular communication, and other functions.

As used herein, the term "fibroblast" generally refers to a cell that synthesizes procollagen and other structural proteins. Fibroblasts are widely distributed in the body and may be present in skin, connective tissue, and other tissues.

The term "fluorescent protein" generally refers to a protein that can be used in genetic engineering techniques as a reporter for expression of an exogenous polynucleotide. When exposed to ultraviolet or blue light, the protein fluoresces and emits bright visible light. The green-emitting proteins include Green Fluorescent Protein (GFP) and the red-emitting proteins include Red Fluorescent Protein (RFP).

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, the gelatin solution forms a reversible gel for use in food, cosmetics, pharmaceuticals, industrial products, medical products, laboratory growth media, and many other applications.

As used herein, the term "gene" generally refers to a polynucleotide encoding a particular protein, and it may refer to the coding region alone or may include regulatory sequences preceding (5 'non-coding sequences) and following (3' non-coding sequences) the coding sequence.

The term "histidine tag" generally refers to 2-30 consecutive histidine residues on the recombinant polypeptide (SEQ ID NO: 54).

The term "host cell" generally refers to a cell engineered to express an introduced exogenous polynucleotide.

The term "keratinocytes" generally refers to cells that produce keratin, tropoelastin, and other cellular components present in the epidermis of the skin.

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

As used herein, the term "non-naturally occurring" generally refers to genes, polypeptides, or proteins that are not normally found in nature, e.g., collagen or elastin polypeptides. Non-naturally occurring collagen or elastin polypeptides may be recombinantly produced (e.g., by expression by a recombinant host cell). The non-naturally occurring collagen or elastin polypeptide may be a recombinant polypeptide (e.g., produced by a recombinant host cell). The non-naturally occurring collagen or elastin polypeptide may be truncated. Other non-naturally occurring collagen or elastin polypeptides include chimeric collagens or chimeric elastin.

Chimeric collagen may be a polypeptide in which a portion of the collagen polypeptide is linked to a second portion of the collagen polypeptide. In another embodiment, the non-naturally occurring collagen comprises a fusion polypeptide comprising additional amino acids, such as a secretion tag, a histidine tag, a green fluorescent protein, and/or a protease cleavage site, a GEK repeat, a GDK repeat, and/or a beta-lactamase.

Similarly, a chimeric elastin may be a polypeptide in which a portion of the elastin polypeptide is linked to a second portion of the elastin polypeptide. For example, a molecule comprising a portion of human elastin linked to a portion of another human polypeptide may be a chimeric elastin. In another embodiment, the non-naturally occurring elastin comprises a fusion polypeptide comprising additional amino acids, such as a secretion tag, a histidine tag, a green fluorescent protein, and/or a protease cleavage site.

In some aspects, the collagen or elastin polypeptides provided herein have a non-naturally occurring amount of hydroxyproline. In some cases, the collagen or elastin polypeptides provided herein lack hydroxyproline. In some cases, the collagen or elastin polypeptides provided herein comprise less hydroxyproline than naturally occurring collagen or elastin. Hydroxyproline includes, but is not limited to, 3-hydroxyproline, 4-hydroxyproline, and 5-hydroxyproline. In some cases, less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10% or less) of the proline present in the amino acid sequence of a collagen or elastin polypeptide provided herein is hydroxyproline. In some cases, a recombinant collagen or elastin polypeptide as provided herein is recombinantly expressed in a host cell (e.g., a bacterial cell) that lacks an enzyme that hydroxylates one or more amino acids (e.g., proline) of the collagen or elastin polypeptide. In some cases, a recombinant collagen or elastin polypeptide as provided herein is recombinantly expressed in a host cell (e.g., a bacterial cell) that lacks prolyl 4-hydroxylase and/or prolyl 3-hydroxylase.

In general, the disclosure provided herein of collagen or elastin or truncated collagen or elastin (e.g., having a particular amino acid sequence) includes polypeptides having or comprising the precise amino acid sequence and homologs thereof. In some cases, homologs of the amino acid sequences provided herein may have longer or shorter sequences, and may have substitution of one or more amino acid residues of such amino acid sequences. Such homologues have particular sequence identity to the listed sequences, for example in the amounts provided herein. For example, to assess percent identity, sequence identity may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligners available on www.ebi.ac.uk/Tools/psa/embos_needle/nucleic acid. Html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment Tools available on blast.ncbi.n lm.gov/blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., the embos Water aligners available on www.ebi.ac.uk/Tools/psa/embos_water/nucleic acid. Html, optionally with default settings). Any suitable parameters of the selected algorithm, including default parameters, may be used to evaluate the optimal alignment. In some cases, the non-naturally occurring collagen or elastin may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a sequence disclosed herein.

The term "protease cleavage site" generally refers to an amino acid sequence that is cleaved by a particular protease.

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

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

Similarly, the term "truncated elastin" generally refers to monomeric polypeptides that are smaller than the full-length elastin, wherein one or more portions of the full-length elastin are absent. The elastin polypeptide may be truncated at the C-terminus relative to the full-length elastin, at the N-terminus relative to the full-length elastin, by removing an internal portion of the full-length elastin polypeptide (e.g., internal truncation), or both at the C-terminus and N-terminus relative to the full-length elastin. In a non-limiting embodiment, the truncated human elastin may comprise an amino acid sequence according to SEQ ID NO. 53 or a homologue thereof. In general, truncated elastin provided herein can have similar or substantially similar functions and/or provide benefits (e.g., as provided herein) to a native or full-length elastin. In some cases, a truncated elastin provided herein can have improved or enhanced function and/or benefit (e.g., as provided herein) compared to a native or full-length elastin.

When used in reference to an amino acid position, "truncated" includes the amino acid position. For example, an N-terminal truncation at amino acid 100 of a full-length protein refers to a truncation of 100 amino acids 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 at amino acid 901 of a full-length protein (assuming 1000 amino acids of the full-length protein) refers to a truncation of 100 amino acids from the C-terminal (i.e., the truncated protein lacks amino acids 901 to 1000 of the full-length protein). Similarly, an internal truncation at amino acids 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).

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

The host bacterial cells may be cultured continuously or discontinuously; in a batch process, a fed-batch process or a repeated fed-batch process.

In general, the signal sequence may be a component of an expression vector, or it may be part of a foreign gene inserted into the vector. The selected signal sequence may be a sequence that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For bacterial host cells that do not recognize and process the native signal sequence of the exogenous gene, the signal sequence may be replaced with any commonly known bacterial signal sequence. In some embodiments, the recombinantly produced polypeptide may be targeted to the periplasmic space using the DsbA signal sequence. Dinh and Bernhardt, J Bacteriol, month 9 2011, 4984-4987.

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

The truncated collagen disclosed herein may comprise a truncation relative to full length collagen. In some embodiments, the truncated collagen disclosed herein may comprise a truncation relative to full length human type 21 collagen α1. In some embodiments, the truncated collagen disclosed herein may comprise a truncation relative to full length human type 1 collagen α2. In some embodiments, the truncated collagen disclosed herein comprises a truncation relative to full length jellyfish (hydroid) collagen. Non-limiting examples of full length collagen are provided in table 1 below.

TABLE 1 full-length collagen amino acid sequence

In some cases, a truncated collagen as described herein may comprise amino acids 1 to 548 of SEQ ID NO. 31; amino acids 1 to 553; amino acids 1 to 558; amino acids 1 to 563; amino acids 1 to 568; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 573. In some cases, a truncated collagen as described herein may comprise amino acids 726 to 957 of SEQ ID No. 31; amino acids 731 to 957; amino acids 736 to 957; amino acids 741 to 957; amino acids 746 to 957; amino acids 751 to 957; or a C-terminal truncation at any amino acid position from amino acid 756 to 957. In some cases, a truncated collagen as described herein can comprise both an N-terminal truncation and a C-terminal truncation. For example, a truncated collagen as described herein may comprise amino acids 1 to 548 of SEQ ID NO. 31; amino acids 1 to 553; amino acids 1 to 558; amino acids 1 to 563; amino acids 1 to 568; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 573; and amino acids 726 to 957; amino acids 731 to 957; amino acids 736 to 957; amino acids 741 to 957; amino acids 746 to 957; amino acids 751 to 957; or a C-terminal truncation at any amino acid position from amino acid 756 to 957. In particular embodiments, the truncated collagen disclosed herein may comprise an N-terminal truncation at amino acid 558 of SEQ ID NO. 31; and a C-terminal truncation at amino acid 746 of SEQ ID NO. 31.

In some cases, a truncated collagen as described herein may comprise amino acids 1 to 401 of SEQ ID No. 32; amino acids 1 to 406; amino acids 1 to 411; amino acids 1 to 416; amino acids 1 to 421; amino acids 1 to 426; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 431. In some cases, a truncated collagen as described herein may comprise amino acids 585 to 1366 of SEQ ID No. 32; amino acids 590 to 1366; amino acids 595 to 1366; amino acids 600 to 1366; amino acids 605 to 1366; amino acids 610 to 1366; amino acids 615 to 1366; or a C-terminal truncation at any amino acid position from amino acid 620 to 1366. In some cases, a truncated collagen as described herein can comprise both an N-terminal truncation and a C-terminal truncation. For example, a truncated collagen as described herein may comprise amino acids 1 to 401 of SEQ ID NO. 32; amino acids 1 to 406; amino acids 1 to 411; amino acids 1 to 416; amino acids 1 to 421; amino acids 1 to 426; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 431; and amino acids 585 to 1366 of SEQ ID NO. 32; amino acids 590 to 1366; amino acids 595 to 1366; amino acids 600 to 1366; amino acids 605 to 1366; amino acids 610 to 1366; amino acids 615 to 1366; or a C-terminal truncation at any amino acid position from amino acid 620 to 1366. In particular embodiments, a truncated collagen as provided herein may comprise an N-terminal truncation at amino acid 416 of SEQ ID NO. 32; and a C-terminal truncation at amino acid 605 of SEQ ID NO. 32.

In some cases, a truncated collagen as described herein may comprise amino acids 1 to 101 of SEQ ID No. 32; amino acids 1 to 106; amino acids 1 to 111; amino acids 1 to 116; amino acids 1 to 121; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 126. In some cases, a truncated collagen as described herein may comprise amino acids 276 to 1366 of SEQ ID NO. 32; amino acids 281 to 1366; amino acids 286 to 1366; amino acids 291 to 1366; amino acids 296 to 1366; amino acids 301 to 1366; or a C-terminal truncation at any amino acid position from amino acid 306 to 1366. In some cases, a truncated collagen as described herein can comprise both an N-terminal truncation and a C-terminal truncation. For example, a truncated collagen as described herein may comprise amino acids 1 to 101 of SEQ ID NO. 32; amino acids 1 to 106; amino acids 1 to 111; amino acids 1 to 116; amino acids 1 to 121; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 126; and amino acids 276 to 1366 of SEQ ID NO. 32; amino acids 281 to 1366; amino acids 286 to 1366; amino acids 291 to 1366; amino acids 296 to 1366; amino acids 301 to 1366; or a C-terminal truncation at any amino acid position from amino acid 306 to 1366. In particular embodiments, a truncated collagen as provided herein may comprise an N-terminal truncation at amino acid 111 of SEQ ID NO. 32; and a C-terminal truncation at amino acid 291 of SEQ ID NO. 32.

In some cases, a truncated collagen as described herein may comprise amino acids 16 to 240 of SEQ ID NO. 33; amino acids 16 to 245; amino acids 16 to 250; amino acids 16 to 255; amino acids 16 to 260; amino acids 16 to 265; amino acids 6 to 255; amino acids 11 to 255; amino acids 21 to 255; amino acids 26 to 255; amino acids 31 to 255; amino acids 21 to 250; amino acids 21 to 245; amino acids 26 to 250; amino acids 26 to 245; amino acids 31 to 250; or an internal truncation at any amino acid position from amino acid 31 to 245. In particular embodiments, a truncated collagen as described herein may comprise an internal truncation at amino acids 16 to 255 of SEQ ID NO. 33.

In some cases, the truncated collagen can comprise any of the amino acid sequences provided in table 2 below. In some cases, the truncated collagen may consist of any of the amino acid sequences provided in table 2 below. In some cases, the truncated collagen may consist essentially of any of the amino acid sequences provided in table 2 below. In specific embodiments, the 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, the 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.

TABLE 2 non-limiting examples of truncated collagens

In some cases, the truncated collagen may be 100 to 300 amino acids, 150 to 250 amino acids, 160 to 220 amino acids, 170 to 200 amino acids, 180 to 190 amino acids, or 185 to 190 amino acids in length.

In one aspect, non-naturally occurring elastin is provided. In some cases, the non-naturally occurring elastin is a recombinant polypeptide produced by a host cell (e.g., a recombinant cell). In some cases, the non-naturally occurring elastin may be human elastin. In some cases, the non-naturally occurring elastin may be a truncated elastin. The truncated elastin may be truncated relative to the full-length elastin (e.g., having an amino acid sequence according to SEQ ID NO: 53). The truncation may be an internal truncation relative to the full-length elastin, an N-terminal truncation relative to the full-length elastin, a C-terminal truncation relative to the full-length elastin, or both the C-and N-terminal truncations relative to the full-length elastin.

The truncated length of the truncated elastin relative to the full length elastin may be 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. In another embodiment, the truncated elastin may be truncated by about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 650, about 700, or about 750 amino acids relative to the full-length elastin. The truncated elastin may be encoded by a portion of the polynucleotide sequences disclosed herein or the entire polynucleotide sequence.

In some embodiments, a truncated elastin (e.g., of a polypeptide provided herein) (e.g., of an amino acid sequence thereof) can be truncated at the C-terminus (relative to a full-length elastin) by any suitable number of amino acid residues, e.g., up to about 10, about 10 to about 800, about 10 to about 700, about 10 to about 500, about 10 to about 400, about 10 to about 300, about 10 to about 200, about 10 to about 100, about 50 to about 800, about 50 to about 700, about 50 to about 600, about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 200, about 50 to about 100, etc. In some cases, the truncated elastin may be truncated at the C-terminus (relative to the full-length elastin) by about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 670, about 680, about 690, about 720, about 700, about 740, about 760, about 770, or more amino acids.

In some embodiments, a truncated elastin (e.g., of a polypeptide provided herein) (e.g., of an amino acid sequence thereof) can be truncated at the N-terminus (relative to a full-length elastin) by any suitable number of amino acid residues, e.g., up to 10, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, etc. In some cases, the truncated elastin may be truncated (relative to a full-length elastin) at the N-terminus by about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 670, about 680, about 690, about 720, about 700, about 740, about 760, about 770, or more amino acids.

In some embodiments, a truncated elastin (e.g., of a polypeptide provided herein) (e.g., of an amino acid sequence thereof) may be truncated at both the N-terminus and the C-terminus relative to a full-length elastin. In some cases, the truncated elastin may be truncated (relative to the full length elastin) at the N-terminus by any suitable number of amino acid residues, e.g., up to 10, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, etc.; and any suitable number of amino acid residues may be truncated (relative to full-length elastin) at the C-terminus, e.g., up to 10, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, etc. In some of the cases where the number of the cases, the truncated elastin may be truncated (relative to full-length elastin) at the N-terminus by about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 720, about 710, about 730, about 760, about 780, or more amino acids 770; and may be truncated (relative to full-length elastin) at the C-terminus by about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 760, about 770, or more amino acids 770.

In some embodiments, a truncated elastin (e.g., of a polypeptide provided herein) (e.g., of an amino acid sequence thereof) can be internally truncated (relative to a full-length elastin) by any suitable number of amino acid residues, e.g., up to 10, 10 to 800, 10 to 700, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, etc. In some cases, the truncated elastin may be internally truncated (relative to a full-length elastin) by about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 720, about 730, about 760, about 780, or more amino acids.

The truncated elastin disclosed herein may comprise a truncation relative to a full-length elastin. In some embodiments, the truncated elastin disclosed herein may comprise a truncation relative to a full length human elastin. In some cases, the full-length human elastin has an amino acid sequence according to SEQ ID NO. 53 provided in Table 3 below.

TABLE 3 amino acid sequence of full-length elastin

In some cases, a truncated elastin as described herein may comprise amino acids 1 to 68 of SEQ ID No. 53; amino acids 1 to 73; amino acids 1 to 78; amino acids 1 to 83; amino acids 1 to 88; amino acids 1 to 93; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 98. In some cases, a truncated elastin as described herein may comprise amino acids 213 to 760 of SEQ ID No. 53; amino acids 218 to 760; amino acids 223 to 760; amino acids 228 to 760; amino acids 233 to 760; amino acids 238 to 760; or a C-terminal truncation at any amino acid position from amino acid 243 to amino acid 760. In some cases, a truncated elastin as described herein may comprise both an N-terminal truncation and a C-terminal truncation. For example, a truncated elastin as described herein may comprise amino acids 1 to 68 of SEQ ID NO. 53; amino acids 1 to 73; amino acids 1 to 78; amino acids 1 to 83; amino acids 1 to 88; amino acids 1 to 93; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 98; and amino acids 213 to 760 of SEQ ID NO 53; amino acids 218 to 760; amino acids 223 to 760; amino acids 228 to 760; amino acids 233 to 760; amino acids 238 to 760; or a C-terminal truncation at any amino acid position from amino acid 243 to amino acid 760. In particular embodiments, the truncated elastin disclosed herein may comprise an N-terminal truncation at amino acid 83 of SEQ ID NO. 53; and a C-terminal truncation at amino acid 228 of SEQ ID NO. 53.

In some cases, a truncated elastin as described herein may comprise amino acids 1 to 68 of SEQ ID No. 53; amino acids 1 to 73; amino acids 1 to 78; amino acids 1 to 83; amino acids 1 to 88; amino acids 1 to 93; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 98. In some cases, a truncated elastin as described herein may comprise amino acids 331 to 760 of SEQ ID No. 53; amino acids 336 to 760; amino acids 341 to 760; amino acids 346 to 760; 351 to 760 amino acids; 356 th to 760 th amino acids; or a C-terminal truncation at any amino acid position from amino acid 361 to 760. In some cases, a truncated elastin as described herein may comprise both an N-terminal truncation and a C-terminal truncation. For example, a truncated elastin as described herein may comprise amino acids 1 to 68 of SEQ ID NO. 53; amino acids 1 to 73; amino acids 1 to 78; amino acids 1 to 83; amino acids 1 to 88; amino acids 1 to 93; or an N-terminal truncation at any amino acid position from amino acid 1 to amino acid 98; and amino acids 331 to 760 of SEQ ID NO. 53; amino acids 336 to 760; amino acids 341 to 760; amino acids 346 to 760; 351 to 760 amino acids; 356 th to 760 th amino acids; or a C-terminal truncation at any amino acid position from amino acid 361 to amino acid 760. In particular embodiments, the truncated elastin disclosed herein may comprise an N-terminal truncation at amino acid 83 of SEQ ID NO. 53; and a C-terminal truncation at amino acid 346 of SEQ ID NO. 53.

In some cases, the truncated elastin may comprise any of the amino acid sequences provided in table 4 below. In some cases, the truncated elastin may consist of any of the amino acid sequences provided in table 4 below. In some cases, the truncated elastin may consist essentially of any of the amino acid sequences provided in table 4 below. In specific embodiments, the non-naturally occurring elastin is or comprises the amino acid sequence of any one of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO: 52. In some embodiments, the truncated elastin comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO: 52.

In some embodiments, the truncated elastin may be a truncate of any of SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, or SEQ ID NO. 52 (e.g., a polypeptide having at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 amino acids); or may be a truncate (e.g., a polypeptide having at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 amino acids) of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any of SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, or SEQ ID NO. 52. For example, the truncated elastin may have an N-terminal truncation, a C-terminal truncation, and/or an internal truncation relative to any of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO: 52; or at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to any one of SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50 or SEQ ID NO. 52.

TABLE 4 non-limiting examples of truncated elastin

In various aspects, the truncated human elastin does not comprise two or more contiguous amino acid sequences of VGVAPG (SEQ ID NO: 34). In some cases, the truncated human elastin does not comprise VGVAPGVGVAPG (SEQ ID NO: 35). In some cases, the truncated human elastin does not comprise VGVAPGVGVAPGVGVAPG (SEQ ID NO: 36). In some cases, the non-naturally occurring truncated human elastin reduces or does not cause extracellular matrix degradation.

In some cases, the truncated elastin may be 100 to 150 amino acids, 100 to 200 amino acids, 100 to 300 amino acids, 140 to 250 amino acids, 140 to 200 amino acids, 150 to 250 amino acids, 160 to 220 amino acids, 170 to 200 amino acids, 180 to 190 amino acids, or 185 to 190 amino acids in length.

In some aspects, the truncated human elastin may have a molecular weight of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50kDa. In some embodiments, the molecular weight is less than about 10, 15, 20, 25, 30, 35, 40, 45, or 50kDa. In some embodiments, the molecular weight is 1kDa to 60kDa, 5kDa to 55kDa, 5kDa to 50kDa, 5kDa to 45kDa, 5kDa to 40kDa, 5kDa to 35kDa, 5kDa to 30kDa, 5kDa to 25kDa, 5kDa to 20kDa, 5kDa to 15kDa, 5kDa to 10kDa, 10kDa to 40kDa, 10kDa to 35kDa, 10kDa to 30kDa, 10kDa to 25kDa or 10kDa to 20kDa.

In some embodiments, the non-naturally occurring collagen or elastin may further comprise an amino acid sequence comprising a secretion tag. The secretion tag can direct collagen to the periplasmic space of the host cell. In certain embodiments, 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 a non-naturally occurring collagen or elastin. In another aspect, the secretion tag can be cleaved from non-naturally occurring collagen or elastin.

In some embodiments, the non-naturally occurring collagen or elastin comprises a histidine (or polyhistidine) tag. In a specific embodiment, the histidine tag or polyhistidine tag is or comprises a sequence of 2 to 20 histidine residues (SEQ ID NO: 55), which is attached to collagen or elastin. 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 to purify proteins by chromatographic methods using nickel primary chromatographic media.

In some embodiments, the non-naturally occurring collagen or elastin further comprises a fluorescent protein. 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 or elastin comprises GFP and/or RFP. In one embodiment, the superfolder GFP is fused to a non-naturally occurring collagen. The superfolder GFP may be a GFP that can be properly folded even if fused to a poorly folded polypeptide. In one aspect, a histidine tag may be attached to non-naturally occurring collagen. In another aspect, the histidine tag can be cleaved from non-naturally occurring collagen or elastin.

In some embodiments, the non-naturally occurring collagen or elastin further comprises a protease cleavage site. Protease cleavage sites can be used to cleave recombinantly produced collagen to remove one or more portions of the polypeptide. Removable polypeptide moieties include secretion tags, histidine tags, fluorescent protein tags, and/or beta-lactamases. Proteases may include endo-, exo-, serine-, cysteine-, threonine-, aspartic-, glutamic-, and metallo-proteases. Exemplary protease cleavage sites include amino acids cleaved by thrombin, TEV protease, factor Xa, enterokinase and rhinovirus 3C protease. In one aspect, the cleavage tag is attached to a non-naturally occurring collagen or elastin. In another aspect, the cleavage tag is removed from the non-naturally occurring collagen or elastin by a suitable protease.

In some embodiments, the non-naturally occurring collagen or elastin further comprises an enzyme that is a beta-lactamase. Beta-lactamases may be used as selectable markers. In one aspect, the beta-lactamase is attached to a non-naturally occurring collagen or elastin. In another aspect, the beta-lactamase is cleaved from a non-naturally occurring collagen or elastin.

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

In one aspect, the composition comprising non-naturally occurring collagen or elastin may be a personal care product (e.g., a cosmetic). In some embodiments, the composition is formulated for topical administration. The composition may contain other cosmetic ingredients suitable for human use. The personal care products are useful for preventing or treating ultraviolet radiation damage to human skin or hair. Personal care products can be used to enhance skin firmness, elasticity, brightness, hydration, tactile or visual texture, and/or to stimulate collagen production. Personal care products can be used to reduce redness of the skin. The personal care product may be applied to the skin or hair. Compositions include, for example, face masks, skin cleansers (e.g., soaps), cleansing creams, cleansing lotions, facial cleansers, cleansing milks, cleansing pads, facial and body creams and moisturizers, facial essences, facial and body films, lotions and sprays, eye creams and eye care products, exfoliating formulas, lipsticks and lipsticks, shampoos, hair conditioners and body washes, hair and scalp essences, hair sprays and gels, eye shadows, concealers, mascaras, and other color cosmetics.

Compositions comprising non-naturally occurring collagen or elastin may also comprise at least one additional ingredient comprising a topical carrier or preservative. The topical carrier may comprise a material selected from the group consisting of liposomes, biodegradable microcapsules, lotions, sprays Topical carriers for fog, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oils, silicone oils, glycerol monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane and water. The preservative may comprise a compound selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1, 3-diol, cis-isomer 1- (3-chloroallyl) -3,5, 7-triaza-1-azonia adamantane chloride, glutaraldehyde, 4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, phenoxyethanol, butanediol, 1,2 hexanediol, methyl p-hydroxybenzoate, sorbic acid,II. Rosemary extract and EDTA.

In further embodiments, compositions comprising non-naturally occurring collagen or elastin may be formulated as injectable agents, for example, as soft tissue fillers based on Hyaluronic Acid (HA) and pharmaceutically acceptable salts of HA, such as dermal and subcutaneous fillers. Such formulations may be injected under one or more layers of skin.

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

The methods provided herein encompass the use of the compositions for the treatment shown in the methods, e.g., by the steps provided herein. In embodiments, the invention provides the use of a composition provided herein in a method for reducing skin damage, promoting repair of damaged skin, protecting skin from UV damage, and/or protecting skin cells from exposure to urban dust (e.g., by applying a composition provided herein to the skin of a subject). In embodiments, the present invention provides the use of a composition provided herein in a method for improving the firmness, elasticity, brightness, hydration, tactile or visual texture of skin and/or stimulating collagen production.

In some embodiments, truncated collagens or elastin as provided herein can 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 reading of collagen production) can be measured. In some cases, an in vitro MatTek full-layer human skin tissue model (see, e.g., example 6) can be used to assess type I procollagen C-peptide levels. In some cases, the type I collagen levels can be measured or determined by an enzyme-linked immunosorbent assay (ELISA). In some cases, truncated collagen or elastin as provided herein can stimulate higher levels of type I collagen production than untreated cells, cells treated with retinol, and/or cells treated with vitamin B3.

In some embodiments, truncated collagen or elastin as provided herein can stimulate fibroblasts to overexpress extracellular matrix genes (see, e.g., example 4). In some cases, the level of extracellular matrix genes can be measured by RNA sequencing. In some cases, truncated collagen or elastin as provided herein can stimulate fibroblasts to overexpress one or more of the collagen type I gene (COL 1A), elastin gene (ELN), and fibronectin gene (FN 1). In some cases, fibroblasts treated with truncated collagens or elastin provided herein may produce higher levels of extracellular matrix genes than untreated fibroblasts or fibroblasts treated with retinol. In some cases, fibroblasts treated with the truncated collagens provided herein may produce extracellular matrix gene levels similar to or higher than those produced by fibroblasts treated with vitamin C.

In some embodiments, truncated collagen or elastin as provided herein can reduce 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 truncated collagen as provided herein. In some cases, inflammation can be measured by measuring the level of IL-1 a produced by UVB-irradiated keratinocytes (e.g., by ELISA). In some cases, UVB-irradiated keratinocytes can produce lower levels of IL-1 a when treated with the truncated collagens provided herein as compared to untreated keratinocytes.

In some embodiments, truncated collagen or elastin as provided herein can increase the viability of keratinocytes irradiated with UVB light (see, e.g., example 4). In some cases, keratinocytes can be pretreated (prior to UVB irradiation) and post-treated (after UVB irradiation) with truncated collagens or elastin provided herein. In some cases, cell viability may be measured using an MTT metabolic colorimetric assay. In some cases, keratinocytes treated with the truncated collagens or elastin provided herein may exhibit greater cell viability after UVB irradiation than untreated keratinocytes.

In some embodiments, truncated collagen or elastin as provided herein can reduce DNA damage in keratinocytes after exposure to UVB light (see, e.g., example 6). In some cases, DNA damage can be assessed by measuring the level of thymine dimer (TT-dimer). In a non-limiting example, an OxiSelect UV-induced DNA damage ELISA kit can be used to measure TT-dimer levels. In some cases, UVB-irradiated keratinocytes treated with the truncated collagens or elastin provided herein can exhibit lower levels of TT-dimer than untreated keratinocytes.

In some embodiments, truncated collagen or elastin as provided herein may have antioxidant capacity (see, e.g., example 4 and example 6). In some cases, an Oxygen Radical Absorbance Capacity (ORAC) assay can be used to measure the oxidative capacity of truncated collagen or elastin. In a non-limiting example, the antioxidant property of the truncated collagen or elastin in the form of a 0.1% solution can be at least 10 μm Trolox (vitamin E) equivalent (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.

In some embodiments, truncated collagen or elastin as provided herein can increase cell viability of keratinocytes exposed to municipal dust pollution as compared to untreated cells (see, e.g., example 6). In some cases, cell viability can be measured by an MTT metabolic colorimetric assay.

In some embodiments, topical administration of the truncated collagen or elastin provided herein to the subject's face 1 week, 2 weeks, 4 weeks, 8 weeks, or more after treatment can result in increased facial skin elasticity compared to baseline (see, e.g., example 5 and example 7). In some cases, facial skin elasticity may be measured by a cutoff.

In some embodiments, topical administration of the truncated collagen or elastin provided herein to the subject's face 1 week, 2 weeks, 4 weeks, 8 weeks, or more after treatment can result in an increase in facial skin collagen content compared to baseline (see, e.g., example 5). In some cases, facial skin collagen content can be measured by SIAscope.

In some embodiments, topical application of the truncated collagen or elastin provided herein can result in reduced facial skin redness (erythema) at 1 week, 2 weeks, 4 weeks, 8 weeks, or more after treatment compared to baseline (see, e.g., example 5). In some cases, facial skin redness (erythema) may be scored by a blind clinical classifier (e.g., using a 5-order ordinal scale as provided in table 6).

In some embodiments, topical administration of truncated collagen or elastin provided herein can result in a reduction of facial wrinkles at 1 week, 2 weeks, 4 weeks, 8 weeks, or more after treatment compared to baseline (see, e.g., example 5). In some cases, facial wrinkles may be scored by a blind clinical sizer.

In some embodiments, topical application of the truncated collagen or elastin provided herein can result in increased facial skin moisture at 1 week, 2 weeks, 4 weeks, 8 weeks, or more post-treatment compared to baseline (see, e.g., example 7). In some cases, topical application of truncated collagen or elastin provided herein can result in increased facial skin moisture as compared to topical application of marine collagen or elastin. In some cases, skin hydration may be measured by a corneometer.

One aspect of the invention provides polynucleotides encoding non-naturally occurring collagens. The polynucleotide may encode collagen from jellyfish or humans. The polynucleotide may encode full length or truncated collagen. In various embodiments, the polynucleotide may comprise 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 a homologue 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 may be codon optimized (e.g., for expression in a host cell).

In some embodiments, the non-naturally occurring elastin may be encoded by a polynucleotide (e.g., a non-naturally occurring elastin; e.g., for expression in a host cell). The polynucleotide may encode a full length elastin or a truncated elastin. In various embodiments, the polynucleotide may comprise (e.g., have at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to) a polynucleotide according to any one of SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, or a homologue thereof. In some cases, the polynucleotide may be codon optimized (e.g., for expression in a host cell).

In another aspect, the invention provides polynucleotides encoding collagen or elastin fusion proteins. The fusion protein may comprise a secretion tag, a histidine tag, a fluorescent protein tag, a protease cleavage site, a beta-lactamase and/or a GEK amino acid trimer repeat and/or a GDK amino acid trimer repeat, and collagen or elastin.

In one aspect, vectors comprising collagen or elastin encoding polynucleotides may be used to transform host cells and express the polynucleotides. The polynucleotide may also comprise a nucleic acid encoding an enzyme that allows the host organism to grow in the presence of the selective agent. The selective agent may include certain sugars (including galactose-containing sugars) or antibiotics (including ampicillin, hygromycin, G418, and the like). Enzymes that may be used to confer resistance to the selective agent include beta-galactosidase or beta-lactamase.

In one aspect, host cells expressing polynucleotides of the invention are provided. The host cell may be any host cell, including a gram-negative bacterial cell, a gram-positive bacterial cell, a yeast cell, an insect cell, a mammalian cell, a plant cell, or any other cell for expressing an exogenous polynucleotide. An exemplary gram negative host cell is E.coli.

In addition to the carbon, nitrogen and inorganic phosphate sources, any desired or necessary supplements may also be included in suitable concentrations, either alone or as a mixture with another supplement or medium (e.g., a complex nitrogen source). In certain embodiments, the medium further comprises one or more components selected from the group consisting of: ammonium chloride, ammonium sulfate, calcium chloride, casamino acid, iron (II) sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate and yeast extract.

Beta-lactamase is an enzyme that confers resistance to lactam antibiotics in prokaryotic cells. Typically, when the beta-lactamase is expressed in a bacterial host cell, the expressed beta-lactamase protein also includes a targeting sequence (secretion tag) that directs the beta-lactamase protein to the periplasmic space of the cell. Beta-lactamase does not work unless it is transported to the periplasmic space of the cell. Beta-lactamases are provided that target the periplasmic space of the cell without the use of an independent secretion tag that targets the enzyme to the periplasmic space of the cell. By producing fusion proteins in which a periplasmic secretion tag is added to the N-terminus of a protein (e.g., GFP, collagen, or elastin, or GFP/collagen (or elastin) chimeras), the functionality of beta-lactamase lacking the native secretion tag can be used to select for complete translation and secretion of the N-terminal fusion protein. Using this approach, the DsbA-GFP-collagen-beta-lactamase fusion can be used to select for truncated products in the target collagen or elastin that facilitate translation and secretion.

Another embodiment provides a method of producing a polypeptide such as provided herein. In some embodiments, the method comprises the steps of: inoculating a culture medium with a recombinant host cell comprising a polynucleotide encoding the polypeptide, culturing the host cell, and isolating the polypeptide from the host cell.

A process for the fermentative preparation of polypeptides (or proteins) is provided. The process comprises the following steps: (a) Culturing a recombinant gram-negative bacterial cell in a medium comprising magnesium salts, wherein the concentration of magnesium ions in the medium is at least about 6mM, and wherein the bacterial cell comprises an exogenous gene encoding a protein; and

(b) The protein is harvested from the culture medium.

The bacteria may be cultivated in any suitable way, for example in a continuous cultivation, as described for example in WO 05/021772, or in a batch process (batch cultivation) or in a fed-batch or repeated fed-batch process, for the production of the target protein. In some embodiments, protein production is performed on a large scale. Various large scale fermentation procedures are available for the production of recombinant proteins. The capacity of the large scale fermentation is at least 1,000 liters, preferably about 1,000 to 100,000 liters. In some cases, the fermentor uses a stirrer impeller to dispense oxygen and nutrients, particularly glucose (preferred carbon/energy source). Small scale fermentation generally refers to fermentation in a fermenter having a volumetric capacity of no more than about 20 liters.

To accumulate the target protein, the host cell may be cultured under conditions sufficient to accumulate the target protein. Such conditions include, for example, temperature, nutrient, and cell density conditions, which allow protein expression and accumulation by cells. Furthermore, as known to those skilled in the art, such conditions may be those under which the cell is capable of transcription, translation and protein transfer from one cellular compartment to another to secrete the essential cellular functions of the protein.

Any suitable bacterial cell is optionally used in the methods provided herein. The bacterial cells may be cultured at any suitable temperature. In a specific embodiment, the bacterial cell is an E.coli cell. For E.coli growth, for example, a typical temperature range is about 20℃to about 39 ℃. In one embodiment, the temperature is from about 20 ℃ to about 37 ℃. In another embodiment, the temperature is about 30 ℃. In one embodiment, host cells in the non-switching state or switching state may be cultured at one temperature and switched to a different temperature to induce protein production. The host cells may be first cultured at one temperature to propagate the cells, and then the cells may be cultured at a lower temperature to induce protein production. The first temperature may be about 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, or 37 ℃. The second temperature may be about 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, or 36 ℃. The culturing at the second temperature may be performed for 1 hour to 100 hours, 5 hours to 90 hours, 5 hours to 80 hours, 5 hours to 70 hours, 10 hours to 70 hours, 15 hours to 65 hours, 15 hours to 60 hours, 20 hours to 55 hours, 20 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.

The pH of the medium may be any pH between about 5 and 9, depending primarily on the host organism. For E.coli, the pH may be 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.

To induce gene expression, the cells may generally be cultured until a certain optical density is reached, e.g., an OD600 of about 1.1, at which time induction begins (e.g., by adding an inducer, by depleting a repressor, inhibitor or medium component, etc.) to induce expression of the exogenous gene encoding the target protein. In some embodiments, expression of the exogenous gene may be induced by an inducer selected from, for example, isopropyl- β -d-1-thiogalactoside, lactose, arabinose, maltose, tetracycline, anhydrous tetracycline, vavlysin, xylose, copper, zinc, and the like. Induction of gene expression may also be achieved by reducing the level of dissolved oxygen during fermentation. The dissolved oxygen level of fermentation during cell proliferation may be 10% to 30%. To induce gene expression, the dissolved oxygen level may be reduced to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%. In a host cell in a physiological state or a transitional state, protein production can be induced by lowering the fermentation temperature as disclosed herein.

Combinations of collagen or elastin polypeptides with other active substances

In certain aspects, provided herein are compositions or formulations (e.g., formulated for application to skin or hair of a subject) comprising collagen or elastin polypeptides (e.g., as described herein) and one or more of hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). In some embodiments, the collagen or elastin polypeptide is any collagen or elastin polypeptide disclosed herein (e.g., full length polypeptide, truncated polypeptide), such as any collagen or elastin polypeptide having one or more of the actions described herein (e.g., synergistic action, e.g., in combination with one or more of hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid)). In some embodiments, the collagen or elastin polypeptide is any collagen or elastin polypeptide (e.g., full length or truncated) disclosed in U.S. provisional patent application No. 62/965,700, the disclosure of which is incorporated herein by reference.

In some cases, the collagen polypeptide is a (e.g., full length) human type 21 collagen polypeptide or a truncate thereof (e.g., truncated relative to a full length human type 21 collagen polypeptide). In some cases, the composition comprises any truncated human type 21 collagen polypeptide as described herein. In some cases, the composition comprises a human type 21 collagen polypeptide comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) sequence identity to SEQ ID No. 31. In some cases, the composition comprises a human type 21 collagen polypeptide comprising the amino acid sequence of SEQ ID NO. 31. In some cases, the composition comprises a human type 21 collagen polypeptide consisting of the amino acid sequence of SEQ ID NO. 31.

In some aspects, the compositions comprise a polypeptide comprising an amino acid sequence that is truncated relative to a full-length human type 21 collagen polypeptide (e.g., SEQ ID NO: 31). In some cases, the polypeptide comprises an amino acid sequence having a C-terminal truncation, an N-terminal truncation, an internal truncation, or any combination thereof, relative to a full-length human type 21 collagen polypeptide (e.g., SEQ ID NO: 31). In some cases, the polypeptide comprises an amino acid sequence having both a C-terminal and an N-terminal truncation relative to a full-length human type 21 collagen polypeptide (e.g., SEQ ID NO: 31).

In some cases, the polypeptide comprises a truncated amino acid sequence having 10 amino acids to 800 amino acids relative to full length human type 21 collagen (e.g., SEQ ID NO: 31). In some cases, the polypeptide comprises an amino acid sequence having 10 amino acids to 800 amino acids, 10 amino acids to 750 amino acids, 10 amino acids to 700 amino acids, 10 amino acids to 650 amino acids, 10 amino acids to 600 amino acids, 10 amino acids to 550 amino acids, 10 amino acids to 500 amino acids, 10 amino acids to 450 amino acids, 10 amino acids to 400 amino acids, 10 amino acids to 350 amino acids, 10 amino acids to 300 amino acids, 10 amino acids to 250 amino acids, 10 amino acids to 200 amino acids, 10 amino acids to 150 amino acids, or 10 amino acids to 100 amino acids. In another embodiment, the polypeptide may be truncated by about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 650, about 660, about 670, about 680, about 700, about 710, about 720, about 730, about 740, about 760, about 780, or about 790.

In some cases, the polypeptide comprises an amino acid sequence that has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%) sequence identity to the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 16. In some cases, the polypeptide comprises SEQ ID NO:14 or SEQ ID NO:16, and a sequence of amino acids. In some cases, the polypeptide consists of SEQ ID NO:14 or SEQ ID NO:16, and a polypeptide comprising the amino acid sequence of 16.

In some aspects, the composition comprises a polypeptide as described herein and hyaluronic acid. In some cases, the hyaluronic acid has a molecular weight of about 1kDa to about 50MDa. In some cases, the hyaluronic acid is a low molecular weight hyaluronic acid. In some cases, the hyaluronic acid has a molecular weight of less than about 50kDa (e.g., less than about 40kDa, less than about 30kDa, less than about 20kDa, less than about 10kDa, less than about 5 kDa). In some cases, the hyaluronic acid has a molecular weight greater than about 50kDa. In some cases, the hyaluronic acid has a molecular weight of about 15kDa to about 40kDa. In some cases, the hyaluronic acid has a molecular weight of about 50kDa to about 200kDa. In some cases, the hyaluronic acid has a molecular weight of about 200kDa to about 1MDa. In some cases, the hyaluronic acid has a molecular weight of about 1MDa to about 5MDa.

In some aspects, the composition comprises a polypeptide as described herein and ascorbic acid. In some aspects, the composition comprises a polypeptide as described herein and retinol. In some aspects, the composition comprises a polypeptide as described and salicylic acid. In some aspects, the composition comprises a polypeptide as described herein and benzoyl peroxide. In some aspects, the composition comprises a polypeptide as described herein and nicotinamide. In some aspects, the composition comprises a polypeptide as described herein and an alpha hydroxy acid (e.g., glycolic acid, lactic acid).

In some embodiments, the compositions provide any suitable amount of polypeptide and one or more of hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid), e.g., in an amount suitable to provide a benefit when administered or administered to an individual or cell. In some embodiments, the composition comprises an amount of the polypeptide and one or more of hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid), which are suitable for providing a benefit to the skin or hair of an individual when applied to the skin or hair of the individual.

In a specific aspect, the composition comprises from about 0.001% w/w to about 30% w/w of the polypeptide provided herein. In more specific aspects, the composition comprises about 0.001% w/w to about 20% w/w of the polypeptide provided herein, about 0.001% w/w to about 10% w/w of the polypeptide provided herein, about 0.001% w/w to about 5% w/w of the polypeptide provided herein, about 0.001% w/w to about 2% w/w of the polypeptide provided herein, about 0.001% w/w to about 1% w/w of the polypeptide provided herein, about 0.001% w/w to about 0.5% w/w of the polypeptide provided herein, or about 0.001% w/w to about 0.2% w/w of the polypeptide provided herein. In some aspects, the composition comprises about 0.01% w/w to about 1% w/w of the polypeptide provided herein.

In various aspects, the composition comprises from about 0.01% w/w to about 50% w/w ascorbic acid. For example, the composition may comprise from about 0.01% w/w to about 25% w/w, from about 0.01% w/w to about 15% w/w, from about 0.01% w/w to about 10% w/w, from about 0.01% w/w to about 5% w/w, from about 0.1% w/w to about 25% w/w, from about 0.1% w/w to about 10% w/w, from about 1% w/w to about 50% w/w, or from about 1% w/w to about 25% w/w of ascorbic acid. In some cases, the composition comprises from about 0.1% w/w to about 25% w/w ascorbic acid.

In various aspects, the composition comprises from about 0.001% w/w to about 10% w/w hyaluronic acid. For example, the composition may comprise from about 0.001% w/w to about 5% w/w, from about 0.001% w/w to about 1% w/w, from about 0.1% w/w to about 10% w/w, from about 0.1% w/w to about 5% w/w, from about 0.1% w/w to about 1%, from about 1% w/w to about 10% w/w, or from about 1% w/w to about 5% w/w hyaluronic acid. In some cases, the composition comprises about 0.01% w/w to about 5% w/w hyaluronic acid.

In various aspects, the composition comprises about 0.001% w/w to about 5% w/w retinol. For example, the composition may comprise about 0.001% w/w to about 2.5% w/w, about 0.001% w/w to about 1% w/w, about 0.01% w/w to about 5% w/w, about 0.01% w/w to about 2.5% w/w, about 0.01% w/w to about 1% w/w, about 1% w/w to about 5% w/w, or about 1% w/w to about 2.5% w/w of retinol. In some cases, the composition comprises about 0.01% w/w to about 5% w/w.

In various aspects, the composition comprises from about 0.1% w/w to about 50% w/w salicylic acid. For example, the composition may comprise from about 0.1% w/w to about 30% w/w, from about 0.1% w/w to about 15% w/w, from about 0.1% w/w to about 5% w/w, from about 1% w/w to about 30% w/w, from about 1% w/w to about 15% w/w, from about 1% w/w to about 5% w/w, from about 10% w/w to about 50% w/w, from about 10% w/w to about 30% w/w, or from about 10% w/w to about 20% w/w salicylic acid. In some cases, the composition comprises from about 2% w/w to about 30% w/w salicylic acid.

In various aspects, the composition comprises about 0.1% w/w to about 20% w/w benzoyl peroxide. For example, the composition may comprise about 0.1% w/w to about 10% w/w, about 0.1% w/w to about 5% w/w, about 0.1% w/w to about 1% w/w, about 1% w/w to about 15% w/w, about 1% w/w to about 10% w/w, about 1% w/w to about 5% w/w, about 5% w/w to about 15% w/w, or about 5% w/w to about 10% w/w benzoyl peroxide.

In various aspects, the composition comprises about 0.1% w/w to about 50% w/w nicotinamide. For example, the composition may comprise about 0.1% w/w to about 25% w/w, about 0.1% w/w to about 15% w/w, about 0.1% w/w to about 5% w/w, about 1% w/w to about 30% w/w, about 1% w/w to about 20% w/w, about 1% w/w to about 10% w/w, about 5% w/w to about 40% w/w, about 5% w/w to about 20% w/w, about 5% w/w to about 15% w/w, about 10% w/w to about 50% w/w, or about 10% w/w to about 25% w/w nicotinamide.

In various aspects, the composition comprises about 0.1% w/w to about 50% w/w of an alpha hydroxy acid (e.g., glycolic acid, lactic acid). For example, the composition may comprise from about 0.1% w/w to about 25% w/w, from about 0.1% w/w to about 15% w/w, from about 0.1% w/w to about 5% w/w, from about 1% w/w to about 30% w/w, from about 1% w/w to about 20% w/w, from about 1% w/w to about 10% w/w, from about 5% w/w to about 40% w/w, from about 5% w/w to about 20% w/w, from about 5% w/w to about 15% w/w, from about 10% w/w to about 50% w/w, or from about 10% w/w to about 25% w/w of an alpha hydroxy acid (e.g., glycolic acid, lactic acid).

Also provided herein are formulations comprising any of the compositions described herein (e.g., collagen polypeptide or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid). In some cases, the formulation is a personal care product. The personal care product may be formulated for application to the skin (e.g., topical application) and/or hair of an individual. In some cases, the personal care product comprises any of the compositions described herein (e.g., collagen polypeptide or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid)), and one or more carriers or excipients. The personal care product may contain other ingredients suitable for human use. Personal care products provided herein may include, but are not limited to, masks, skin cleansers (e.g., soaps), cleansing creams, cleansing lotions, facial cleansers, cleansing milks, cleansing pads, facial milks, facial and body creams and moisturizers, facial essences, facial and body films, lotions and sprays, eye creams and eye care products, exfoliating formulas, lipsticks and lipsticks, shampoos, hair conditioners and body washes, hair and scalp essences, hair sprays and gels, eye shadows, concealers, mascaras, and other color cosmetics. In some aspects, the personal care product is a cosmetic.

In various embodiments, the formulations and compositions described herein comprise a collagen and/or elastin polypeptide in combination with additional active ingredients that provide benefits to the skin or hair. Such active ingredients may include retinoids or vitamin a compounds, such as retinol and/or retinoic acid, for example, for repairing uv radiation damaged skin and/or reducing fine lines and wrinkles; vitamin C (ascorbic acid) as an antioxidant, for example, for protecting skin from uv radiation and/or reducing fine lines and wrinkles; alpha Hydroxy Acids (AHAs), such as glycolic acid, citric acid, and lactic acid, as exfoliating agents, e.g., formulations and compositions for stimulating skin turnover and absorption applied to the skin; hyaluronic acid, for example, for improving skin hydration; coenzyme Q10, e.g., for reducing fine lines and wrinkles and/or protecting skin from uv radiation; tea extracts, such as green tea, black tea and oolong tea extracts having antioxidant and/or anti-inflammatory properties, for example, for improving skin texture and/or reducing fine lines and wrinkles; grape seed extract having antioxidant and/or anti-inflammatory properties, for example, for promoting collagen production; niacinamide with antioxidant properties, for example, for improving skin hydration, skin brightness and skin elasticity; and antibacterial or acne-preventing active ingredients, such as benzoyl peroxide, salicylic acid or sulfur-containing ingredients. In various aspects, the personal care product may further comprise one or more additional ingredients. The one or more additional ingredients may be selected from: water, oil glycerol polyether-8 esters, glycerol, coconut alkanes, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, pentanediol, disodium EDTA, octanoyl glycol, chlorophenyl glyoxyl, phenoxyethanol, and vegetable oils (e.g., olive oil).

In various aspects, the personal care product may further comprise one or more topical carriers (e.g., suitable for topical application). The one or more topical carriers may be selected from: liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, olive oil, vegetable oil, glycerol monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and water.

In various aspects, the personal care product may further comprise one or more preservatives. The one or more preservatives may be selected from: tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1, 3-diol, cis-isomer 1- (3-chloroallyl) -3,5, 7-triaza-1-azonia adamantane chloride, glutaraldehyde, 4-dimethyloxazolidine, 7-ethylbicyclo-oxazolidine, phenoxyethanol, butanediol, 1, 2-hexanediol, methyl p-hydroxybenzoate, sorbic acid,II. Rosemary extract and ethylenediamine tetraacetic acid (EDTA).

In various aspects, the personal care product may comprise ingredients for hydrogel formation of the composition, alone or in combination with cross-linking materials (e.g., enzymes, polysaccharides, or small molecules). In various aspects, the personal care product may have film forming capabilities.

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) when applied to human skin cell cultures, human skin equivalent cultures, or ex vivo human skin) are capable of increasing viability of skin cells, increasing proliferation of skin cells, or both (e.g., as measured by MTT viability) (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with human type 21 collagen polypeptide or truncate thereof or elastin polypeptide or truncate thereof alone, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with human type 21 collagen polypeptide or a truncate thereof or elastin polypeptide or a truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) when applied to human skin cell cultures, human skin equivalent cultures, or ex vivo human skin) are capable of increasing production of extracellular matrix (ECM) components (e.g., collagen, elastin, fibronectin, fibrillin, hyaluronic acid), reducing production of matrix degradation proteins (e.g., matrix Metalloproteinases (MMPs), proteases), or both (e.g., as measured by transcriptome analysis, as measured by enzyme-linked immunosorbent assay (ELISA)), e.g., as compared to pretreatment, compared to untreated (or vehicle) controls, compared to treatment with human type 21 collagen polypeptide or truncations thereof alone, compared to treatment with elastin or elastin truncate alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, comparison to treatment with hydroxyamide alone, for example, comparison to treatment with niacinamide alone. In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with human type 21 collagen polypeptide or a truncate thereof, elastin polypeptide or a truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) when applied to skin cells (e.g., keratinocytes, fibroblasts) or human skin equivalent cultures) are capable of increasing wound healing capacity (e.g., as measured by wound healing score assays) (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptide or truncations thereof alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha-hydroxy acids (e.g., glycolic acid, lactic acid) when applied to skin cells (e.g., keratinocytes, fibroblasts) or human skin equivalent cultures after exposure to photodamage (e.g., UV irradiation, blue light exposure) are capable of improving the viability of the cells (e.g., as measured by MTT viability assay), improving the proliferation of the cells (e.g., as measured by MTT viability assay), reducing the cytotoxicity of the cells (e.g., as measured by fluorescence microscopy), reducing the apoptosis of the cells (e.g., as measured by fluorescence microscopy), improving the DNA repair of the cells (e.g., as measured by thymidine-dimer ELISA assay), reducing inflammation (e.g., as measured by Luminex assay of cytokines), reducing Reactive Oxidative Stress (ROS) (e.g., as measured by CM-H2DCFDA assay) or as compared to, for example, e.g., by CM-H2 assay) or pre-fda pre-treatment alone, compared to the treatment with either the same, the use of the same, the photo-oxidative agent, the same, or the pro-oxidative agent, alone, the photo-oxidative agent, or the same compared to the photo-oxidative agent, alone, or the photo-oxidative agent, and/or the pro-oxidative agent, alone compared to the treatment, and/or the pro-oxidative agent, when compared to the composition, and/, as compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone. In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha-hydroxy acids (e.g., glycolic acid, lactic acid) when applied to skin cells (e.g., keratinocytes, fibroblasts) or human skin equivalent cultures after exposure to contamination (e.g., urban dust exposure) are capable of improving the viability of the cells (e.g., as measured by MTT viability assay), improving the proliferation of the cells (e.g., as measured by MTT viability assay), reducing the cytotoxicity of the cells (e.g., as measured by MTT viability assay), reducing the apoptosis of the cells (e.g., as measured by fluorescence microscopy), improving the DNA repair of the cells (e.g., as measured by thymidine-dimer detection), reducing inflammation (e.g., as measured by ELISA assay or Luminex detection of cytokines), reducing reactive oxidative stress (e.g., as measured by CM-H2 dcdetection) or any combination thereof, e.g., as compared to that of either the pre-fda, alone, as compared to the treatment with the collagen, the photo-sloped acid alone, the photo-sloped acid, or the like, alone, as compared to the photo-sloped acid, or alone, or as compared to the treatment As compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone. In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha-hydroxy acid (e.g., glycolic acid, lactic acid)) are capable of improving the viability of the cells (e.g., as measured by MTT viability assay), improving the proliferation of the cells (e.g., as measured by MTT viability assay), reducing the cytotoxicity of the cells (e.g., as measured by MTT viability assay), reducing the apoptosis of the cells (e.g., as measured by fluorescence microscopy), improving the DNA repair of the cells (e.g., as measured by thymidine-dimer ELISA assay), reducing inflammation (e.g., as measured by lumine assay or cytokine Luminex assay), reducing reactive oxidative stress (e.g., as measured by CM-H assay or alpha-hydroxy acid), compared to pretreatment (e.g., dch-CM 2) alone, as compared to prior to treatment with either the prior art, e.g., to treatment with water, prior to the treatment with either the same, the treatment with the same, or a separate comparison to the treatment with water, the same, the treatment with water, the same comparison with the same alone, or an equivalent culture medium when applied to the same culture as measured by the same, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone. In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid)) when applied to the skin of a subject are capable of improving the appearance and/or quality of the skin of the subject (e.g., compared to pretreatment, compared to untreated (or vehicle) control, compared to treatment with collagen or elastin polypeptide or truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid alone (e.g., glycolic acid, lactic acid). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising a collagen polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) when applied to the skin of a subject) are capable of reducing wrinkles, reducing fine lines, reducing skin redness, reducing skin pigmentation/hyperpigmentation, improving skin brightness, reducing pore size, reducing skin roughness, reducing acne (e.g., as measured using a CLARITY assay), or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptide alone or truncations thereof, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone. In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, niacinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) when applied to the skin of a subject) are capable of increasing skin elasticity, increasing skin firmness, increasing skin hydration, increasing skin barrier function, increasing skin collagen content, increasing skin elastin content, increasing dermal density, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) control, as compared to treatment with collagen or elastin polypeptide alone or truncations thereof, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with niacinamide alone, as compared to treatment with alpha hydroxy acids alone (e.g., glycolic acid, lactic acid). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) when applied to the skin of a subject) are capable of improving the skin microflora (e.g., compared to pretreatment, compared to untreated (or vehicle) control, compared to treatment with human type 21 collagen polypeptide or truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) when applied to the skin of a subject) are capable of reducing the abundance of harmful microorganisms on the skin, promoting beneficial microorganisms on the skin, increasing the diversity of microorganisms on the skin, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptides or truncations thereof alone, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acids alone (e.g., glycolic acid, lactic acid). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) when applied to subject hair) are capable of improving the appearance and/or quality of subject hair (e.g., compared to pretreatment, compared to untreated (or vehicle) control, compared to treatment with collagen or elastin polypeptide or truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptide and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) when applied to hair of a subject) are capable of improving hair growth, increasing thickness of hair fiber diameter, increasing combability, reducing hair loss, increasing growth rate, increasing hair tensile strength, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) control, as compared to treatment with collagen or elastin polypeptide or truncate thereof alone, as compared to treatment with ascorbic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acid alone (e.g., glycolic acid, lactic acid). In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, the compositions and/or formulations provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) when applied to hair of a subject) are capable of improving hair fiber thickness, improving hair fiber density, increasing moisture, increasing hydrophobicity, reducing split ends, reducing frizziness, improving static control, improving fiber alignment, improving shine, increasing wet comb, increasing dry comb, increasing hair breakage resistance, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptide alone or truncate thereof, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone. In some cases, the compositions and/or formulations may be capable of synergistically or additively producing any of the above-described effects (e.g., compared to treatment with a collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

Also provided herein are methods of using the compositions and/or formulations (e.g., personal care products) provided herein (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). In some cases, the methods involve applying a composition or formulation provided herein (e.g., a personal care product) (e.g., comprising collagen or elastin polypeptides or truncations thereof) and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) to the skin of a subject. In some cases, the methods involve applying a composition or formulation provided herein (e.g., a personal care product) (e.g., comprising collagen or elastin polypeptides and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha-hydroxy acids (e.g., glycolic acid, lactic acid)) to the hair of a subject.

In various aspects, a method is provided for treating skin of a subject, the method comprising applying any composition and/or formulation (e.g., a personal care product) as described herein (e.g., comprising one or more of a collagen or elastin polypeptide or truncate thereof, hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) to skin of a subject (e.g., topical application), thereby treating skin of a subject. In some cases, the treatment includes cosmetic treatment (e.g., improving or restoring the appearance of the individual).

In various aspects, the methods result in improved appearance and/or quality of the subject's skin (e.g., compared to pretreatment, compared to untreated (or vehicle) controls, compared to treatment with collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. In various aspects, the method results in reduced wrinkles, reduced fine lines, reduced skin redness, reduced skin pigmentation/hyperpigmentation, increased skin brightness, reduced pore size, reduced skin roughness, reduced acne (e.g., as measured using a CLARITY assay), or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptide alone or truncations thereof, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acid alone (e.g., glycolic acid, lactic acid). In various aspects, the method results in increased skin elasticity, increased skin firmness, increased skin hydration, increased skin barrier function, increased skin collagen content, increased skin elastin content, increased dermis density, or any combination thereof (e.g., compared to pretreatment, compared to untreated (or vehicle) control, compared to treatment with collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone. In various aspects, the method results in an improvement in skin microbiology (e.g., compared to pretreatment, compared to untreated (or vehicle) control, compared to treatment with collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone). In various aspects, the method results in a decrease in abundance of harmful microorganisms on the skin, an increase in the diversity of microorganisms on the skin, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptides or truncations thereof alone, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone). In some cases, the method can synergistically or additively produce any of the above results (e.g., compared to treatment with a collagen or elastin polypeptide or truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In various aspects, a method is provided for treating hair in a subject, the method comprising administering to hair in a subject any of the compositions and/or formulations (e.g., personal care products) as described herein (e.g., comprising a collagen polypeptide (e.g., collagen or elastin polypeptide or truncations thereof) and one or more of hyaluronic acid, ascorbic acid, retinol, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid), thereby treating hair in a subject. In some cases, the treatment includes cosmetic treatment (e.g., improving or restoring the appearance of the individual).

In various aspects, the methods result in improved appearance and/or quality of hair in a subject (e.g., compared to pretreatment, compared to untreated (or vehicle) control, compared to treatment with collagen or elastin polypeptide or truncations thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. In various aspects, the method results in improved hair growth, thickened hair fiber diameter, increased combability, reduced hair loss, increased growth rate, increased hair tensile strength, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptide or truncations thereof alone, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. In various aspects, the method results in improved hair fiber thickness, improved hair fiber density, increased moisture, increased hydrophobicity, reduced hair pin bifurcation, reduced frizz, increased static control, improved fiber alignment, improved shine, increased wet comb, increased dryability, greater resistance to hair breakage, or any combination thereof (e.g., as compared to pretreatment, as compared to untreated (or vehicle) controls, as compared to treatment with collagen or elastin polypeptide alone or truncates thereof, as compared to treatment with ascorbic acid alone, as compared to treatment with hyaluronic acid alone, as compared to treatment with retinol alone, as compared to treatment with salicylic acid alone, as compared to treatment with benzoyl peroxide alone, as compared to treatment with nicotinamide alone, as compared to treatment with alpha hydroxy acid alone (e.g., glycolic acid, lactic acid). In some cases, the method can synergistically or additively produce any of the above results (e.g., compared to treatment with a collagen or elastin polypeptide or truncate thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with hyaluronic acid alone, compared to treatment with retinol alone, compared to treatment with salicylic acid alone, compared to treatment with benzoyl peroxide alone, compared to treatment with nicotinamide alone, compared to treatment with an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone).

In another aspect, a method for increasing type I collagen production in skin cells (e.g., keratinocytes, fibroblasts) is provided, the method comprising: contacting the skin cells with a composition and/or formulation comprising collagen or elastin polypeptides or truncations thereof, and hyaluronic acid, thereby increasing the production of type I collagen in the skin cells. In some cases, the methods involve contacting the skin of a subject in need thereof (e.g., containing skin cells (e.g., keratinocytes, fibroblasts)) with a composition and/or formulation comprising collagen or elastin polypeptides or truncations thereof and hyaluronic acid to increase production of type I collagen in the skin cells. The subject in need thereof may be an individual in need of increased type I collagen production in the skin. The subject in need thereof may be an individual in need of improvement in one or more of skin aging, skin elasticity, skin firmness, or wrinkles and fine lines. In some cases, the increase in type I collagen production can be synergistic or additive (e.g., compared to treatment with collagen or elastin polypeptide alone or truncates thereof, compared to treatment with hyaluronic acid alone).

In another aspect, a method for increasing fibronectin production in skin cells (e.g., keratinocytes, fibroblasts) is provided, the method comprising: contacting the skin cells with a composition and/or formulation comprising a collagen or elastin polypeptide or truncate thereof, and hyaluronic acid, thereby increasing production of fibronectin in the skin cells. In some cases, the methods involve contacting the skin of a subject in need thereof (e.g., containing skin cells (e.g., keratinocytes, fibroblasts)) with a composition and/or formulation comprising collagen or elastin polypeptides or truncations thereof and hyaluronic acid to increase production of fibronectin in the skin cells. The subject in need thereof may be an individual in need of increased fibronectin production in skin. The subject in need thereof may be an individual in need of improvement in one or more of skin aging, skin elasticity, skin firmness, or wrinkles and fine lines. In some cases, the increase in fibronectin production can be synergistic or additive (e.g., compared to treatment with collagen or elastin polypeptide alone or truncates thereof, compared to treatment with hyaluronic acid alone).

In another aspect, a method for increasing elastin production in skin cells (e.g., keratinocytes, fibroblasts) is provided, the method comprising: contacting the skin cells with a composition and/or formulation comprising a collagen or elastin polypeptide or truncate thereof, and ascorbic acid and/or retinol, thereby increasing the production of elastin in the skin cells. In some cases, the methods involve contacting the skin of a subject in need thereof (e.g., containing skin cells (e.g., keratinocytes, fibroblasts)) with a composition and/or formulation comprising a collagen or elastin polypeptide or truncate thereof to increase production of elastin in the skin cells. The subject in need thereof may be an individual in need of increased elastin production in the skin. The subject in need thereof may be an individual in need of improvement in one or more of skin aging, skin elasticity, skin firmness, or wrinkles and fine lines. In some cases, the increase in elastin production can be synergistic or additive (e.g., compared to treatment with collagen or elastin polypeptide or truncates thereof alone, compared to treatment with ascorbic acid alone, compared to treatment with retinol alone).

In another aspect, a method for increasing type III collagen production in skin cells (e.g., keratinocytes, fibroblasts) is provided, the method comprising: contacting the skin cells with a composition and/or formulation comprising collagen or elastin polypeptides or truncations thereof, and ascorbic acid, thereby increasing the production of type III collagen in the skin cells. In some cases, the methods involve contacting the skin of a subject in need thereof (e.g., containing skin cells (e.g., keratinocytes, fibroblasts)) with a composition and/or formulation comprising collagen or elastin polypeptides or truncations thereof and ascorbic acid to increase production of type III collagen in the skin cells. The subject in need thereof may be an individual in need of increased type III collagen production in the skin. The subject in need thereof may be an individual in need of improvement in one or more of skin aging, skin elasticity, skin firmness, or wrinkles and fine lines. In some cases, the increase in type III collagen production can be synergistic or additive (e.g., compared to treatment with collagen or elastin polypeptide alone or truncates thereof, compared to treatment with ascorbic acid alone).

Examples

Examples1. Production of truncated collagen

A codon-optimized DNA sequence was synthesized and expressed, optimized for expression in E.coli, encoding jellyfish collagen with 240 internal amino acid truncations (relative to full-length jellyfish collagen (SEQ ID NO: 33)). The DNA sequence is shown in SEQ ID NO. 1 below. In SEQ ID NO. 1, the DsbA secretion tag is encoded by nucleotides 1 to 72 and encodes amino acids 1 to 24 of SEQ ID NO. 2. A histidine tag comprising 9 histidine residues is encoded by nucleotide numbers 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 by amino acids 34-37 of SEQ ID NO. 2. The thrombin cleavage site is encoded by nucleotides 112 to 135 of SEQ ID NO. 1 and by amino acids 38 to 45 of SEQ ID NO. 2. Truncated collagen is encoded by nucleotides 136 to 822 of SEQ ID NO. 1 and by amino acids 46 to 274 of SEQ ID NO. 2.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATCACCATCACCACCACCACCATCACCACTCTGGCTCGAGCCTGGTGCCGCGCGGCAGCCATATGGGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTGAAAAAGGAGAACAGGGACGTACAGGTGCAGCAGGTAAACAGGGCAGCCCGGGTGCCGATGGTGCCCGTGGCCCGCTGGGTAGCATTGGTCAGCAGGGTGCAAGAGGCGAACCGGGCGATCCGGGTAGTCCGGGCCTGCGTGGTGATACGGGTCTGGCCGGTGTTAAAGGCGTTGCAGGTCCTTCAGGTCGTCCAGGTCAACCGGGTGCAAATGGTCTGCCGGGTGTTAATGGTCGTGGCGGTCTGGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:1)

The truncated collagen is about 54% full length jellyfish collagen (SEQ ID NO: 33) and is disclosed in SEQ ID NO:2, infra.

MKKIWLALAGLVLAFSASAAQYEDHHHHHHHHHSGSSLVPRGSHMGPQGVVGADGKDGTPGEKGEQGRTGAAGKQGSPGADGARGPLGSIGQQGARGEPGDPGSPGLRGDTGLAGVKGVAGPSGRPGQPGANGLPGVNGRGGLERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:2)

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

GTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTGAAAAAGGAGAACAGGGACGTACAGGTGCAGCAGGTAAACAGGGCAGCCCGGGTGCCGATGGTGCCCGTGGCCCGCTGGGTAGCATTGGTCAGCAGGGTGCAAGAGGCGAACCGGGCGATCCGGGTAGTCCGGGCCTGCGTGGTGATACGGGTCTGGCCGGTGTTAAAGGCGTTGCAGGTCCTTCAGGTCGTCCAGGTCAACCGGGTGCAAATGGTCTGCCGGGTGTTAATGGTCGTGGCGGTCTGGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:3)

A truncated jellyfish collagen amino acid sequence free of DsbA secretion tag, histidine tag, linker and thrombin cleavage site is disclosed in SEQ ID NO. 4.

GPQGVVGADGKDGTPGEKGEQGRTGAAGKQGSPGADGARGPLGSIGQQGARGEPGDPGSPGLRGDTGLAGVKGVAGPSGRPGQPGANGLPGVNGRGGLERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:4)

The polynucleotide of SEQ ID NO. 1 was codon optimized and synthesized by internal synthesis of Gen9 DNA (now Ginkgo Bioworks). The overlap between pET28 vector and SEQ ID NO. 1 was designed to be 30 to 40bp long, and PCR was usedGXL polymerase (www.clontech.com/US/Products/PCR/GC_Rich/PrimeSTAR_GXL_DNA_PolymerasesItex=10020:22372:US) was added. Then use SGI Gibson

(us.vwr.com/store/product/17613857/gibson-assembly-hifi-1-step-kit-system-genes-inc) the cut pET28a vector was assembled together with the insert DNA (SEQ ID NO: 1) into the final plasmid. Then by Eurofins Genomics

(www.eurofinsgenomics.com) plasmid sequences were verified by Sanger sequencing.

Transformed cells were cultured in minimal medium and frozen with glycerol in a ratio of 50:50 cells to glycerol in 1.5ml aliquots. One vial of frozen culture was resuscitated overnight in 50ml of minimal medium at 37℃and 200 rpm. Cells were transferred to 300ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

A bioreactor was prepared with 2.7L minimal medium+glucose and 300ml of culture with an OD600 of 5-10 was added to give a starting volume of 3L. Cells were grown at 28 ℃ and pH 7, with dissolved oxygen maintained at 20% saturation using a continuous process containing stirring, air and oxygen. The pH was controlled using a 28% w/w ammonium hydroxide solution. Once the initial bolus of 40g/L was exhausted over about 13 hours, the fermentation was performed in fed-batch mode using a DO-stat based fed-batch algorithm. After 24-26 hours of initial growth, the OD600 reaches over 100. At this point, 300mL of 500g/L sucrose was added and the temperature was reduced to 25 ℃. High density cultures were induced for protein production using 1mM IPTG. Fermentation was continued for an additional 20-24 hours and cells were harvested using a bench centrifuge at 9000rcf, 15 ℃ for 60 minutes. The cell pellet recovered by centrifugation was suspended in pH 8 buffer containing 0.5M NaCl and 0.1M KH2PO4 at a weight specific gravity of 2 Xbuffer to 1 Xcells.

The harvested cells were disrupted in 2 portions in a homogenizer at 14,000psi pressure. The resulting slurry contains collagen and other proteins.

The fermentation is carried out at various temperatures ranging from 25 ℃ to 28 ℃. For some fermentations, the fermentation temperature is maintained at a constant temperature and the collagen is purified immediately after the fermentation is completed (OD 600 of 5-10). For other fermentations, the fermentation temperature is maintained for a desired period of time and when the cell density reaches an OD600 of 5-10, the temperature is reduced to induce protein production. Typically, the temperature is reduced from 28 ℃ to 25 ℃. After fermentation was continued at 25℃for 40-60 hours, collagen was isolated.

Collagen was purified by acid treatment of homogenized cell broth. In addition, non-homogenized whole cells recovered from the bioreactor after centrifugation were acid treated and resuspended in the above buffer. The pH of the homogenized slurry or resuspended whole cells was reduced to pH 3 using 6M hydrochloric acid. The acidified cell slurry was mixed and incubated overnight at 4 ℃ and then centrifuged. The supernatant of the acidified slurry was tested on a polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitation. The salt content of the collagen slurry thus obtained is high. To obtain volume and salt reduction, concentration and diafiltration steps were performed using an EMD Millipore tangential flow filtration system with 0.1m2 each ultrafiltration cassette. Using 2 parallel cartridges, the total area of filtration was 0.2m2. A volume reduction of 5x and a salt reduction of 19x was achieved during the TFF stage. The final collagen slurry was run on SDS-PAGE gels to confirm the presence of collagen. The slurry was dried using a multi-tray lyophilizer for 3 days to give a white fluffy collagen powder.

Purified truncated collagen obtained from homogenized cell broth or non-homogenized cells was analyzed on SDS-PAGE gels and thick and clear bands were observed at the expected size of 27 kilodaltons. Purified collagen was also analyzed by mass spectrometry, confirming that the 27 kilodalton protein was jellyfish collagen.

Alternative purification methods for full length and truncated collagens are provided below.

The fermentation broth was mixed with 0.3% -0.5% w/v Polyethylenimine (PEI). After 15 minutes incubation with PEI, the broth 9000rcf was centrifuged for 15 minutes to recover the supernatant containing collagen. Discarding cell-containing pellet, mixing PEI-treated collagen-containing supernatant with sodium bentonite (final 0.2% w/v)Wyoming Bentonite) are mixed and centrifuged. Discarding the bentonite-containing precipitateAnd recovering the supernatant.

Bentonite treated supernatants were concentrated 3-6 fold on tangential flow filtration system (TFF) (EMD Millipore) using a 5kDa cassette. Collagen is retained in the permeate stream with little loss. To remove salts, the retentate from the concentration step was diafiltered using the same TFF device. Final conductivity of protein solution<10 milliSiemens. Typical conductivities are 400 micro Siemens to 1.5 milliSiemens. The high concentration collagen solution has a higher conductivity approaching 4 millisiemens. The skilled artisan will appreciate that conductivities above 10 millisiemens can be observed depending on the concentration of collagen. Next, the desalted and concentrated protein was treated with activated carbon using W-L9000 10X 40 granular resin (Carbon Activated Corporation). 5% w/v of carbon resin was mixed with the collagen-containing protein feed and mixed with gentle agitation at 45-50 ℃. The carbon-treated slurry was filtered using a buchner funnel lined with an Ertel filter press pad M-953 (Ertel Alsop) with or without a filter aid such as diatomaceous earth (Sigma Aldrich). After filtration, the collagen solution was filtered through a 0.2 micron filter, followed by sodium bentonite (final 0.2% w/v) Wyoming Bentonite) for one to several hours and 9000rcf centrifuged for 15-30 minutes to give a high purity, clear and particle-free collagen solution. When removal of endotoxin proteins is desired, the proteins are passed through a chromatographic filter (e.g., sartobind-Q (Sartorius-Stedim)) to specifically remove endotoxin proteins.

Purified collagen was analyzed on SDS-PAGE gels, and thick and clear bands were observed at 30 kilodaltons. The increase in size is due to the structure of the collagen molecule and the high glycine/proline amino acid content. Purified collagen was also analyzed by mass spectrometry, confirming that the 30 kilodalton protein was truncated collagen.

Truncated collagen was further analyzed by HPLC using Agilent 1100 series HPLC. The column is a 50mm Agilent PLRP-S reverse phase column having an inner diameter of 4.6mm, a particle size of μm and a pore size of 1000 angstroms.

Samples were prepared by 1:1 dilution in an HPLC grade aqueous solution of 0.04% sodium azide. 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 diluted appropriately with an aqueous HPLC grade solution of 20mM ammonium acetate buffer at pH about 4.5. After mixing the samples, they were transferred to 300 μl microcircuits and then placed in an autosampler. Analytical parameters such as sample flow rate, column temperature, mobile phase flow rate, mobile phase composition, etc. can be varied using ChemStation (software for operating HPLC). In one exemplary but non-limiting analysis, the parameters are: the flow rate of the sample is 1mL/min, the column temperature is 80 ℃, the column pressure is 60-70bar, and the mobile phase composition is 97.9% water/1.9% acetonitrile and 0.2% trifluoroacetic acid; the UV wavelength used for analysis was 214.4nm, the injection volume was 10. Mu.L, and the sample run time was 10 minutes.

Under these conditions, the elution time of truncated jellyfish collagen of SEQ ID NO. 5 is about 5.4 minutes. ChemStation quantifies the peak area of the elution peak and calculates the protein concentration using a calibration curve that directly relates the peak area to the protein concentration. Calibration curves were generated using known collagen solutions serially diluted to a concentration range containing 0.06mg/mL to 1.00mg/mL collagen.

Truncated collagen without His tag-linker-thrombin cleavage site

Truncated jellyfish collagens without His tag, linker and thrombin cleavage site are disclosed below. The codon-optimized nucleotide sequence encoding the 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 to 72 of SEQ ID NO. 6 and encodes amino acids 1 to 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.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTAATGCAGGTCAGAAAGGTCCGTCAGGTGAACCTGGCAGCCCTGGTAAAGCAGGTAGTGCCGGTGAGCAGGGTCCGCCGGGCAAAGATGGTAGTAATGGTGAGCCGGGTAGCCCTGGCAAAGAAGGTGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:6)

MKKIWLALAGLVLAFSASAAQYEDGPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:7)

A polynucleotide encoding truncated jellyfish collagen without His tag, linker and thrombin cleavage site is disclosed in SEQ ID NO. 8.

GGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACCCCGGGTAATGCAGGTCAGAAAGGTCCGTCAGGTGAACCTGGCAGCCCTGGTAAAGCAGGTAGTGCCGGTGAGCAGGGTCCGCCGGGCAAAGATGGTAGTAATGGTGAGCCGGGTAGCCCTGGCAAAGAAGGTGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCCGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAAACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTCGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGGTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCGGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGTTTACGTTAA(SEQ ID NO:8)

Truncated jellyfish collagen without His tag, linker and thrombin cleavage site is disclosed in SEQ ID NO. 5.

GPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLR(SEQ ID NO:5)

Truncated collagen and GFP β -lactamase fusion with DsbA secretion tag-His tag-linker-thrombin cleavage site (form 1):

Jellyfish collagen and GFP β -lactamase fusion with DsbA secretion tag-His tag-linker-thrombin cleavage site are disclosed below. The codon-optimized nucleotide sequence encoding the 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 to 72 of SEQ ID NO. 9 and encodes amino acids 1 to 24 of SEQ ID NO. 10. The His tag is encoded by nucleotides 73 to 99 of SEQ ID NO. 9 and encodes the 9 histidine tag of amino acids 25 to 33 of SEQ ID NO. 10. The linker is encoded by nucleotides 100-111 of SEQ ID NO. 9 and by amino acids 34-37 of SEQ ID NO. 10. The thrombin cleavage site is encoded by nucleotides 112 to 135 of SEQ ID NO. 9 and by amino acids 38 to 45 of SEQ ID NO. 10. A Green Fluorescent Protein (GFP) with a linker is encoded by nucleotide 136 to 873 of SEQ ID NO. 9 and by amino acids 46 to 291 of SEQ ID NO. 10. The truncated collagen sequence is encoded by nucleotides 874 to 1440 of SEQ ID NO. 9 and by amino acids 292 to 480 of SEQ ID NO. 10. The beta-lactamase with linker is encoded by nucleotides 1441-2232 of SEQ ID NO. 9 and by amino acids 481-744 of SEQ ID NO. 10. Beta-lactamases target correctly the periplasmic space of cells even if the polypeptide does not have an independent secretion tag. The DsbA secretion tag directs the entire transcript (truncated collagen with DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP β -lactamase fusion protein) to the periplasmic space of the cell and β -lactamase functions normally.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATCACCATCACCACCACCACCATCACCACTCTGGCTCGAGCCTGGTGCCGCGCGGCAGCCATATGTCTGGCTCGAGCAGTAAAGGTGAAGAACTGTTCACCGGTGTTGTTCCGATCCTGGTTGAACTGGATGGTGATGTTAACGGCCACAAATTCTCTGTTCGTGGTGAAGGTGAAGGTGATGCAACCAACGGTAAACTGACCCTGAAATTCATCTGCACTACCGGTAAACTGCCGGTTCCATGGCCGACTCTGGTGACTACCCTGACCTATGGTGTTCAGTGTTTTTCTCGTTACCCGGATCACATGAAGCAGCATGATTTCTTCAAATCTGCAATGCCGGAAGGTTATGTACAGGAGCGCACCATTTCTTTCAAAGACGATGGCACCTACAAAACCCGTGCAGAGGTTAAATTTGAAGGTGATACTCTGGTGAACCGTATTGAACTGAAAGGCATTGATTTCAAAGAGGACGGCAACATCCTGGGCCACAAACTGGAATATAACTTCAACTCCCATAACGTTTACATCACCGCAGACAAACAGAAGAACGGTATCAAAGCTAACTTCAAAATTCGCCATAACGTTGAAGACGGTAGCGTACAGCTGGCGGACCACTACCAGCAG

AACACTCCGATCGGTGATGGTCCGGTTCTGCTGCCGGATAACCA

CTACCTGTCCACCCAGTCTAAACTGTCCAAAGACCCGAACGAAA

AGCGCGACCACATGGTGCTGCTGGAGTTCGTTACTGCAGCAGGT

ATCACGCACGGCATGGATGAACTCTACAAATCTGGCGCGCCGG

GCGGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTAC

CCCGGGTAATGCAGGTCAGAAAGGTCCGTCAGGTGAACCTGGC

AGCCCTGGTAAAGCAGGTAGTGCCGGTGAGCAGGGTCCGCCGG

GCAAAGATGGTAGTAATGGTGAGCCGGGTAGCCCTGGCAAAGA

AGGTGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGC

CGCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTA

AACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGT

CGTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCG

GTCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGC

GGTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGC

CTGGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCA

GCTGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGC

GCACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGG

GTTTACGTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA

AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCA

ACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT

CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATT

ATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA

CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA

AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGT

GCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT

GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCAC

AACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT

GCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGC

GAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT

GGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC

CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGT

GGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC

CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACT

ATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA(SEQ ID NO:9)

MKKIWLALAGLVLAFSASAAQYEDHHHHHHHHHSGSSLVPRGSHMSGSSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSGAPGGPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLRHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW(SEQ ID NO:10)

The polynucleotide of SEQ ID NO. 9 was constructed by assembling several DNA fragments. The collagen-containing sequences were codon optimized and synthesized by internal synthesis of Gen9 DNA (now Ginkgo Bioworks). GFP was also synthesized by Gen 9. PCR and UsingGXL polymerase (www.clontech.com/US/Products/PCR/GC_Rich/PrimeSTAR_GXL_DNA_PolymerasesItex=10020:22372:US) was isolated from plasmid pKD46

(cgsc2.biology.yale.edu/Strain.phpID＝68099) Beta-lactamase was cloned. The overlap between pET28 vector, GFP, collagen and beta-lactamase was designed to be 30 to 40bp long, and PCR was usedGXL polymerase is added. Then use SGI Gibson->

(us.vwr.com/store/product/17613857/gibson-assembly-hifi-1-step-kit-system-genetics-inc) the cut pET28a vector was assembled together with the insert into the final plasmid. Plasmid sequences were then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

Collagen was purified by acid treatment of non-homogenized whole cells recovered from the bioreactor after centrifugation and resuspended in the above buffer. The pH of the resuspended suspension was reduced to 3 using 6M hydrochloric acid. The acidified cell slurry was mixed and incubated overnight at 4 ℃ and then centrifuged. The pH was then raised to 9 using 10N NaOH and the supernatant of the slurry was tested on a polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitation. The salt content of the collagen slurry thus obtained is high. To obtain volume and salt reduction, concentration and diafiltration steps were performed using an EMD Millipore tangential flow filtration system with 0.1m2 each ultrafiltration cassette. Using 2 parallel cartridges, the total area of filtration was 0.2m2. A volume reduction of 5x and a salt reduction of 19x was achieved during the TFF stage. The final collagen slurry was run on SDS-PAGE gels to confirm the presence of collagen. The slurry was dried using a multi-tray lyophilizer for 3 days to give a white fluffy collagen powder.

Purified collagen-GFP-beta-lactamase fusion proteins were analyzed on SDS-PAGE gels and running at an apparent molecular weight of 90 kilodaltons was observed. The expected size of the fusion protein is 85kDa. The 90kDa band was confirmed by mass spectrometry to be the correct collagen fusion protein.

Truncated collagen and GFP β -lactamase fusion with DsbA secretion tag-His tag-linker-thrombin cleavage site (form 2):

jellyfish collagen and GFP β -lactamase fusion with DsbA secretion tag-His tag-linker-thrombin cleavage site are disclosed below. The codon-optimized nucleotide sequence encoding the 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 to 72 of SEQ ID NO. 11 and encodes amino acids 1 to 24 of SEQ ID NO. 12. The His tag is encoded by nucleotides 73 to 99 of SEQ ID NO. 11 and encodes the 9 histidine tag of amino acids 25 to 33 of SEQ ID NO. 12. The linker is encoded by nucleotides 100-111 of SEQ ID NO. 11 and by amino acids 34-37 of SEQ ID NO. 12. The thrombin cleavage site is encoded by nucleotides 112 to 135 of SEQ ID NO. 11 and by amino acids 38 to 45 of SEQ ID NO. 12. A Green Fluorescent Protein (GFP) with a linker is encoded by nucleotide 136 to 873 of SEQ ID NO. 11 and by amino acids 46 to 291 of SEQ ID NO. 12. The truncated collagen sequence is encoded by nucleotides 874-1440 of SEQ ID NO. 11 and by amino acids 292-480 of SEQ ID NO. 12. The beta-lactamase with linker is encoded by nucleotides 1441-2232 of SEQ ID NO. 11 and by amino acids 481-744 of SEQ ID NO. 12.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGC

GTTTAGCGCATCGGCGGCGCAGTATGAAGATCACCATCACCACC

ACCACCATCACCACTCTGGCTCGAGCCTGGTGCCGCGCGGCAGC

CATATGTCTGGCTCGAGCAGTAAAGGTGAAGAACTGTTCACCG

GTGTTGTTCCGATCCTGGTTGAACTGGATGGTGATGTTAACGGC

CACAAATTCTCTGTTCGTGGTGAAGGTGAAGGTGATGCAACCAA

CGGTAAACTGACCCTGAAATTCATCTGCACTACCGGTAAACTGC

CGGTTCCATGGCCGACTCTGGTGACTACCCTGACCTATGGTGTT

CAGTGTTTTTCTCGTTACCCGGATCACATGAAGCAGCATGATTT

CTTCAAATCTGCAATGCCGGAAGGTTATGTACAGGAGCGCACC

ATTTCTTTCAAAGACGATGGCACCTACAAAACCCGTGCAGAGGT

TAAATTTGAAGGTGATACTCTGGTGAACCGTATTGAACTGAAAG

GCATTGATTTCAAAGAGGACGGCAACATCCTGGGCCACAAACT

GGAATATAACTTCAACTCCCATAACGTTTACATCACCGCAGACA

AACAGAAGAACGGTATCAAAGCTAACTTCAAAATTCGCCATAA

CGTTGAAGACGGTAGCGTACAGCTGGCGGACCACTACCAGCAG

AACACTCCGATCGGTGATGGTCCGGTTCTGCTGCCGGATAACCA

CTACCTGTCCACCCAGTCTaaaCTGTCCAAAGACCCGAACGAAAA

GCGCGACCACATGGTGCTGCTGGAGTTCGTTACTGCAGCAGGTA

TCACGCACGGCATGGATGAACTCTACAAATCTGGCGCGCCGGG

CGGTCCGCAGGGTGTTGTTGGTGCAGATGGTAAAGACGGTACC

CCGGGTAATGCAGGTCAGAAAGGTCCGTCAGGTGAACCTGGCA

GCCCTGGTAAAGCAGGTAGTGCCGGTGAGCAGGGTCCGCCGGG

CAAAGATGGTAGTAATGGTGAGCCGGGTAGCCCTGGCAAAGAA

GGTGAACGTGGTCTGGCAGGACCGCCGGGTCCTGATGGTCGCC

GCGGTGAAACGGGTTCACCGGGTATTGCCGGTGCCCTGGGTAA

ACCAGGTCTGGAAGGTCCGAAAGGTTATCCTGGTCTGCGCGGTC

GTGATGGTACCAATGGCAAACGTGGCGAACAGGGCGAAACCGG

TCCAGATGGTGTTCGTGGTATTCCGGGTAACGATGGTCAGAGCG

GTAAACCGGGCATTGATGGTATTGATGGCACCAATGGTCAGCCT

GGCGAAGCAGGTTATCAGGGTGGTCGCGGTACCCGTGGTCAGC

TGGGTGAAACAGGTGATGTTGGTCAGAATGGTGATCGCGGCGC

ACCGGGTCCGGATGGTAGCAAAGGTAGCGCCGGTCGTCCGGGT

TTACGTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAG

ATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC

AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCC

AATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT

CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA

CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAA

AGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC

TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA

CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA

CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGC

TGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCC

TGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAA

CTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA

GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGG

CTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGG

TCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTC

CCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGG

ATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA(SEQ ID NO:11)

MKKIWLALAGLVLAFSASAAQYEDHHHHHHHHHSGSSLVPRGSHMSGSSSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSGAPGGPQGVVGADGKDGTPGNAGQKGPSGEPGSPGKAGSAGEQGPPGKDGSNGEPGSPGKEGERGLAGPPGPDGRRGETGSPGIAGALGKPGLEGPKGYPGLRGRDGTNGKRGEQGETGPDGVRGIPGNDGQSGKPGIDGIDGTNGQPGEAGYQGGRGTRGQLGETGDVGQNGDRGAPGPDGSKGSAGRPGLRHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW(SEQ ID NO:12)

EXAMPLE 2 human collagen

Human 21 type collagen alpha 1

Truncated human type 21 collagen α1 without His tag, linker and thrombin cleavage site (truncated relative to full length human type 21 collagen α1 (SEQ ID NO: 31)) is disclosed below. The codon-optimized nucleotide and amino acid sequences encoding the collagen are disclosed below. The DsbA secretion tag is encoded by nucleotides 1 to 72 of SEQ ID NO. 13 and encodes amino acids 1 to 24 of SEQ ID NO. 14. The truncated collagen sequence is encoded by nucleotides 73 to 633 of SEQ ID NO. 13 and encodes amino acids 25 to 211 of SEQ ID NO. 14.

The codon-optimized nucleotide sequence encoding the collagen is provided in SEQ ID NO. 13.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGCAGGTTTTCCGGGTCTGCCTGGTCCGGCAGGCGAACCGGGTCGTCATGGTAAAGATGGTCTGATGGGTAGTCCGGGTTTTAAAGGTGAAGCAGGTTCACCGGGTGCACCTGGTCAGGATGGCACCCGTGGTGAACCGGGTATTCCGGGATTTCCGGGTAATCGTGGCCTGATGGGTCAGAAAGGTGAAATTGGTCCGCCTGGTCAGCAGGGTAAAAAAGGCGCACCGGGTATGCCAGGACTGATGGGTTCAAATGGCAGTCCGGGTCAGCCAGGCACACCGGGTTCAAAAGGTAGCAAAGGCGAACCTGGTATTCAGGGTATGCCTGGTGCAAGCGGTCTGAAAGGCGAGCCAGGTGCCACCGGTTCTCCGGGTGAACCAGGTTATATGGGTCTGCCAGGTATCCAAGGCAAAAAAGGTGATAAAGGTAATCAGGGCGAAAAAGGCATTCAGGGCCAGAAAGGCGAAAATGGCCGTCAGGGTATTCCAGGCCAGCAGGGCATCCAGGGTCATCATGGTGCAAAAGGTGAACGTGGTGAAAAGGGCGAACCAGGTGTTCGTTTA(SEQ ID NO:13)

The amino acid sequence is disclosed in SEQ ID NO. 14.

MKKIWLALAGLVLAFSASAAQYEDAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATGSPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVR(SEQ ID NO:14)

A codon-optimized nucleotide sequence encoding truncated human type 21 collagen alpha 1 without DsbA secretion tag collagen is provided in SEQ ID NO. 15.

TGCAGGTTTTCCGGGTCTGCCTGGTCCGGCAGGCGAACCGGGTCGTCATGGTAAAGATGGTCTGATGGGTAGTCCGGGTTTTAAAGGTGAAGCAGGTTCACCGGGTGCACCTGGTCAGGATGGCACCCGTGGTGAACCGGGTATTCCGGGATTTCCGGGTAATCGTGGCCTGATGGGTCAGAAAGGTGAAATTGGTCCGCCTGGTCAGCAGGGTAAAAAAGGCGCACCGGGTATGCCAGGACTGATGGGTTCAAATGGCAGTCCGGGTCAGCCAGGCACACCGGGTTCAAAAGGTAGCAAAGGCGAACCTGGTATTCAGGGTATGCCTGGTGCAAGCGGTCTGAAAGGCGAGCCAGGTGCCACCGGTTCTCCGGGTGAACCAGGTTATATGGGTCTGCCAGGTATCCAAGGCAAAAAAGGTGATAAAGGTAATCAGGGCGAAAAAGGCATTCAGGGCCAGAAAGGCGAAAATGGCCGTCAGGGTATTCCAGGCCAGCAGGGCATCCAGGGTCATCATGGTGCAAAAGGTGAACGTGGTGAAAAGGGCGAACCAGGTGTTCGTtaa(SEQ ID NO:15)

The amino acid sequence of truncated human type 21 collagen alpha 1 without the DsbA secretion tag is disclosed in SEQ ID No. 16.

AGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGSNGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATGSPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVR(SEQ ID NO:16)

The polynucleotide of SEQ ID NO. 13 was synthesized by a Twist Bioscience. The overlap between pET28 vector and SEQ ID NO. 15 and SEQ ID NO. 16 was designed to be 20 to 30bp long, and PCR were usedGXL polymerase (www.takarabio.com/products/pcr/gc-rich-pcr/primestar-GXL-dna-polymerase) was added. The cut pET28a vector was then assembled with insert DNA (SEQ ID NO: 13) into the final plasmid using seamless cloning (www.takarabio.com/products/cloning/in-fusion-cloning). Plasmid sequences were then verified by Sanger sequencing by Genewiz (www.genewiz.com/en).

Transformed cells were cultured in minimal medium and frozen with plant glycerol in a ratio of 50:50 cells to glycerol in 1.5ml aliquots. One vial of frozen culture was resuscitated overnight in 50ml of minimal medium at 37℃and 200 rpm. Cells were transferred to 300ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

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

1) 5L of deionized water solution of glucose syrup with the concentration of 550g/kg is autoclaved. (VWR, product # 97061-170).

2) Autoclave 3946mL of deionized water:

20g(NH ₄ ) ₂ HPO ₄ (VWR, product # 97061-932);

66.5g KH ₂ PO ₄ (VWR, product # 97062-348);

22.5g H ₃ C ₆ H ₅ O ₇ (VWR, product #bdh9228-2.5 KG);

8.85g MgSO ₄ .7H ₂ o (VWR, product # 97062-134);

10mL of 1000x trace metal formulation (Table 5)

After autoclaving, add

118g of (1) to (2);

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

using 28% NH ₄ OH (VWR, product #bdh3022) adjusts the pH to 6.1.

TABLE 5 trace metal formulation

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

The fermentation is carried out at various temperatures ranging from 25 ℃ to 28 ℃. For some fermentations, the temperature of the fermentation is maintained at a constant temperature and the collagen is purified immediately after the fermentation is completed. For other fermentations, the fermentation temperature is maintained for a desired period of time and when the cell density reaches an OD600 of 10-20, the temperature is reduced to induce protein production. Typically, the temperature is reduced from 28 ℃ to 25 ℃. Fermentation was continued at 25℃for 40-60 hours.

Collagen was purified as follows: the pH of the fermentation broth is reduced to 3-3.5 using 5% -50% sulfuric acid. The cells were then separated using centrifugation. The supernatant of the acidified broth was tested on polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitation. The salt content of the collagen slurry thus obtained is high. To obtain volume and salt reduction, concentration and diafiltration steps were performed using an EMD Millipore tangential flow filtration system with 0.1m2 each ultrafiltration cassette. Using 2 parallel cartridges, the total area of filtration was 0.2m2. A volume reduction of 5x and a salt reduction of 19x was achieved during the TFF stage. The final collagen slurry was run on SDS-PAGE gels to confirm the presence of collagen.

Purified collagen was analyzed on an SDS-PAGE gel and thick and clear bands were observed at the expected size of 25 kilodaltons. Quantification of collagen titer and purity was performed using reversed phase and size exclusion HPLC chromatography. The titer is generally 3 to 8 g/l. Purified collagen was further analyzed by mass spectrometry and confirmed to match the published sequence of human type 21 collagen.

Truncating human type 1 collagen alpha 2 (1)

Truncated human type 1 collagen a 2 without His tag, linker and thrombin cleavage site (truncated relative to full length human type 1 collagen a 2 (SEQ ID NO: 32)) is disclosed below. The codon-optimized nucleotide and amino acid sequences are disclosed below. The DsbA secretion tag is encoded by nucleotides 1 to 72 of SEQ ID NO. 17 and encodes amino acids 1 to 24 of SEQ ID NO. 18. The truncated collagen sequence is encoded by nucleotides 73 to 636 of SEQ ID NO. 17 and encodes amino acids 25 to 212 of SEQ ID NO. 18.

The codon-optimized nucleotide sequence encoding the collagen is provided in SEQ ID NO. 17.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATATGGGTCCGCCTGGTAGCCGTGGTGCAAGTGGTCCGGCAGGCGTTCGTGGTCCGAATGGTGATGCAGGTCGTCCGGGTGAACCGGGTCTGATGGGTCCTCGTGGTCTGCCTGGTTCACCGGGTAATATTGGTCCTGCAGGTAAAGAAGGTCCGGTTGGTCTGCCAGGTATTGATGGCCGTCCGGGTCCGATTGGTCCAGCCGGTGCACGTGGTGAACCTGGCAATATTGGTTTTCCGGGTCCTAAAGGTCCGACCGGTGATCCGGGTAAAAATGGTGATAAAGGTCATGCAGGTCTGGCAGGCGCACGCGGTGCACCTGGTCCGGATGGTAATAATGGTGCACAGGGTCCACCGGGTCCGCAGGGTGTTCAAGGTGGTAAAGGCGAACAGGGTCCTGCCGGTCCTCCGGGTTTTCAGGGACTGCCTGGTCCGAGCGGTCCTGCGGGTGAAGTTGGTAAACCTGGTGAACGCGGTCTGCATGGTGAATTTGGCCTGCCTGGGCCTGCAGGTCCGCGTGGCGAACGTGGTCCGCCAGGTGAAAGCGGTGCAGCAGGTCCGACAGGTTAA(SEQ ID NO:17)

The amino acid sequence is disclosed in SEQ ID NO. 18.

MKKIWLALAGLVLAFSASAAQYEDMGPPGSRGASGPAGVRGPNGDAGRPGEPGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPAGPPGFQGLPGPSGPAGEVGKPGERGLHGEFGLPGPAGPRGERGPPGESGAAGPTG(SEQ ID NO:18)

The nucleotide sequence of truncated human type 1 collagen a 2 (1) without the DsbA secretion tag is disclosed in SEQ ID NO 19.

ATGGGTCCGCCTGGTAGCCGTGGTGCAAGTGGTCCGGCAGGCGTTCGTGGTCCGAATGGTGATGCAGGTCGTCCGGGTGAACCGGGTCTGATGGGTCCTCGTGGTCTGCCTGGTTCACCGGGTAATATTGGTCCTGCAGGTAAAGAAGGTCCGGTTGGTCTGCCAGGTATTGATGGCCGTCCGGGTCCGATTGGTCCAGCCGGTGCACGTGGTGAACCTGGCAATATTGGTTTTCCGGGTCCTAAAGGTCCGACCGGTGATCCGGGTAAAAATGGTGATAAAGGTCATGCAGGTCTGGCAGGCGCACGCGGTGCACCTGGTCCGGATGGTAATAATGGTGCACAGGGTCCACCGGGTCCGCAGGGTGTTCAAGGTGGTAAAGGCGAACAGGGTCCTGCCGGTCCTCCGGGTTTTCAGGGACTGCCTGGTCCGAGCGGTCCTGCGGGTGAAGTTGGTAAACCTGGTGAACGCGGTCTGCATGGTGAATTTGGCCTGCCTGGGCCTGCAGGTCCGCGTGGCGAACGTGGTCCGCCAGGTGAAAGCGGTGCAGCAGGTCCGACAGGTTAA(SEQ ID NO:19)

The amino acid sequence of truncated human type 1 collagen a 2 (1) without the DsbA secretion tag is disclosed in SEQ ID No. 20.

MGPPGSRGASGPAGVRGPNGDAGRPGEPGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPAGPPGFQGLPGPSGPAGEVGKPGERGLHGEFGLPGPAGPRGERGPPGESGAAGPTG(SEQ ID NO:20)

Truncating human type 1 collagen alpha 2 (2)

Truncated human type 1 collagen a 2 without His tag, linker and thrombin cleavage site (truncated relative to full length human type 1 collagen a 2 (SEQ ID NO: 32)) is disclosed below. The codon-optimized nucleotide and amino acid sequences are disclosed below. The DsbA secretion tag is encoded by nucleotides 1 to 72 of SEQ ID NO. 21 and encodes amino acids 1 to 24 of SEQ ID NO. 22. The truncated collagen sequence is encoded by nucleotides 73 to 609 of SEQ ID NO. 21 and encodes amino acids 25 to 203 of SEQ ID NO. 22.

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

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGGTTTTCAGGGTCCTGCCGGTGAACCGGGTGAACCTGGTCAGACAGGTCCGGCAGGCGCACGTGGTCCTGCAGGTCCTCCTGGTAAAGCCGGTGAAGATGGTCATCCGGGTAAACCGGGTCGTCCTGGTGAACGTGGTGTTGTTGGTCCGCAGGGTGCCCGTGGTTTTCCGGGTACTCCGGGTCTGCCAGGTTTTAAAGGTATTCGTGGTCATAATGGTCTGGATGGTCTGAAAGGTCAGCCTGGTGCACCGGGTGTTAAAGGTGAACCAGGTGCTCCGGGTGAAAATGGCACACCGGGTCAGACCGGTGCGCGTGGTCTGCCTGGCGAACGCGGTCGTGTTGGTGCACCTGGTCCAGCCGGTGCACGCGGTAGTGATGGTAGCGTTGGTCCGGTTGGTCCAGCGGGTCCGATTGGTAGCGCAGGTCCACCGGGTTTTCCAGGCGCACCGGGTCCGAAAGGTGAAATTGGTGCAGTTGGTAATGCAGGCCCTGCCGGTCCAGCAGGACCGCGTGGTGAAGTTGGCCTGCCTGGTCTGTAA(SEQ ID NO:21)

The amino acid sequence is disclosed in SEQ ID NO. 22.

MKKIWLALAGLVLAFSASAAQYEDGFQGPAGEPGEPGQTGPAGARGPAGPPGKAGEDGHPGKPGRPGERGVVGPQGARGFPGTPGLPGFKGIRGHNGLDGLKGQPGAPGVKGEPGAPGENGTPGQTGARGLPGERGRVGAPGPAGARGSDGSVGPVGPAGPIGSAGPPGFPGAPGPKGEIGAVGNAGPAGPAGPRGEVGLPGL(SEQ ID NO:22)

The nucleotide sequence of truncated human type 1 collagen a 2 (2) without the DsbA secretion tag is disclosed in SEQ ID NO. 23.

GGTTTTCAGGGTCCTGCCGGTGAACCGGGTGAACCTGGTCAGACAGGTCCGGCAGGCGCACGTGGTCCTGCAGGTCCTCCTGGTAAAGCCGGTGAAGATGGTCATCCGGGTAAACCGGGTCGTCCTGGTGAACGTGGTGTTGTTGGTCCGCAGGGTGCCCGTGGTTTTCCGGGTACTCCGGGTCTGCCAGGTTTTAAAGGTATTCGTGGTCATAATGGTCTGGATGGTCTGAAAGGTCAGCCTGGTGCACCGGGTGTTAAAGGTGAACCAGGTGCTCCGGGTGAAAATGGCACACCGGGTCAGACCGGTGCGCGTGGTCTGCCTGGCGAACGCGGTCGTGTTGGTGCACCTGGTCCAGCCGGTGCACGCGGTAGTGATGGTAGCGTTGGTCCGGTTGGTCCAGCGGGTCCGATTGGTAGCGCAGGTCCACCGGGTTTTCCAGGCGCACCGGGTCCGAAAGGTGAAATTGGTGCAGTTGGTAATGCAGGCCCTGCCGGTCCAGCAGGACCGCGTGGTGAAGTTGGCCTGCCTGGTCTGTAA(SEQ ID NO:23)

The amino acid sequence of truncated human type 1 collagen a 2 (2) without the DsbA secretion tag is disclosed in SEQ ID No. 24.

GFQGPAGEPGEPGQTGPAGARGPAGPPGKAGEDGHPGKPGRPGERGVVGPQGARGFPGTPGLPGFKGIRGHNGLDGLKGQPGAPGVKGEPGAPGENGTPGQTGARGLPGERGRVGAPGPAGARGSDGSVGPVGPAGPIGSAGPPGFPGAPGPKGEIGAVGNAGPAGPAGPRGEVGLPGL(SEQ ID NO:24)

The polynucleotides of SEQ ID NO. 13, 17 or 21 were subcloned into vector pET28a as described herein to prepare transformation vectors. Host cells were transformed with the vector and the polynucleotide was expressed as described in example 1.

After fermentation was completed, the truncated human collagen was purified from the fermentation broth using the procedure disclosed in example 2. Purified truncated human collagen was analyzed using SDS-PAGE and HPLC as disclosed in example 2.

In SDS-PAGE analysis, all three truncated human collagens were run at the expected molecular weight. The standard curve of jellyfish collagen of example 1 was used in the analysis of truncated human collagen using HPLC. The retention time of human collagen is slightly different from jellyfish collagen. The retention time of SEQ ID NO. 16 was 5.645 min, the retention time of SEQ ID NO. 20 was 5.631 min, and SEQ ID NO. 24 was run at the two peaks with retention times of 5.531 and 5.7 min. Truncated human type 1 collagen alpha 2 truncations 5 with DsbA secretion and FLAG tag

The amino acid sequence of truncated human type 1 collagen alpha 2 truncated 5 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO 25. The DsbA secretion tag is encoded by nucleotides 1 to 57 of SEQ ID NO. 26 and the amino acid sequence is amino acids 1 to 19 of SEQ ID NO. 25. The collagen nucleotide sequence is nucleotide 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.

MKKIWLALAGLVLAFSASAGDQGPVGRTGEVGAVGPPGFAGEKGPSGEAGTAGPPGTPGPQGLLGAPGILGLPGSRGERGLPGVAGAVGEPGPLGIAGPPGARGPPGAVGSPGVNGAPGEAGRDGNPGNDGPPGRDGQPGHKGERGYPGNIGPVGAAGAPGPHGPVGPAGKHGNRGETGPSGPVGPAGAVGPRGPSGPQGIRGDKGEPGEKGPRGLPGLGDYKDDDDK(SEQ ID NO:25)

The nucleotide sequence of truncated human type 1 collagen alpha 2 truncated 5 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO 26.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGGTGATCAGGGTCCGGTTGGTCGTACCGGTGAAGTTGGTGCAGTCGGGCCGCCGGGTTTTGCGGGTGAAAAAGGCCCGTCAGGTGAAGCAGGCACCGCTGGCCCTCCTGGCACGCCTGGCCCACAGGGTTTACTGGGCGCACCTGGAATTCTGGGACTGCCGGGCAGCCGTGGAGAACGCGGTTTACCAGGTGTTGCCGGTGCCGTTGGTGAACCTGGTCCACTGGGCATTGCAGGGCCGCCTGGCGCACGGGGACCGCCTGGTGCTGTTGGTAGTCCGGGTGTGAATGGTGCTCCGGGTGAAGCCGGTCGTGACGGTAATCCGGGAAATGACGGCCCGCCAGGCCGCGATGGTCAGCCGGGTCATAAAGGTGAGCGTGGTTACCCAGGTAATATTGGTCCAGTCGGTGCCGCCGGTGCGCCGGGTCCTCATGGCCCTGTCGGTCCAGCCGGTAAACATGGTAATCGCGGTGAGACAGGTCCGTCAGGACCAGTGGGCCCTGCTGGCGCAGTCGGTCCGCGCGGGCCGAGTGGCCCTCAGGGTATTCGTGGCGATAAAGGGGAACCGGGCGAAAAAGGGCCGCGGGGTCTGCCAGGCCTGGGTGACTACAAAGACGACGACGACAAATAA(SEQ ID NO:26)

The polynucleotide of SEQ ID NO. 26 is subcloned into vector pET28a, expressed in a host E.coli cell, and the truncated collagen purified as described herein. Purified collagen produced clear bands on SDS-PAGE and anti-FLAG western was observed at about 100 kilodaltons. In the case where the protein is not expressed, no band appears at this position on the gel.

Truncated human type 1 collagen alpha 2 truncations 6 with DsbA secretion and FLAG tag

The amino acid sequence of truncated human type 1 collagen alpha 2 truncated 6 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO 27. The DsbA secretion tag is encoded by nucleotides 1 to 57 of SEQ ID NO. 28 and the amino acid sequence is amino acids 1 to 19 of SEQ ID NO. 27. The collagen nucleotide sequence is nucleotide 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.

MKKIWLALAGLVLAFSASAKGHNGLQGLPGIAGHHGDQGAPGSVGPAGPRGPAGPSGPAGKDGRTGHPGTVGPAGIRGPQGHQGPAGPPGPPGPPGPPGVSGGGYDFGYDGDFYRADQPRSAPSLRPKDYEVDATLKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFSTGETCIRAQPENIPAKNWYRSSKDGDYKDDDDK(SEQ ID NO:27)

The nucleotide sequence of truncated human type 1 collagen alpha 2 truncated 6 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO. 28.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGAAAGGTCACAATGGACTGCAAGGCCTGCCAGGTATTGCAGGTCATCATGGTGATCAAGGTGCCCCGGGAAGCGTTGGTCCGGCGGGGCCGAGAGGCCCTGCGGGACCTTCAGGTCCGGCAGGCAAAGATGGTCGGACAGGCCATCCGGGCACCGTTGGCCCTGCAGGAATTCGTGGACCGCAGGGTCATCAGGGACCTGCTGGTCCGCCAGGTCCCCCGGGCCCTCCGGGACCACCGGGTGTTAGTGGTGGTGGTTATGATTTTGGCTATGATGGTGATTTTTATCGTGCAGATCAGCCGCGTAGCGCACCGAGCCTGCGTCCTAAAGATTATGAAGTTGATGCAACCCTGAAAAGCCTGAATAATCAGATTGAAACACTGCTGACACCGGAAGGTAGCCGTAAAAATCCGGCCCGTACCTGTCGTGATCTGCGTCTGAGCCACCCGGAATGGAGCAGCGGTTATTATTGGATTGATCCGAATCAAGGTTGTACCATGGATGCAATTAAAGTTTATTGTGATTTTAGCACAGGTGAAACATGTATCCGTGCACAGCCGGAAAATATTCCGGCCAAAAATTGGTATCGTAGTAGCAAAGATGGTGACTACAAAGACGACGACGACAAATAA(SEQ ID NO:28)

The polynucleotide of SEQ ID NO. 28 is subcloned into vector pET28a, expressed in a host E.coli cell, and the truncated collagen purified as described herein. Purified collagen produced clear bands on SDS-PAGE and anti-FLAG western was observed at about 25 kilodaltons. In the case where the protein is not expressed, no band appears at this position on the gel.

Truncated human type 1 collagen alpha 2 truncations 7 with DsbA secretion and FLAG tag

The amino acid sequence of truncated human type 1 collagen alpha 2 truncations 7 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO 29. The DsbA secretion tag is encoded by nucleotides 1 to 57 of SEQ ID NO. 30 and the amino acid sequence is amino acids 1 to 19 of SEQ ID NO. 29. The collagen nucleotide sequence is nucleotide 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 nucleotide 760 to 786 of SEQ ID NO. 30 and the amino acid sequence is amino acids 254 to 262 of SEQ ID NO. 29.

MKKIWLALAGLVLAFSASAYEVDATLKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFSTGETCIRAQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVEGVTSKEMATQLAFMRLLANYASQNITYHCKNSIAYMDEETGNLKKAVILQGSNDVELVAEGNSRFTYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFKGDYKDDDDK(SEQ IDNO:29)

The nucleotide sequence of truncated human type 1 collagen alpha 2 truncations 7 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO. 30.

TGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGTATGAAGTTGATGCAACCCTGAAAAGCCTGAATAATCAGATTGAAACACTGCTGACACCGGAAGGTAGCCGTAAAAATCCGGCCCGTACCTGTCGTGATCTGCGTCTGAGCCACCCGGAATGGAGCAGCGGTTATTATTGGATTGATCCGAATCAAGGTTGTACCATGGATGCAATTAAAGTTTATTGTGATTTTAGCACAGGTGAAACATGTATCCGTGCACAGCCGGAAAATATTCCGGCCAAAAATTGGTATCGTAGTAGCAAAGATAAAAAACATGTGTGGCTGGGTGAAACCATTAATGCAGGTAGCCAGTTTGAATACAATGTTGAAGGTGTTACCAGCAAAGAAATGGCAACACAGCTGGCATTTATGCGTCTGCTGGCAAATTATGCAAGCCAGAATATTACATATCATTGTAAAAATAGCATTGCATATATGGATGAAGAAACCGGTAATCTGAAAAAAGCAGTTATTCTGCAGGGTAGCAATGATGTTGAACTGGTTGCCGAAGGTAATAGCCGTTTTACATATACCGTTCTGGTTGATGGTTGTAGCAAAAAAACCAATGAATGGGGTAAAACCATCATTGAATATAAAACCAACAAACCGAGCCGTCTGCCGTTTCTGGATATCGCTCCGCTGGATATTGGTGGTGCCGATCAGGAATTTTTTGTCGATATCGGTCCTGTGTGTTTTAAAGGTGACTACAAAGACGACGACGACAAATAA(SEQ ID NO:30)

The polynucleotide of SEQ ID NO. 30 is subcloned into vector pET28a, expressed in a host E.coli cell, and the truncated collagen purified as described herein. Purified collagen produced clear bands on SDS-PAGE and anti-FLAG western was observed at about 30 kilodaltons. In the case where the protein is not expressed, no band appears at this position on the gel.

EXAMPLE 3 human clinical study of truncated human type 21 collagen

Clinical studies were performed using human subjects to determine the efficacy of topical skin care products containing truncated human type 21 collagen (SEQ ID NO: 16). Research is conducted in accordance with the U.S. and international standards (FDA and ICH guidelines) for the clinical trial administration of pharmaceuticals and applicable government regulations.

A base formulation (control formulation) made from water, olive oil glycerol polyether-8 esters, glycerol, coconut alkane, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, pentanediol, disodium EDTA, octanoyl glycol, chlorophenylglycol, phenoxyethanol was prepared. Formulations containing truncated human type 21 collagen were prepared by adding sufficient collagen to prepare a topical formulation containing 0.1% w/w collagen.

Professional hierarchical assessment of qualification and efficacy

Visual Analog Scales (VAS) are commonly used in clinical studies to measure the intensity or frequency of various symptoms, subjective features, or attitudes that cannot be measured directly. VAS is a reliable scale that is more sensitive to small changes than a simple ordinal scale. (A. Paul-Dauphin, F.Guillemin, J.Virion and S. Briencon, "Bias and precision in visual analog scales: A randomized controlled trial," American Journal of Epidemiology, vol.150, stage 10, pages 1117-27, 1999). When responding to VAS projects, the specialty ranker specifies the level at which they agree with the statement by indicating a position along a line (10 cm) between two endpoints or anchor responses. Simple VAS was used to evaluate efficacy parameters, where the end of the 10cm horizontal line was defined as the limit in the left (best) to right (worst) direction. The signs of photo-aging can be categorized as follows: mild = 1-3.9cm, moderate = 4-6.9cm, severe = 7-10cm.

The following VAS was used:

* Criteria for inclusion

Ordinal numbers tables allow the direct and objective attachment of numbers to the quality of a given attribute. When responding to ordinal scale items, the specialty ranker specifies their level of agreement with the statement by selecting a set level or level.

The appearance qualification of each subject's facial skin redness (erythema) was assessed at baseline by a professional classifier using the following five-order ordinal scale (table 6). If eligible, each subject will receive further erythema assessment at weeks 2, 4, 6 and 8.

TABLE 6 five-order ordinal scale for erythema assessment

The corneometer CM 820 (courage+khazaka, germany) measures the degree of relative hydration of the skin surface by applying an alternating current to the skin with a pair of closely spaced electrodes and measuring capacitance. The change in skin moisture content changes the conductance of the capacitive circuit.

The corneometer is capable of reproducibly detecting slight changes in hydration levels with a measurement time of only about one second. The depth of measurement is small (stratum corneum of about 10-20 μm), which ensures that the assessment is not affected by deeper layers of skin.

All subjects performed facial corneometer measurements at baseline, immediately after initial application, and at weeks 2, 4, and 8. Three measurements were made for each time point and averaged. The measured positions are recorded on the face map for evaluation consistency at each time point.

Cutometer MPA 580 (Coura+Khazaka, germany) measures the viscoelasticity of the skin by applying suction to the skin surface, sucking the skin into the holes of the probe and determining the penetration depth using an optical measurement system.

The resistance (compactness) of the skin sucked up by the negative pressure and its ability to return to the initial position (elasticity) are calculated and displayed as a curve. The Cutometer output includes a number of parameters that measure different parts of the curve, including R0 (Uf, compactness), R2 (Ua/Uf, total elasticity), R5 (Ur/Ue, net elasticity), R7 (Ur/Uf, elastic parts), and R9 (R3 [ last maximum amp ] -R0[ Uf ], fatigue).

All subjects performed a Cutometer measurement (following the prepared randomization code) on the left or right cheek at baseline, immediately after initial application, and weeks 2, 4, and 8. Skin elasticity was reported using the R5 (Ur/Ue) and R2 (Ua/Uf) parameters. This value will increase as the skin becomes more elastic. Skin firmness was reported using the R0 (Uf) parameter. As the skin becomes more compact, this value will decrease. The evaluation position is recorded on the face map of each object for measurement consistency between accesses.

COSMETRICS ^TM SIAScope (Astron clinical, toft, UK) is a non-invasive optical skin imaging instrument that uses spectrophotometry Pi Nafen analysis (SIA) or chromophore mapping. The technique is based on a unique combination of percutaneous angiography and contact back spectrophotometry (contact remittance spectrophotometry). The hardware consists of a handheld imaging probe attached to a laptop. The unit is placed in contact with the skin surface and the skin is illuminated with discrete wavelength high intensity LEDs of 400 to 1000nm (spanning a small range of the visible and near infrared spectrum). A digital image is captured for each wavelength. Three parameters chromophore profiles were retrieved up to 2mm deep, zhou Changduo up to 11mm deep, one for each of the following parameters: epidermal melanin, dermal hemoglobin, and dermal collagen.

For this study, dermal collagen will be measured on the left or right cheek at baseline and weeks 2, 4 and 8 according to the prepared randomization codes. The evaluation location will be recorded on the face map of each subject for measurement consistency between visits.

DermaScan C USB (Cortex Technology ApS, hadsund, denmark) is a compact high resolution ultrasound scanner. The use of a 20MHz, high definition 60x150 μm, 13mm penetration probe provides linear scanning, high precision operation, and true position detection for image definition and sharpness.

The instrument is provided by cyberDERM, inc. (Broomall, pa., USA). All subjects underwent ultrasound assessment of the face at baseline and weeks 2, 4 and 8. The evaluation position at each visit is the same and will be recorded on the face map. After obtaining the ultrasound scans, they were sent to cyberDERM, inc for analysis of dermis thickness (density).

All clinical photographs were performed according to the SOP of the IRSI to ensure reproducibility of high quality images during the study. Imaging was performed in a designated photographic suite with a matt black wall and blocking all natural light. In order to prepare the subject for clinical photography, the subject is required to remove all jewelry, including earrings, necklaces, and any face jewelry. Trained technicians examine subjects under the light amplification circuit to ensure that no residual makeup or skin care is visible on the face, eyes, or lips. A black cape and black headpiece are provided to the subject and instructed to be placed to ensure that all hair is pulled back neatly and covered.

Clarity ^TM The 2D study System Ti (Clarity) (BrightTex Bio-Photonics (BTBP), san Jose CA, USA) captured high quality frontal, left and right images of the full face. Three cameras within the system allow for capturing an 18 megapixel SLR image at 16 bits using both real-time dynamic display and automated facial alignment inspection for baseline images to ensure reproducibility.

Multispectral illumination (diffuse white light, cross-polarization, blue, and parallel polarization) reveals skin conditions above and below the surface layer of the skin. The system uses skin feature recognition to apply automated skin segmentation and region mapping to allow subsequent skin analysis. The image is analyzed for attributes related to pigmentation, subsurface pigmentation, lighting, skin color, redness, wrinkles, skin texture, pores, acne, and/or lips.

All subjects captured forward looking, left looking and right looking facial images at baseline and weeks 2, 4 and 8 with standard light and parallel polarized light.

Subjective questionnaires allow sponsors to evaluate subjects' skin, products tested, and their effects. The question will require the subject to agree to a statement of the five-level scale and an open answer.

Fourteen female subjects were included in the study. The inclusion criteria were caucasian female subjects of Fitzpatrick type III skin well-being and aged between 35 and 65 years (including at the time of inclusion). The inclusion criteria also included signs of facial aging determined by the professional classifier at baseline: a) The score of the 10cm scale of lines/wrinkles is more than or equal to 2cm and less than or equal to 6; and b) a 5-order ordinal number scale score of 1.ltoreq.3 for facial redness (erythema). Table 7 discloses demographics of study participants.

TABLE 7 demographic profile

Results of professional clinical sizer evaluations after two weeks of treatment for lines/wrinkles, firmness (vision), elasticity (touch), brightness, texture/softness (touch), texture/smoothness (vision) and erythema are shown in table 8. All the characteristics tested were improved. The scores for brightness, texture/softness (tactile), texture/smoothness (visual) and erythema have statistically significant improvements.

TABLE 8 professional clinical classifier evaluation-unary, compared to baseline

* Indicating a statistically significant improvement over baseline, p.ltoreq.0.05

The instrument used by the corneometer and the cutoff to evaluate hydration, firmness, and elasticity is shown in table 9. The improvement in skin hydration, firmness and elasticity is statistically significant. In addition, table 9 shows stimulation of collagen production by skin cells as demonstrated by Spectrophotometric Intradermal Analysis (SIA).

TABLE 9 Instrument assessment-unary, compared to baseline

Ni=no improvement

* Represents a statistically significant deterioration from baseline, p.ltoreq.0.05

The results demonstrate that truncated human type 21 collagen shows statistically significant improvements in skin elasticity, brightness, hydration, tactile texture, or visual texture. Furthermore, the results indicate that truncated human type 21 collagen shows a statistically significant reduction in visible lines or wrinkles as well as a significant reduction in erythema.

EXAMPLE 4 in vitro investigation of human type 21 truncated collagen on skin cells

Truncated human type 21 collagen alpha 1 stimulates fibroblasts to produce type I collagen

A series of in vitro experiments were performed to assess the effect of truncated human type 21 collagen on human skin fibroblasts and keratinocytes. In a first experiment, human primary fibroblasts were evaluated for type I collagen secretion. Fibroblasts were incubated with 0.03% of the polypeptide according to SEQ ID NO. 16 for 48 hours. The culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) for procollagen type I C-peptide, which is a reading of total secreted type I collagen. As shown in FIG. 1, cells treated with the SEQ ID NO 16 polypeptide secreted higher levels of type I collagen (FIG. 1; "B") than untreated cells (FIG. 1; "A") or cells treated with retinol (FIG. 1; "C").

Gene for stimulating fibroblast to produce extracellular matrix protein by truncated human type 21 collagen alpha 1

RNA sequencing was performed to analyze overall gene expression. After 48 hours of exposure, the fibroblasts were incubated with 0.03% of the polypeptide according to SEQ ID NO. 16. These fibroblasts express higher levels of several extracellular matrix genes than cells incubated in medium alone. As shown in FIG. 2A, fibroblasts treated with the SEQ ID NO 16 polypeptide (FIG. 2A, "C") up-regulated the type I collagen gene (COL 1A) relative to untreated cells (FIG. 2A; "A") or fibroblasts treated with retinol (FIG. 2A; "B"). This response was similar to that of fibroblasts treated with vitamin C (FIG. 2A; "D"). As shown in FIG. 2B, fibroblasts treated with the SEQ ID NO 16 polypeptide (FIG. 2B; "B") upregulated the elastin gene (ELN) relative to untreated cells (FIG. 2B; "A") and various marine collagens (FIG. 2B; "C", "D", "E", and "F"). As shown in FIG. 2C, fibroblasts treated with the polypeptide of SEQ ID NO 16 (FIG. 2C; "B") up-regulated the fibronectin gene (FN 1) relative to untreated cells (FIG. 2C; "A"), retinol (FIG. 2C; "C"), and vitamin C (FIG. 2C; "D").

Truncating human type 21 collagen α1 reduced inflammation of UVB-irradiated keratinocytes.

With 40mJ/cm ² The human primary keratinocytes were irradiated with UVB light and then treated with 0.1% of the SEQ ID NO. 16 polypeptide for 24 hours. The levels of the pro-inflammatory cytokine IL-1a were determined by ELISA. As shown in FIG. 3, UVB-irradiated keratinocytes treated with the polypeptide of SEQ ID NO 16 (FIG. 3; "B") expressed lower levels of IL-1α than untreated UVB-irradiated keratinocytes (FIG. 3; "A").

Truncated human type 21 collagen α1 has antioxidant capacity.

An Oxygen Radical Absorbance Capacity (ORAC) assay was used to evaluate the antioxidant potential of the SEQ ID NO 16 polypeptide. The ORAC assay is a cell-free assay that uses fluorescent readings to measure the antioxidant capacity of a product. Data are reported as Trolox (vitamin E) equivalents. As shown in FIG. 4, a 0.1% solution of the polypeptide of SEQ ID NO:16 has an antioxidant property equivalent to 190. Mu.M Trolox.

Truncating human type 21 collagen α1 increases the cell viability of UVB-irradiated keratinocytes.

To further evaluate the effect of treatment with the SEQ ID NO 16 polypeptide on UVB-irradiated keratinocytes, pre-irradiation and post-irradiation treatment experiments were performed. Human primary keratinocytes were pretreated with 0.1% of the SEQ ID NO:16 polypeptide for 24 hours with 40mJ/cm ² UVB light irradiation was followed by a further treatment with 0.1% of the SEQ ID NO. 16 polypeptide for 24 hours. Cell viability was assessed using an MTT metabolic colorimetric assay. As shown in FIG. 5, UVB-irradiated keratinocytes treated with the SEQ ID NO 16 polypeptide (FIG. 5; "B") showed higher cell viability than UVB-irradiated keratinocytes without such treatment (FIG. 5; "A").

Example 5 human clinical study of truncated human type 21 collagen.

Local application of truncated human type 21 collagen alpha 1 is associated with increased facial skin elasticity

In clinical studies (n=15 subjects), subjects used 8 weeks after a washout period of 1 week protein-free basal facial essence, a topical facial essence containing 0.1% of the SEQ ID NO:16 polypeptide. The topical application of the SEQ ID NO. 16 polypeptide was associated with increased skin elasticity as measured using a cutometer. As shown in fig. 6, 100% of subjects showed improvement in skin elasticity at 2 weeks (fig. 6; "B") and 4 weeks (fig. 6; "C") compared to baseline (fig. 6; "a").

Topical application of human type 21 collagen alpha 1 is associated with increased facial skin collagen content

In clinical studies (n=15 subjects), subjects used 8 weeks after a washout period of 1 week protein-free basal facial essence, a topical facial essence containing 0.1% of the SEQ ID NO:16 polypeptide. The topical application of the SEQ ID NO. 16 polypeptide correlates with increased skin collagen as measured by SIAscope. As shown in FIG. 7, skin collagen content increased at 2 weeks (FIG. 7; "B") and 8 weeks (FIG. 7; "C") compared to baseline (FIG. 7; "A").

Topical application of human type 21 collagen alpha is associated with reduced facial skin redness

In clinical studies (n=15 subjects), subjects used 8 weeks after a washout period of 1 week protein-free basal facial essence, a topical facial essence containing 0.1% of the SEQ ID NO:16 polypeptide. As shown in FIG. 8, topical application of the SEQ ID NO:16 polypeptide correlated with a reduction in redness of the skin at 4 weeks (FIG. 8; "B") and 8 weeks (FIG. 8; "C") compared to baseline (FIG. 8; "A").

Topical application of human type 21 collagen alpha 1 is associated with reduction of facial wrinkles

In clinical studies (n=15 subjects), subjects used 8 weeks after a washout period of 1 week protein-free basal facial essence, a topical facial essence containing 0.1% of the SEQ ID NO:16 polypeptide. As shown in FIG. 9, local application of the SEQ ID NO:16 polypeptide correlated with reduction of facial wrinkles at 4 weeks (FIG. 9; "B") and 8 weeks (FIG. 9; "C") compared to baseline (FIG. 9; "A").

EXAMPLE 6 in vitro investigation of truncated jellyfish collagen on skin cells

Truncated jellyfish collagen stimulates skin cells to produce type I collagen

A series of in vitro experiments were performed to assess the effect of truncated jellyfish collagen on human skin fibroblasts and keratinocytes. After 48 hours of treatment with the polypeptide of SEQ ID NO. 5, collagen type I secretion was assessed in an in vitro full-thickness human skin tissue model (MatTek) containing fibroblasts and keratinocytes. The tissue model was then rinsed and incubated with fresh medium for an additional 48 hours (96 hour time point). Culture supernatants were analyzed by ELISA for type I procollagen C-peptide (reading of total secreted type I collagen). As shown in FIG. 10, the tissue model treated with the SEQ ID NO 5 polypeptide (FIG. 10; "A") secreted higher levels of type I collagen than the untreated tissue model (FIG. 10; "B") or the tissue model treated with the positive control vitamin B3 (FIG. 10; "C").

Truncating jellyfish collagen reduces DNA damage in keratinocytes after exposure to UVB light

In a further study, 25mJ/cm was used ² The human primary keratinocytes were irradiated with UVB light and then incubated overnight in a medium containing 0.03% of the polypeptide of SEQ ID NO. 5. DNA was extracted from cells and assayed for levels of thymidine dimer (an indicator of DNA damage) using an oxselect UV-induced DNA damage ELISA kit. As shown in FIG. 11, cells treated with the SEQ ID NO. 5 polypeptide (FIG. 11; "B") showed lower levels of thymidine dimer than untreated cells (FIG. 11; "A"), and therefore less DNA damage.

Truncating jellyfish collagen increases cell viability of UVB-irradiated keratinocytes

Human primary keratinocytes were irradiated with 40mJ/cm2 UVB light and then incubated in a medium containing 0.03% of the polypeptide of SEQ ID No. 5 for 48 hours. Cell viability was assessed using an MTT metabolic colorimetric assay. As shown in FIG. 12, UVB-irradiated keratinocytes treated with the polypeptide of SEQ ID NO:5 (FIG. 12; "B") showed higher cell viability than untreated UVB-irradiated keratinocytes (FIG. 12; "A").

Truncating jellyfish collagen increases cell viability of keratinocytes exposed to urban dust pollution

To test protection against municipal dust, human primary keratinocytes were pretreated with 0.03% of the SEQ ID NO:5 polypeptide for 24 hours and then exposed to 2mg/ml municipal dust (NIST 1649B) for 24 hours. Cell viability was assessed using an MTT metabolic colorimetric assay. As shown in FIG. 13, keratinocytes pretreated with the SEQ ID NO:5 polypeptide (FIG. 13; "B") showed higher cell viability after urban dust exposure than untreated keratinocytes exposed to urban dust (FIG. 13; "A").

Shortening jellyfish collagen reduces inflammation of UVB-irradiated keratinocytes

In a further study of the in vitro full-thickness human skin tissue model (MatTek), 300mJ/cm was used ² UVB light irradiates the MatTek tissue model and is then treated with 0.01% of the SEQ ID No. 5 polypeptide for 24 hours. The levels of the pro-inflammatory cytokine IL-1α were determined by ELISA. As shown in FIG. 14, the tissue model treated with the SEQ ID NO 5 polypeptide (FIG. 14; "A") showed lower levels of IL-1α than the untreated UVB-irradiated control tissue model (FIG. 14; "B").

Truncated jellyfish collagen has antioxidant effect

The polypeptide of SEQ ID No. 5 was also evaluated in the ORAC assay. As shown in FIG. 15, a 0.1% solution of the SEQ ID NO:5 polypeptide has antioxidant properties equivalent to 193. Mu.M Trolox.

EXAMPLE 7 human clinical study of truncated jellyfish collagen

Topical application of truncated jellyfish collagen is associated with facial skin moisture increase

In clinical studies (n=18 subjects), subjects used topical facial extracts containing 0.05% of the SEQ ID NO:5 polypeptide for 2 weeks. As shown in FIG. 16, local application of the SEQ ID NO:5 polypeptide correlated with increased skin hydration at 1 week (FIG. 16; "A2") and 2 weeks (FIG. 16; "A3") compared to baseline (FIG. 16; "A1"). It was also demonstrated that topical application of the SEQ ID NO 5 polypeptide increased skin hydration relative to topical application of marine collagen at baseline (FIG. 16; "B1"), 1 week (FIG. 16; "B2"), and 2 weeks (FIG. 16; "B3").

Local application of truncated jellyfish collagen is associated with an increase in facial skin elasticity

In clinical studies (n=18 subjects), subjects used topical facial extracts containing 0.05% of the SEQ ID NO:5 polypeptide for 2 weeks. As shown in FIG. 17, local application of the SEQ ID NO:5 polypeptide correlated with increased skin elasticity measured using a cutometer at 1 week (FIG. 17; "B") and 2 weeks (FIG. 17; "C") compared to baseline (FIG. 17; "A").

Example 8 in vitro abrasion of skin cells by human type 21 collagen in combination with retinol, ascorbic acid and hyaluronic acid Study of

Human type 21 collagen α1 in combination with hyaluronic acid synergistically increases type I collagen production

Human full-thickness skin equivalents containing keratinocytes and fibroblasts (MatTek Epiderm FT co-cultured tissue model) were treated with a combination of 0.1% w/w truncated human type 21 collagen polypeptide (having the amino acid sequence of SEQ ID NO: 16) ("C21"; FIG. 18) with retinol (10. Mu.M), ascorbic acid (2% w/w) or hyaluronic acid (0.1% w/w) (low molecular weight, 15-40 kDa). The medium was changed every 2 days. On day 4, the supernatant was collected and analyzed for type I procollagen C-peptide by enzyme-linked immunosorbent assay (ELISA), which is a reading of total secreted type I collagen. As shown in fig. 18, cells treated with truncated human type 21 collagen polypeptide, retinol, ascorbic acid or hyaluronic acid alone increased type I collagen production compared to untreated cells. The combination of truncated human type 21 collagen polypeptide with hyaluronic acid increased type I collagen production by 171% relative to an untreated control. This appears to be a synergy, as treatment with truncated human type 21 collagen polypeptide alone increased type I collagen production by 64%, while treatment with hyaluronic acid alone increased type I collagen production by 72%.

Combination of human type 21 collagen α1 with hyaluronic acid increases fibronectin production

Human full-thickness skin equivalents containing keratinocytes and fibroblasts (MatTek Epiderm FT co-cultured tissue model) were treated with a combination of 0.1% w/w truncated human type 21 collagen polypeptide (having the amino acid sequence of SEQ ID NO: 16) ("C21"; FIG. 19) with retinol (10. Mu.M), ascorbic acid (2% w/w) or hyaluronic acid (0.1% w/w) (low molecular weight, 15-40 kDa). The medium was changed every 2 days. On day 4, supernatants were collected and analyzed for fibronectin by enzyme-linked immunosorbent assay (ELISA). As shown in fig. 19, cells treated with truncated human type 21 collagen polypeptide, retinol, ascorbic acid or hyaluronic acid alone increased fibronectin production compared to untreated cells. The combination of truncated human type 21 collagen polypeptide with hyaluronic acid increased fibronectin production by 103% relative to an untreated control. This appears to be additive because treatment with truncated human type 21 collagen polypeptide increased fibronectin production by 67% while treatment with hyaluronic acid alone increased fibronectin production by 30%.

Human type 21 collagen alpha 1 in combination with ascorbic acid or retinol increases elastin production

Human full-thickness skin equivalents containing keratinocytes and fibroblasts (MatTek Epiderm FT co-cultured tissue model) were treated with a combination of 0.1% w/w truncated human type 21 collagen polypeptide (having the amino acid sequence of SEQ ID NO: 16) ("C21"; FIG. 20) with retinol (10. Mu.M), ascorbic acid (2% w/w) or hyaluronic acid (0.1% w/w) (low molecular weight, 15-40 kDa). On day 2, supernatants were collected and analyzed for elastin by enzyme-linked immunosorbent assay (ELISA). As shown in fig. 20, cells treated with truncated human type 21 collagen polypeptide, retinol, ascorbic acid or hyaluronic acid alone increased elastin production compared to untreated cells. The combination of truncated human type 21 collagen polypeptide with retinol increased elastin production by 45% relative to the untreated control. This appears to be a synergy, as treatment with truncated human type 21 collagen polypeptide alone increases elastin production by 20% whereas treatment with retinol alone increases elastin production by 17%. The combination of truncated human type 21 collagen polypeptide with ascorbic acid increased elastin production by 33% relative to the untreated control. This appears to be a synergy in that treatment with truncated human type 21 collagen polypeptide alone increases elastin production by 20% whereas treatment with ascorbic acid alone increases elastin production by 5%. Combination of human type 21 collagen α1 with ascorbic acid increases type III collagen production

Human full-thickness skin equivalents containing keratinocytes and fibroblasts (MatTek Epiderm FT co-cultured tissue model) were treated with a combination of 0.1% w/w truncated human type 21 collagen polypeptide (having the amino acid sequence of SEQ ID NO: 16) ("C21"; FIG. 21) with retinol (10. Mu.M), ascorbic acid (2% w/w) or hyaluronic acid (0.1% w/w) (low molecular weight, 15-40 kDa). On day 2, the supernatant was collected and analyzed for procollagen type III N-peptide by enzyme-linked immunosorbent assay (ELISA), which is a reading of total secreted type III collagen. As shown in fig. 21, cells treated with truncated human type 21 collagen polypeptide, retinol, ascorbic acid or hyaluronic acid alone increased type III collagen production compared to untreated cells. The combination of truncated human type 21 collagen polypeptide with ascorbic acid increased type III collagen production by 122% relative to untreated controls, which was greater than treatment with truncated human type 21 collagen polypeptide alone (82%) or ascorbic acid (80%).

EXAMPLE 9 truncating human type 21 collagen polypeptide and hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzene peroxide In vitro test results of combinations of formyl, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid)

The combination of truncated human type 21 collagen polypeptides with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) increases the viability and proliferation of keratinocytes and fibroblasts.

In vitro studies, cultures of (e.g., human) skin cells, (e.g., human) skin equivalent cultures or ex vivo (e.g., human) skin are treated with truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Treatment with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation of keratinocytes and fibroblasts (e.g., as measured by, for example, MTT viability assays) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids alone.

The combination of truncated human type 21 collagen polypeptides with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) increases the production of extracellular matrix components and reduces the production of matrix degrading proteins.

In vitro studies, cultures of (e.g., human) skin cells, (e.g., human) skin equivalent cultures or ex vivo (e.g., human) skin are treated with truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergistically, additively) the production of extracellular matrix components (e.g., collagen, elastin, fibronectin, fibrillin, hyaluronic acid) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone (e.g., as measured by, for example, transcriptome analysis, ELISA). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) the production of matrix degrading proteins (e.g., matrix metalloproteinases, proteases) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone (e.g., glycolic acid, lactic acid) (e.g., as measured by, e.g., transcriptome analysis, ELISA).

Combination of truncated human type 21 collagen polypeptides with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) improves wound healing

In vitro studies, cultures of (e.g., human) skin cells, (e.g., human) skin equivalent cultures or ex vivo (e.g., human) skin are treated with truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) enhances (e.g., synergizes, accumulates) the wound healing capacity (e.g., as measured by, for example, a wound healing scratch assay) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone.

Combination of truncated human type 21 collagen polypeptides with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) has improved results following photodamage challenge

In vitro studies, cultures of (e.g., human) skin cells are challenged with light damage (e.g., UV irradiation, blue light exposure), cultures of (e.g., human) skin equivalents, or ex vivo (e.g., human) skin, and then treated with truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). In contrast to treatment with truncated human type 21 collagen alone, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid), treatment with truncated human type 21 collagen polypeptides (e.g.,the amino acid sequence having SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation (e.g., as measured by, for example, MTT viability assay) of skin cells (e.g., keratinocytes, fibroblasts). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) cytotoxicity and apoptosis (e.g., as measured by, e.g., fluorescence microscopy) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone. Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) enhances (e.g., synergizes, accumulates) DNA repair (e.g., as measured by, e.g., thymidine dimer ELISA) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone. Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) inflammation (e.g., as measured by, for example, ELISA assays or Luminex detection of cytokines) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. Relative to truncated human type 21 collagen alone, ascorbic acid, retinol, and transdermal agent Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergistically, cumulatively) reactive oxidative stress (e.g., as by, for example, CM-H ₂ Measured by DCFDA detection).

Combination of truncated human type 21 collagen polypeptides with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) has improved results after contamination challenge

In vitro studies, cultures of (e.g., human) skin cells, cultures of (e.g., human) skin equivalents, or isolated (e.g., human) skin are challenged with contamination (e.g., urban dust exposure), and then treated with truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation (e.g., as measured by, for example, MTT viability assays) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone. Treatment with truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid alone (e.g. Such as glycolic acid, lactic acid) reduces (e.g., cooperates with, accumulates) cytotoxicity and apoptosis (e.g., as measured by, e.g., fluorescence microscopy) of skin cells (e.g., keratinocytes, fibroblasts). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) enhances (e.g., synergizes, accumulates) DNA repair (e.g., as measured by, e.g., thymidine dimer ELISA) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone. Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) inflammation (e.g., as measured by, for example, ELISA assays or Luminex detection of cytokines) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergistically, accumulates) reactive oxidative stress (e.g., as by, for example, CM-H ₂ Measured by DCFDA detection).

The combination of truncated human type 21 collagen polypeptides with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) has improved results after challenge with a highly irritating skin active substance.

In vitro studies, skin cell cultures (e.g., human), skin equivalent cultures (e.g., human), or ex vivo (e.g., human) skin are challenged with a skin active substance that is highly irritating (e.g., retinoic acid or retinol, benzoyl peroxide, salicylic acid), and then treated with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation (e.g., as measured by, for example, MTT viability assays) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone. Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) cytotoxicity and apoptosis (e.g., as measured by, e.g., fluorescence microscopy) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid alone. Treatment with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) provides for the extraction relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone DNA repair (e.g., as measured by, for example, thymidine dimer ELISA detection) of high (e.g., synergistic, additive) skin cells (e.g., keratinocytes, fibroblasts). Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) inflammation (e.g., as measured by, for example, ELISA assays or Luminex detection of cytokines) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone. Treatment with a truncated human type 21 collagen polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergistically, accumulates) reactive oxidative stress (e.g., as by, for example, CM-H ₂ Measured by DCFDA detection).

EXAMPLE 10 truncated human collagen type 21 polypeptide, hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzene peroxide Clinical test results of combinations of formyl, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid)

Topical application of truncated human type 21 collagen polypeptides in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) improves skin appearance and quality

In clinical studies, subjects used topical formulations of truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Subjects treated with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) exhibit reduced wrinkles and fine lines, reduced skin redness and pigmentation/hyperpigmentation, increased skin brightness, reduced pore size, reduced skin roughness, and/or reduced acne relative to subjects treated with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids alone (e.g., glycolic acid, lactic acid).

Subjects treated with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) exhibit increased skin elasticity, increased skin firmness, increased skin hydration, increased skin barrier function, increased skin collagen and elastin content, and/or increased dermis density relative to subjects treated with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone.

Subjects treated with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) exhibited an improvement in the microflora of the skin (e.g., decreased abundance of harmful microorganisms, promotion of beneficial microorganisms, and/or increased diversity of microorganisms) relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone.

EXAMPLE 11 truncated human collagen type 21 polypeptide, hyaluronic acid, retinol, ascorbic acid, salicylic acid, benzene peroxide Improvement of combinations of formyl, nicotinamide or alpha hydroxy acids (e.g. glycolic acid, lactic acid)Hair quality and appearance.

In clinical studies, subjects used compositions of truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) formulated for hair alone or in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Subjects treated with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) exhibited improved hair growth, thickened hair fiber diameter, increased combability, reduced hair loss, increased growth rate, and/or increased hair tensile strength relative to subjects treated with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone.

Subjects treated with truncated human type 21 collagen polypeptides (e.g., having the amino acid sequence of SEQ ID NO: 16) in combination with ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) exhibited improved hair fiber thickness and density, increased moisture, increased hydrophobicity, reduced hair tip bifurcation, reduced frizz/improved static control, fiber alignment/gloss, increased wet comb/dry comb and/or greater resistance to hair breakage relative to treatment with truncated human type 21 collagen, ascorbic acid, retinol, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) alone.

EXAMPLE 12.25kDa truncated human elastin

Non-naturally occurring truncated human elastin without His tag, linker and/or thrombin cleavage site is disclosed below. The non-naturally occurring truncated human elastin is truncated relative to the full length human elastin (SEQ ID NO: 53). The codon-optimized nucleotide and amino acid sequences encoding the elastin are disclosed below. In SEQ ID NO. 37, the DsbA secretion tag is encoded by nucleotides 1 to 57 and encodes amino acids 1 to 19 of SEQ ID NO. 38. In SEQ ID NO. 37, the non-naturally occurring truncated human elastin sequence is encoded by nucleotides 58 to 927 and encodes amino acids 20 to 309 of SEQ ID NO. 38.

The codon-optimized nucleotide sequence encoding the non-naturally occurring truncated elastin is provided in SEQ ID NO. 37.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGGTCCGCAACCTGGGGTTCCGTTAGGTTATCCGATTAAAGCACCGAAACTGCCCGGCGGTTATGGTCTGCCGTACACAACCGGTAAACTGCCGTATGGTTATGGCCCGGGTGGAGTTGCGGGTGCAGCAGGTAAAGCGGGTTATCCTACCGGAACCGGTGTAGGTCCGCAGGCCGCTGCTGCCGCCGCCGCAAAAGCAGCGGCTAAATTTGGCGCCGGAGCAGCGGGTGTTCTGCCTGGAGTTGGTGGTGCGGGCGTGCCAGGGGTACCTGGTGCAATTCCGGGTATTGGTGGTATTGCCGGTGTCGGCACCCCGGCCGCGGCAGCTGCGGCAGCGGCGGCTGCCAAAGCTGCTAAATACGGTGCCGCGGCGGGTCTGGTGCCAGGAGGTCCGGGTTTTGGTCCGGGAGTGGTTGGCGTGCCTGGCGCAGGCGTTCCTGGTGTGGGCGTTCCAGGTGCAGGGATTCCTGTTGTGCCTGGTGCCGGTATTCCCGGCGCGGCCGTTCCGGGGGTGGTTAGCCCGGAAGCCGCAGCGAAGGCTGCGGCAAAGGCAGCAAAGTATGGCGCACGCCCAGGAGTCGGCGTGGGTGGTATCCCGACCTATGGGGTGGGCGCAGGGGGTTTTCCTGGTTTCGGCGTAGGTGTAGGAGGTATACCGGGCGTGGCCGGTGTACCAGGGGTTGGTGGCGTCCCTGGTGTTGGCGGTGTGCCAGGTGTTGGTATTTCACCGGAAGCACAGGCAGCAGCCGCAGCTAAGGCAGCGAAATATGGTGCCGCCGGCGCAGGAGTTTTAGGTGGGCTGGTTCCGGGCCCGCAGGCAGCTGTGCCGGGGGTTCCAGGCACCGGTGGTGTCCCTGGAGTCGGTACGCCGTAA(SEQ ID NO:37)

The amino acid sequence of a 25kDa non-naturally occurring truncated human elastin sequence comprising the DsbA secretion signal is disclosed in SEQ ID NO. 38.

MKKIWLALAGLVLAFSASAGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGPQAAVPGVPGTGGVPGVGTP(SEQ ID NO:38)

A codon-optimized nucleotide sequence encoding a non-naturally occurring truncated human elastin without a DsbA secretion tag is provided in SEQ ID NO 39.

GGTCCGCAACCTGGGGTTCCGTTAGGTTATCCGATTAAAGCACCGAAACTGCCCGGCGGTTATGGTCTGCCGTACACAACCGGTAAACTGCCGTATGGTTATGGCCCGGGTGGAGTTGCGGGTGCAGCAGGTAAAGCGGGTTATCCTACCGGAACCGGTGTAGGTCCGCAGGCCGCTGCTGCCGCCGCCGCAAAAGCAGCGGCTAAATTTGGCGCCGGAGCAGCGGGTGTTCTGCCTGGAGTTGGTGGTGCGGGCGTGCCAGGGGTACCTGGTGCAATTCCGGGTATTGGTGGTATTGCCGGTGTCGGCACCCCGGCCGCGGCAGCTGCGGCAGCGGCGGCTGCCAAAGCTGCTAAATACGGTGCCGCGGCGGGTCTGGTGCCAGGAGGTCCGGGTTTTGGTCCGGGAGTGGTTGGCGTGCCTGGCGCAGGCGTTCCTGGTGTGGGCGTTCCAGGTGCAGGGATTCCTGTTGTGCCTGGTGCCGGTATTCCCGGCGCGGCCGTTCCGGGGGTGGTTAGCCCGGAAGCCGCAGCGAAGGCTGCGGCAAAGGCAGCAAAGTATGGCGCACGCCCAGGAGTCGGCGTGGGTGGTATCCCGACCTATGGGGTGGGCGCAGGGGGTTTTCCTGGTTTCGGCGTAGGTGTAGGAGGTATACCGGGCGTGGCCGGTGTACCAGGGGTTGGTGGCGTCCCTGGTGTTGGCGGTGTGCCAGGTGTTGGTATTTCACCGGAAGCACAGGCAGCAGCCGCAGCTAAGGCAGCGAAATATGGTGCCGCCGGCGCAGGAGTTTTAGGTGGGCTGGTTCCGGGCCCGCAGGCAGCTGTGCCGGGGGTTCCAGGCACCGGTGGTGTCCCTGGAGTCGGTACGCCGTAA(SEQ ID NO:39)

The amino acid sequence of a non-naturally occurring truncated human elastin without a DsbA secretion tag is disclosed in SEQ ID No. 40.

GPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGPQAAVPGVPGTGGVPGVGTP(SEQ ID NO:40)

The polynucleotide of SEQ ID NO. 37 was synthesized by internal DNA synthesis of Gen9 DNA (now Ginkgo Bioworks). The overlap between pET28 vector and SEQ ID NO. 37 and SEQ ID NO. 39 was designed to be 20 to 30bp long, and PCR were usedGXL polymerase (www.takarabio.com/products/pcr/gc-rich-pcr/primestar-GXL-dna-polymerase) was added. The cut pET28a vector was then assembled with insert DNA (SEQ ID NO: 37) into the final plasmid using seamless cloning (www.takarabio.com/products/cloning/in-fusion-cloning). Then by Genewiz (www.genewiz.com/en)Sanger sequencing to verify the sequence of the plasmid.

Transformed cells were cultured in minimal medium and frozen with plant glycerol in a ratio of 50:50 cells to glycerol in 1.5mL aliquots. One vial of frozen culture was resuscitated overnight in 50mL of minimal medium at 37 ℃ at 200 rpm. Cells were transferred to 300mL of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10. Minimal medium was prepared according to the minimal medium formulation described in example 2 above.

The fermentation is carried out at various temperatures ranging from 25 ℃ to 28 ℃. For some fermentations, the temperature of the fermentation is maintained at a constant temperature. For other fermentations, the fermentation temperature is maintained for a desired period of time and when the cell density reaches an OD600 of 10-20, the temperature is reduced to induce protein production. Typically, the temperature is reduced from 28 ℃ to 25 ℃; fermentation was continued at 25℃for 40-60 hours.

Purified non-naturally occurring truncated elastin was analyzed on an SDS-PAGE gel and a clear band of expected size 25 kilodaltons was observed at the position where there was no band in the control strain.

EXAMPLE 13.21kDa truncated human elastin

Non-naturally occurring truncated human elastin was synthesized according to the method of example 12.

The amino acid sequence of a 21kDa non-naturally occurring truncated human elastin truncated at the N-terminus relative to the full length human elastin (SEQ ID NO: 53) is disclosed in SEQ ID NO: 41. The 21kDa non-naturally occurring truncated human elastin has amino acids 1-522 deleted from the full-length human elastin (SEQ ID NO: 53).

GPGGVAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAGADEGVRRSLSPELREGDPSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK(SEQ ID NO:41)。

A codon-optimized polynucleotide sequence encoding a 21kDa non-naturally occurring truncated human elastin is disclosed in SEQ ID NO. 42.

GGTCCGGGCGGTGTCGCAGCAGCAGCTAAAAGCGCGGCGAAAGTTGCGGCCAAAGCCCAACTGCGCGCCGCCGCGGGCCTCGGTGCAGGTATTCCGGGGCTGGGTGTCGGAGTTGGAGTCCCGGGTTTGGGCGTGGGCGCGGGAGTTCCGGGACTGGGAGTGGGTGCCGGAGTTCCTGGCTTTGGTGCAGGCGCAGATGAAGGTGTTCGTCGTAGCCTGAGTCCGGAACTGCGTGAAGGTGATCCGAGTAGCAGCCAGCATCTGCCGAGCACCCCGAGCAGCCCGCGTGTTCCGGGTGCATTAGCTGCAGCAAAAGCCGCCAAGTATGGTGCAGCCGTGCCGGGCGTCTTAGGTGGTCTGGGCGCCCTGGGTGGTGTAGGCATTCCGGGAGGTGTTGTGGGTGCAGGACCGGCCGCCGCAGCTGCGGCCGCCAAAGCAGCTGCAAAAGCGGCCCAGTTTGGTTTAGTGGGCGCCGCAGGTTTAGGCGGTTTAGGTGTGGGTGGACTGGGTGTACCTGGCGTAGGCGGTCTGGGTGGAATTCCGCCCGCAGCGGCCGCGAAAGCGGCAAAATATGGCGCGGCAGGCCTGGGCGGCGTGCTGGGTGGGGCAGGTCAGTTTCCGCTGGGCGGGGTTGCCGCACGTCCGGGATTTGGTCTGAGCCCGATTTTCCCTGGCGGCGCATGTCTGGGTAAAGCATGTGGTCGTAAACGTAAATAA(SEQ ID NO:42)

Cells were cultured and expressed the polynucleotide of SEQ ID NO. 42 as described in example 12.

EXAMPLE 14.60kDa truncated human elastin

The amino acid sequence of a 60.3kDa non-naturally occurring truncated human elastin relative to the internal truncation of the full length human elastin (SEQ ID NO: 53) is disclosed in SEQ ID NO: 43. The 60.3kDa non-naturally occurring truncated human elastin has amino acids 461-527 deleted from the full length human elastin (SEQ ID NO: 53).

GGVPGAIPGGVPGGVFYPGAGLGALGGGALGPGGKPLKPVPGGLAGAGLGAGLGAFPAVTFPGALVPGGVADAAAAYKAAKAGAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGPQAAVPGVPGTGGVPGVGTPAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAGADEGVRRSLSPELREGDPSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK(SEQ ID NO:43)

A codon-optimized polynucleotide sequence encoding a 60.3kDa non-naturally occurring truncated human elastin is disclosed in SEQ ID NO. 44.

GGTGGCGTACCAGGCGCAATTCCTGGGGGTGTCCCAGGCGGTGTTTTTTATCCGGGCGCCGGTCTTGGCGCACTGGGTGGCGGTGCACTGGGCCCGGGCGGCAAACCGCTGAAACCGGTACCAGGTGGTTTAGCAGGCGCCGGCTTAGGCGCAGGTCTGGGAGCATTTCCGGCAGTTACCTTTCCAGGGGCACTGGTTCCTGGAGGTGTGGCCGATGCAGCCGCGGCATATAAAGCCGCTAAAGCCGGTGCGGGTTTAGGAGGCGTCCCAGGTGTCGGTGGCCTGGGTGTTAGCGCCGGTGCAGTTGTTCCGCAGCCGGGAGCAGGGGTTAAACCTGGTAAAGTGCCGGGAGTAGGTCTGCCAGGCGTTTATCCTGGTGGTGTTTTGCCGGGTGCCCGTTTTCCGGGCGTTGGTGTTCTTCCAGGCGTGCCG

ACCGGAGCCGGTGTTAAACCGAAAGCCCCCGGTGTTGGAGGTG

CATTTGCAGGCATCCCGGGAGTTGGCCCGTTTGGTGGTCCGCAA

CCTGGGGTTCCGTTAGGTTATCCGATTAAAGCACCGAAACTGCC

CGGCGGTTATGGTCTGCCGTACACAACCGGTAAACTGCCGTATG

GTTATGGCCCGGGTGGAGTTGCGGGTGCAGCAGGTAAAGCGGG

TTATCCTACCGGAACCGGTGTAGGTCCGCAGGCCGCTGCTGCCG

CCGCCGCAAAAGCAGCGGCTAAATTTGGCGCCGGAGCAGCGGG

TGTTCTGCCTGGAGTTGGTGGTGCGGGCGTGCCAGGGGTACCTG

GTGCAATTCCGGGTATTGGTGGTATTGCCGGTGTCGGCACCCCG

GCCGCGGCAGCTGCGGCAGCGGCGGCTGCCAAAGCTGCTAAAT

ACGGTGCCGCGGCGGGTCTGGTGCCAGGAGGTCCGGGTTTTGGT

CCGGGAGTGGTTGGCGTGCCTGGCGCAGGCGTTCCTGGTGTGGG

CGTTCCAGGTGCAGGGATTCCTGTTGTGCCTGGTGCCGGTATTC

CCGGCGCGGCCGTTCCGGGGGTGGTTAGCCCGGAAGCCGCAGC

GAAGGCTGCGGCAAAGGCAGCAAAGTATGGCGCACGCCCAGGA

GTCGGCGTGGGTGGTATCCCGACCTATGGGGTGGGCGCAGGGG

GTTTTCCTGGTTTCGGCGTAGGTGTAGGAGGTATACCGGGCGTG

GCCGGTGTACCAGGGGTTGGTGGCGTCCCTGGTGTTGGCGGTGT

GCCAGGTGTTGGTATTTCACCGGAAGCACAGGCAGCAGCCGCA

GCTAAGGCAGCGAAATATGGTGCCGCCGGCGCAGGAGTTTTAG

GTGGGCTGGTTCCGGGCCCGCAGGCAGCTGTGCCGGGGGTTCC

AGGCACCGGTGGTGTCCCTGGAGTCGGTACGCCGGCAGCAGCA

GCTAAAAGCGCGGCGAAAGTTGCGGCCAAAGCCCAACTGCGCG

CCGCCGCGGGCCTCGGTGCAGGTATTCCGGGGCTGGGTGTCGG

AGTTGGAGTCCCGGGTTTGGGCGTGGGCGCGGGAGTTCCGGGA

CTGGGAGTGGGTGCCGGAGTTCCTGGCTTTGGTGCAGGCGCAG

ATGAAGGTGTTCGTCGTAGCCTGAGTCCGGAACTGCGTGAAGGT

GATCCGAGTAGCAGCCAGCATCTGCCGAGCACCCCGAGCAGCC

CGCGTGTTCCGGGTGCATTAGCTGCAGCAAAAGCCGCCAAGTATGGTGCAGCCGTGCCGGGCGTCTTAGGTGGTCTGGGCGCCCTGGGTGGTGTAGGCATTCCGGGAGGTGTTGTGGGTGCAGGACCGGCCGCCGCAGCTGCGGCCGCCAAAGCAGCTGCAAAAGCGGCCCAGTTTGGTTTAGTGGGCGCCGCAGGTTTAGGCGGTTTAGGTGTGGGTGGACTGGGTGTACCTGGCGTAGGCGGTCTGGGTGGAATTCCGCCCGCAGCGGCCGCGAAAGCGGCAAAATATGGCGCGGCAGGCCTGGGCGGCGTGCTGGGTGGGGCAGGTCAGTTTCCGCTGGGCGGGGTTGCCGCACGTCCGGGATTTGGTCTGAGCCCGATTTTCCCTGGCGGCGCATGTCTGGGTAAAGCATGTGGTCGTAAACGTAAATAA(SEQ ID NO:44)

Cells were cultured and expressed the polynucleotide of SEQ ID NO. 44 as described in example 12.

EXAMPLE 15 Effect of truncated elastin on fibroblast viability, procollagen Synthesis and tropoelastin Synthesis Sound box

Human fibroblast cultures were used to assess the ability of the non-naturally occurring truncated human elastin molecules of example 12 to affect procollagen and tropoelastin synthesis. Human fibroblast cultures were also used to determine the viability of human fibroblasts following exposure to truncated elastin.

A stock solution of 2% w/w truncated elastin was prepared from the truncated elastin of example 12. Aliquots from the 2% truncated elastin stock solution were then used in the experiments described below.

Preparation of fibroblasts

Fibroblasts were inoculated into 0.5mL of Fibroblast Growth Medium (FGM) in 24-well plate single wells at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate overnight. The next day, the medium was removed via aspiration to remove any non-adherent cells and replaced with 0.5mL fresh FGM. Cells were grown until confluence, with medium changes every 48 to 72 hours. After confluence was reached, the cells were treated with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1.5% Fetal Bovine Serum (FBS) for 24 hours to wash out any effects of growth factors included in normal medium. After a 24 hour elution period, cells were treated with a specified concentration of truncated elastin in FGM with 1.5% fbs. Transforming growth factor beta (TGF-beta) (20 ng/mL) was used as a positive control for collagen and elastin synthesis. Untreated cells (negative control) received DMEM with only 1.5% fbs. Cells were incubated for 48 hours and at the end of the incubation period, cell culture medium was collected and stored frozen (-75 ℃) or assayed immediately. The materials were tested in triplicate.

MTT assay

The MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide, a tetrazolium) assay is a colorimetric assay for determining the metabolic activity of cells. The change in cell number was assessed via an MTT assay. When cells are exposed to MTT, mitochondria in living cells reduce MTT, resulting in the formation of insoluble purple crystalline formazan, which is extracted from the cells with isopropanol and quantified spectrophotometrically. Non-viable cells are unable to reduce MTT and therefore are unable to produce purple crystalline formazan. The intensity of purple is directly proportional to the number of living cells (metabolically active cells). The intensity of purple is directly proportional to the metabolic activity of the cell and inversely proportional to the toxicity of the test material.

After 2 days of incubation as described above, the cell culture medium (see above) was removed and the fibroblasts were washed twice with Phosphate Buffer (PBS) to remove any remaining elastin molecules. After final washing, 500. Mu.L of DMEM supplemented with 0.5mg/ml MTT was added to each well and the cells were incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate for 1 hour. Incubation methodAfter incubation, the DMEM/MTT solution was removed, the cells were washed once more with PBS, and then 0.5mL of isopropanol was added to the wells to extract the purple crystalline formazan. 200 microliters of isopropyl extract was transferred to a 96-well plate and the plate was read at 540nm using isopropyl alcohol as a blank.

The mean MTT absorbance of the negative control cells was calculated and used to represent 100% cell viability. The individual MTT absorbance values of the cells subjected to each treatment were then divided by the average value of the negative control cells and expressed as a percentage to determine the change in cell viability caused by each treatment.

Procollagen synthesis

Fibroblasts are the major source of extracellular matrix peptides, including structural collagen and elastin. Procollagen is a large peptide synthesized by fibroblasts in the dermis layer of the skin and is a precursor of collagen. When the peptide is processed to form mature collagen, the propeptide portion is excised (type I C-peptide). The mature collagen and type I C-peptide fragments are then released into the extracellular environment. When collagen is synthesized, the type I C-peptide fragments accumulate in the tissue culture medium. Because of the 1:1 stoichiometric ratio between the two parts of the procollagen peptide, the determination of type I C-peptide reflects the amount of collagen synthesized. Type I C-peptide can be determined via an enzyme-linked immunosorbent assay (ELISA) based method.

A series of type I C-peptide standards ranging from 0ng/mL to 640ng/mL were prepared. Next, ELISA microplates were prepared by removing any unwanted strips from the plate frame, followed by adding 100. Mu.L of peroxidase-labeled anti-type I procollagen C peptide antibody to each well used for the assay. Then 20. Mu.L of sample (collected tissue culture medium) or standard is added to the appropriate wells, the microplate is covered and incubated for 3.+ -. 0.25 hours at 37 ℃. After incubation, wells were aspirated and washed three times with 400 μl wash buffer. After the last wash removal, 100 μl of peroxidase substrate solution (hydrogen peroxide+tetramethylbenzidine as chromophore) was added to each well and the plate was incubated for 15±5 minutes at room temperature. After incubation, 100 μl of stop solution (1N sulfuric acid) was added to each well and the plate read at 450nm using a microplate reader.

To quantify the amount of each species present, a standard curve is generated using the known concentrations of each species. Regression analysis was performed to establish a line that best fits the data points. The absorbance values of the test material and the untreated samples were used to estimate the amount of each species present in each sample.

Elastin synthesis

Elastin is the major component of the elastic fiber network that imparts to tissue the ability to rebound after brief stretching. The protein is released by fibroblasts (soluble elastin) into the extracellular space, where it then crosslinks with other elastin to form a broad network of fibers and lamellae (insoluble elastin). Soluble elastin can be readily measured from cell culture media via ELISA-based methods.

Soluble α -elastin was dissolved in 0.1M sodium carbonate (pH 9.0) at a concentration of 1.25 μg/mL. Then 150 μl of this solution was applied to wells of a 96-well Maxisorp Nunc plate and the plate was incubated overnight at 4 ℃. The next day, wells were filled with PBS containing 0.25% Bovine Serum Albumin (BSA) and 0.05% tween 20. Plates were then incubated with the blocking solution for 1 hour at 37 ℃ and washed twice with PBS containing 0.05% tween 20.

A set of α -elastin standards ranging from 0 to 100ng/mL was generated. 180. Mu.L of standard or truncated elastin was then transferred to a 650. Mu.L microcentrifuge tube. An anti-elastin antibody solution (antibody 1:100 diluted in PBS containing 0.25% BSA and 0.05% Tween 20) was prepared and 20. Mu.L of this solution was added to the tube. The tubes were then incubated overnight at 4.+ -. 2 ℃. The following day, 150 μl from each tube was transferred to a 96-well elastin ELISA plate and the plate incubated for 1 hour at room temperature. Plates were then washed 3 times with PBS containing 0.05% tween 20. After washing, 200 μl of a solution containing peroxidase-linked secondary antibody diluted in PBS containing 0.25% bsa and 0.05% tween 20 was added and the plate was incubated for 1 hour at room temperature. After washing the plates three times, 200 μl of substrate solution was added and the plates were incubated for 10 to 30 minutes in the dark at room temperature. After this final incubation, plates were read at 460nm using an microplate reader.

EXAMPLE 16 truncationsEffects of elastin on keratinocyte proliferation and UVB protection

Human keratinocyte culture models were used to assess the ability of test materials to exert an effect on cell proliferation. In addition, the effect of the test material on cell viability after UVB exposure was also assessed.

The study was performed in two parts. In the first part, cultured keratinocytes were incubated with the test material for 48 hours, after which the change in the number of living cells was assessed using the MTT assay. In the second part of the study, cultured keratinocytes were irradiated with UVB and then treated with the test material for 48 hours. At the end of the 48 hour period, the number of living cells was again assessed via the MTT assay.

The change in the number of living cells can be determined using an MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide, a tetrazolium) assay. MTT assay is a colorimetric assay of cellular metabolic activity that is a reflection of the number of living cells. Mitochondria in living cells reduce MTT, resulting in the formation of insoluble purple crystalline formazan, which is extracted from cells with isopropanol and quantified spectrophotometrically. The intensity of purple is directly proportional to the number of metabolically active cells.

Proliferation assay

For proliferation assays, keratinocytes were seeded into 96-well plates without growth factors and incubated at 37.+ -. 2 ℃ and 5% ± 1% co2 for 24 hours. After this initial incubation, the medium was replaced with medium supplemented with test material. Normal medium (containing growth factors) was used as positive control. After the addition of the test material, the cells were cultured for 48 hours as described above. At the end of the incubation period, the change in viable cell number was determined using the MTT assay.

UVB protection assay

For UVB protection assays, keratinocytes were seeded into 96-well plates using normal medium and at 37.+ -. 2 ℃ and 5%±1％CO ₂ Incubate for 24 hours. After this initial incubation, the medium was replaced with 100. Mu.L of Phosphate Buffer (PBS) and the cells were exposed to UVB (40 mJ/cm) ² ). After UVB exposure, PBS was replaced with fresh medium supplemented with test material (100. Mu.g/mL ascorbic acid was used as positive control), and cells were incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ The culture was performed for 48 hours. At the end of 48 hour incubation, the MTT assay was used to determine cell viability.

Example 17 Effect of truncated elastin on thymine dimer formation

After exposure to ultraviolet radiation, the thymine dimer (TT dimer) content of the DNA present in the cells increases. Increased TT dimer formation is associated with skin damage and certain types of cell proliferative diseases, including skin cancer.

The truncated elastin of example 12 was tested to determine if it could reduce TT dimer formation in human epidermal keratinocytes. Human keratinocytes were inoculated into 12-well plates using normal medium and incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate for 24 hours. After this initial incubation, the medium was replaced with 100. Mu.L of Phosphate Buffer (PBS) and the cells were exposed to UVB (25 mJ/cm) ² ). After UVB exposure, PBS was replaced with fresh medium supplemented with test material or Trolox (100. Mu.g/ml, positive control), and cells were incubated at 37.+ -. 2 ℃ and 5%. + -. 1% CO ₂ Incubate overnight. At the end of incubation, cellular DNA was extracted and assayed for thymidine dimer content using an ELISA-based method.

After overnight incubation, the cell culture medium was removed from the wells and replaced with 200 μl PBS and 20 μl proteinase K. After rotating the plate to mix PBS and proteinase K, 200 μl of buffer AL was added to each well. After again rotating the plate to mix the reagents, the plate was incubated at 55±2 ℃ for 10 minutes. After cooling the plate to room temperature, 200. Mu.L of 100% ethanol was added to precipitate the DNA. The precipitated DNA mixture was then transferred to a DNeasy adsorption column in a 2mL collection tube and centrifuged at 8,000RPM for 1 minute. The flow-through tube and collection tube were discarded, 500. Mu.L of wash buffer 1 was added to the adsorption column, and the column was placed in a new collection tube and centrifuged at 8,000RPM for 1 minute. The flow-through tube and collection tube were again discarded, 500. Mu.L of wash buffer 2 was added to the adsorption column, and the column was placed in a new collection tube and centrifuged at 14,000RPM for 3 minutes. The adsorption column was then placed in a new 1.5mL centrifuge tube, and 110. Mu.L of ultrapure water was added to the column. The column was incubated for 1 min at room temperature and then centrifuged at 8,000RPM for 1 min.

The extracted DNA was quantified via a fluorometry. mu.L aliquots of DNA samples were mixed with 100. Mu.L Tris-EDTA (TE) buffer in 96-well plates. A series of DNA standards were also transferred to wells of a 96-well plate (in duplicate). Finally, 100 μl of diluted CyQUANT green dye was added to each well, and the fluorescence intensity of each well was determined using an excitation wavelength of 480nm and an emission wavelength of 520 nm.

An OxiSelect can be used ^TM The UV-induced DNA damage ELISA kit was used to determine thymine dimer detection.

An aliquot of the genomic DNA sample or standard was converted to single stranded DNA by incubating the sample at 95 ℃ for 10 minutes, followed by cooling on ice. 100. Mu.L of each sample or standard was transferred to a DNA binding ELISA plate and incubated overnight at 4 ℃. The next day, wells were rinsed once with 100 μl PBS and then blocked with 150 μl assay diluent for one hour at room temperature. After removal of assay diluent, 100 μl of anti-CPD antibody was added to each well and the plate was incubated for one hour at room temperature. After incubation, the plates were washed three times with 250 μl wash buffer per well, and then 150 μl blocking reagent was added to the plates. The plate was closed again for one hour at room temperature and then washed three times as before. Then 100 μl of secondary antibody was added to each well and the plate was incubated for 1 hour at room temperature. After washing the plate again, 100 μl of substrate was added to each well and the plate was incubated for 5-20 minutes to allow color to develop in the plate. The color-generating reaction was stopped by adding 100. Mu.L of stop solution and the plate was read at 460nm using a microplate reader.

To quantify the amount of DNA present, a standard curve is generated using the known concentrations of DNA and their respective fluorescence intensities (measured in RFU or relative fluorescence units). Regression analysis is performed to establish a line of best fit data points. The Relative Fluorescence Units (RFU) of each unknown sample are then used to estimate the amount of DNA.

A series of DNA standards of known thymine dimer content were used to generate a standard curve. The standard curve is used to determine the amount of DNA damage in the sample DNA. Average values for each treatment group were calculated and compared using ANOVA.

EXAMPLE 18 protection of fibroblast cells by truncated human elastin

The effect of non-naturally occurring truncated human elastin on fibroblast viability, procollagen synthesis and elastin synthesis was determined according to the method of example 15.

The effect of non-naturally occurring truncated human elastin on keratinocyte proliferation and UVB protection was determined according to the method of example 16.

The effect of non-naturally occurring truncated human elastin on thymine dimer formation after exposure to UV radiation was determined according to the method of example 17.

Example 19 Effect of truncated elastin on inflammatory cytokines

Keratinocytes and dermal fibroblasts play an important role in the immune response of the skin. Keratinocytes can release a large amount of cytokines in response to irritant chemicals or UV radiation (pro-inflammatory/pro-stimulatory stimulus). These cytokines are thought to help direct immune cells to the site of inflammation. Cytokines released by keratinocytes include TNF alpha, IL-1 beta, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18 and IL-1RA.

The test model used in this study was MatTek EpiDerm. This skin model consists of normal human-derived epidermal keratinocytes which are cultured to form a multi-layered, highly differentiated human epidermis model. Ultrastructural analysis reveals the presence of transparent stratum corneum particles, tensioned microfilaments, desmosomes and multilamellar stratum corneum containing intercellular lamellar lipid layers arranged in a pattern characteristic of the epidermis in the body. Markers of mature epidermis specific differentiation have been located in this model, such as the filaggrin, the K1/K10 cytokeratin pair, endo-coat proteins and type I epidermis transglutaminase. MatTek EpiDerm also has mitotic and metabolic activity.

MatTek EpiDerm tissues were used to evaluate the ability of various test materials to inhibit the release of inflammatory mediator IL-1. Alpha. The test material was compared to non-prescription topical hydrocortisone formulation (positive control), untreated tissue (negative control 1) and untreated non-inflamed tissue (negative control 2). The test is also used to assess the viability of the tissue after exposure to the test material.

IL-1α, IL-6 and IL-8 are synthesized and stored in keratinocytes and have been identified as mediators of skin irritation and inflammation. The release of these cytokines can be measured directly in tissue culture medium via a colorimetric-based enzyme-linked immunosorbent assay (ELISA). Briefly, antibodies covalently attached to a solid support can bind IL-1α, IL-6, or IL-8 present in old media samples. Secondary antibodies covalently attached to acetylcholinesterase can in turn detect specifically bound cytokines. Upon addition of a substrate of suitable color, acetylcholinesterase can produce a colored end product that can be measured spectrophotometrically.

MatTek EpiDerm tissue was purchased from MatTek corporation and stored at 4℃until use. Prior to use, the tissue to be used is removed from the agarose transport tray and placed in a 6-well plate containing 0.9mL of hydrocortisone free test medium (37.+ -. 2 ℃). The tissue was incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate overnight. After this initial incubation, the test medium was replaced with 0.9mL of fresh hydrocortisone free medium (37.+ -. 2 ℃). Three tissues were prepared for each test material.

Inflammatory responses in tissues are triggered via UV irradiation (UVB). 300mJ/cm of tissue was administered using UV lamps ² Dose of UVB radiation. Immediately after the application of the inflammatory stimulus, 50 μl or mg of test material is applied directly onto the tissue surface. An over-the-counter drug hydrocortisone cream was used as positive control. For negative controls, the tissues were exposed to inflammatory stimuli but not treated with any type of anti-inflammatory material. An additional set of tissues was kept from exposure to inflammatory stimuli to provide baseline measurements of cytokines. After exposure to inflammatory stimuli, the tissue is then treatedAt 37+ -2deg.C and 5% + -1% CO ₂ Incubate for 24 hours. After 24 hours incubation, the cell culture medium was collected and stored at-75 ℃ until cytokine analysis.

ELISA plates were prepared by diluting the appropriate capture antibodies in PBS. Subsequently, 100 μl of diluted capture antibody was added to the wells of a 96-well ELISA plate, and the plate was incubated overnight at room temperature. The next day, plates were washed three times with 300 μl of wash buffer (0.05% tween 20 in PBS) and then blocked by adding 300 μl of blocking buffer (1% bsa in PBS) to each well. Plates were incubated with blocking buffer for at least one hour. After incubation, the blocking buffer was removed and the plates were washed three times as described above.

A series of standards were prepared and 100 μl of each of these standards was split into two wells in a suitable 96-well plate (in duplicate). Subsequently, 100 μl of each sample was added to the additional wells and the plates were incubated for two hours at room temperature. After incubation, the plates were washed three times as described above. Once removed by the last wash, 100 μl of biotin-conjugated detection antibody was added. After incubating the plates for two hours at room temperature, the plates were washed again as described above. Then 100 μl HRP-streptavidin was added to each well and the plate incubated for 20 minutes at room temperature. Next, 100 μl of substrate solution (hydrogen peroxide+tetramethylbenzidine as chromophore) was added to each well. Once a sufficient level of coloration has occurred, 50 μl of stop solution (2N sulfuric acid) is added to each well and the plate is read at 460 nm.

After 24 hours incubation, the tissue was rinsed twice with at least 100. Mu.L of phosphate buffer to remove the test material, then transferred to a 6-well plate containing 1.0mL of assay medium supplemented with MTT (1 mg/mL) and incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate 3±0.25 hours. After incubation, the tissues were rinsed at least twice with 100 μl of phosphate buffer, blotted dry, and then placed in 24-well plates containing 2mL of isopropanol per well. The 24-well plate was covered and incubated on a shake table at room temperature for at least 2 hours to extract the reduced MTT from the tissue. After extraction, 200. Mu.L of the isopropanol/MTT mixture sample was transferred to a 96-well plate, using 200. Mu.L of isopropanol as a blank,the absorbance of the sample was read with a microplate reader at 540 nm.

EXAMPLE 20 urban dust protection of truncated elastin

Keratinocyte culture models were used to assess the ability of truncated elastin to protect by promoting cell survival after exposure to municipal dust.

Human epidermal keratinocytes were pretreated with the test material and then exposed to municipal dust. At the end of the treatment period, changes in cell viability were determined via MTT assay.

Keratinocytes were inoculated into 100. Mu.L of medium in individual wells of a 96-well plate and incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate overnight. The next day, the medium was removed via aspiration to remove any non-adherent cells and replaced with 100 μl of fresh medium. Cells were grown until confluence, with medium changes every 48 to 72 hours.

Pretreatment with test material followed by urban dust treatment

Test materials were prepared in cell culture medium at 2x their final desired concentration. Urban dust (NIST 1649B from Sigma Chemicals) was also prepared in 2x solution. For pretreatment, 50 μl of 2x test material was combined with 50 μl of medium and the cells were incubated for 24 hours. At the end of the pretreatment period, the test material containing the medium was removed and replaced with 50 μl of 2x municipal dust and 50 μl of medium. Another group of cells was treated with medium alone (without dust exposure) and used as a reference control to represent 100% cell viability. The cells were then incubated for 24 hours and MTT assays were performed to determine changes in cell viability.

At the end of the treatment period, the cell culture medium was removed and the cells were washed with PBS. After washing, 100. Mu.L of cell culture medium supplemented with 0.5mg/mL MTT was added to each well, and the cells were incubated at 37.+ -. 2 ℃ and 5%.+ -. 1% CO ₂ Incubate for 30 minutes. After incubation, the medium/MTT solution was removed, the cells were washed once more with PBS, and then 100 μl isopropanol was added to the wells to extract purple crystalline formazan. The 96-well plates were then read at 540nm using isopropanol as a blank.

The average MTT absorbance of the cells not exposed to dust was calculated and used to represent 100% of the number of cells. The individual MTT values of the cells subjected to the various treatments were then divided by the average of the cells not exposed to dust and expressed as a percentage to determine the change in cell number caused by each treatment.

EXAMPLE 21.23kDa truncated human elastin

Non-naturally occurring truncated human elastin without His tag, linker and thrombin cleavage site is disclosed below. The codon-optimized nucleotide and amino acid sequences encoding the truncated elastin are disclosed below. In SEQ ID NO. 45, the DsbA secretion tag is encoded by nucleotides 1 to 57 and encodes amino acids 1 to 19 of SEQ ID NO. 46. In SEQ ID NO. 45, the truncated elastin sequence is encoded by nucleotides 58 to 843 and encodes amino acids 20 to 281 of SEQ ID NO. 46.

The codon-optimized nucleotide sequence encoding the non-naturally occurring truncated elastin is provided in SEQ ID NO. 45.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGGCAGCAGCCGCAGCAGCGGCAAAAGCAGCAGCGAAATTTGGTGCGGGTGCAGCCGGTGTGCTGCCAGGCGTAGGTGGCGCTGGTGTACCTGGTGTCCCTGGCGCAATTCCAGGTATTGGCGGTATTGCAGGCGTTGGTACTCCGGCAGCTGCAGCCGCTGCCGCAGCCGCAGCTAAAGCAGCCAAATATGGTGCAGCGGCAGGCCTGGTTCCTGGCGGTCCAGGTTTTGGTCCGGGTGTTGTTGGCGTCCCAGGTGCCGGTGTGCCTGGTGTGGGTGTTCCAGGTGCGGGTATTCCGGTTGTTCCTGGCGCAGGCATTCCGGGTGCCGCAGTTCCGGGTGTAGTTAGCCCGGAATAA(SEQ ID NO:45)

The amino acid sequence of a 23kDa non-naturally occurring truncated human elastin sequence comprising the DsbA secretion signal is disclosed in SEQ ID NO. 46.

MKKIWLALAGLVLAFSASAAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPE(SEQ ID NO:46)

A codon-optimized nucleotide sequence encoding a non-naturally occurring truncated human elastin that is devoid of DsbA secretion tag elastin is provided in SEQ ID NO. 47.

GCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGGCAGCAGCCGCAGCAGCGGCAAAAGCAGCAGCGAAATTTGGTGCGGGTGCAGCCGGTGTGCTGCCAGGCGTAGGTGGCGCTGGTGTACCTGGTGTCCCTGGCGCAATTCCAGGTATTGGCGGTATTGCAGGCGTTGGTACTCCGGCAGCTGCAGCCGCTGCCGCAGCCGCAGCTAAAGCAGCCAAATATGGTGCAGCGGCAGGCCTGGTTCCTGGCGGTCCAGGTTTTGGTCCGGGTGTTGTTGGCGTCCCAGGTGCCGGTGTGCCTGGTGTGGGTGTTCCAGGTGCGGGTATTCCGGTTGTTCCTGGCGCAGGCATTCCGGGTGCCGCAGTTCCGGGTGTAGTTAGCCCGGAATAA(SEQ ID NO:47)

The amino acid sequence of a non-naturally occurring truncated human elastin without a DsbA secretion tag is disclosed in SEQ ID No. 48.

AGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPE(SEQ ID NO:48)

The polynucleotide of SEQ ID NO. 45 is synthesized by Twist DNA. The overlap between pET28 vector and SEQ ID NO. 45 was designed to be 20 to 30bp long, and PCR was used

GXL polymerase (www.takarabio.com/products/pcr/gc-rich-pcr/primestar-GXL-dna-polymerase) was added. The cut pET28a vector was then assembled with insert DNA (SEQ ID NO: 45) into the final plasmid using seamless cloning (www.takarabio.com/products/cloning/in-fusion-cloning). The sequence of the plasmid was then verified by Genewiz (www.genewiz.com/en) using Sanger sequencing.

Transformed cells were cultured in minimal medium of example 12 and frozen with plant glycerol in a ratio of 50:50 cells to glycerol in 1.5mL aliquots. One vial of frozen culture was resuscitated overnight in 50mL of minimal medium at 37 ℃ at 200 rpm. Cells were transferred to 300mL of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

23kDa non-naturally occurring truncated human elastin was detected on a 12% BisTris protein gel. A photograph of the gel is depicted in fig. 22. The correct mass of the 23kDa non-naturally occurring truncated human elastin was confirmed by complete mass spectrometry using liquid chromatography-mass spectrometry.

EXAMPLE 22.13kDa truncated human elastin

Non-naturally occurring truncated humans without His tag, linker and thrombin cleavage site are disclosed below. The codon-optimized nucleotide and amino acid sequences encoding the elastin are disclosed below. In SEQ ID NO. 49, the DsbA secretion tag is encoded by nucleotides 1 to 57 and encodes amino acids 1 to 19 of SEQ ID NO. 50. In SEQ ID NO. 49, the truncated elastin sequence is encoded by nucleotides 58 to 489 and encodes amino acids 20 to 163 of SEQ ID NO. 50.

A codon-optimized nucleotide sequence encoding the elastin is provided in SEQ ID NO. 49.

ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGTAA(SEQ ID NO:49)

The amino acid sequence of a 13kDa non-naturally occurring truncated human elastin sequence comprising the DsbA secretion signal is disclosed in SEQ ID NO. 50.

MKKIWLALAGLVLAFSASAAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQ(SEQ ID NO:50)

A codon-optimized nucleotide sequence encoding a non-naturally occurring truncated human elastin without a DsbA secretion tag is provided in SEQ ID NO. 51.

GCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGTAA(SEQ ID NO:51)

The amino acid sequence of a non-naturally occurring truncated human elastin without a DsbA secretion tag is disclosed in SEQ ID No. 52.

AGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQ(SEQ ID NO:52)

The polynucleotide of SEQ ID NO. 49 is synthesized by Twist DNA. The overlap between pET28 vector and SEQ ID NO. 49 was designed to be 20 to 30bp long, and PCR was used

GXL polymerase (www.takarabio.com/products/pcr/gc-rich-pcr/primestar-GXL-dna-polymerase) was added. Then makeThe cut pET28a vector was assembled with insert DNA (SEQ ID NO: 49) into the final plasmid using seamless cloning (www.takarabio.com/products/cloning/in-fusion-cloning). The sequence of the plasmid was then verified by Sanger sequencing by Genewiz (www.genewiz.com/en).

Transformed cells were cultured in minimal medium and frozen with plant glycerol in a ratio of 50:50 cells to glycerol in 1.5mL aliquots. One vial of frozen culture was resuscitated overnight in 50mL of minimal medium at 37 ℃ at 200 rpm. Cells were transferred to 300mL of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

13kDa non-naturally occurring truncated human elastin was detected on a 12% BisTris protein gel. A photograph of the gel is depicted in fig. 22. The correct mass of the 13kDa non-naturally occurring truncated human elastin was confirmed by complete mass spectrometry using liquid chromatography-mass spectrometry.

EXAMPLE 23.13 purity and homogeneity of non-naturally occurring truncated elastin

The polypeptide having the amino acid sequence according to SEQ ID NO. 52 was analyzed by SDS-PAGE to evaluate purity and uniformity. FIG. 23 shows that the polypeptide having the amino acid sequence according to SEQ ID NO. 52 appears as a single band on SDS-PAGE protein gels, indicating high purity and homogeneity. In contrast, commercially available elastin appears as a hazy band on protein gels, indicating high heterogeneity.

Example 24 in vitro studies of non-naturally occurring truncated human elastin

Non-naturally occurring truncated human elastin stimulates fibroblasts to produce type I collagen

Fibroblasts are the major source of extracellular matrix peptides, including structural collagen and elastin. Human primary fibroblasts were evaluated for type I collagen secretion. The fibroblasts were incubated with the polypeptide according to SEQ ID NO. 52 for 48 hours. Culture supernatants were analyzed by ELISA for type I procollagen C-peptide, which is a reading of total secreted type I collagen. FIG. 24 shows that fibroblasts treated with 0.1% w/w solution (FIG. 24, "B"), 0.05% w/w solution (FIG. 24, "C") and 0.025% w/w solution (FIG. 24, "D") of the polypeptide according to SEQ ID NO. 52 secrete higher levels of type I collagen than untreated control fibroblasts (FIG. 24, "A").

Non-naturally occurring truncated human elastin shows antioxidant capacity

An Oxygen Radical Absorption Capacity (ORAC) assay was performed to analyze the antioxidant capacity of the polypeptide according to SEQ ID NO: 52. The ORAC assay is a cell-free assay that uses fluorescent readings to measure antioxidant capacity. Data are reported as Trolox (vitamin E) equivalent (TE). As shown in FIG. 25, 0.0125% w/w solution (FIG. 25, "E"), 0.025% w/w solution (FIG. 25, "D"), 0.05% w/w solution (FIG. 25, "C"), 0.1% w/w solution (FIG. 25, "B"), and 0.2% w/w solution (FIG. 25, "A") of the polypeptide according to SEQ ID NO. 52 each exhibit antioxidant properties. For example, a 0.2% w/w polypeptide solution according to SEQ ID NO:52 shows antioxidant properties corresponding to 228. Mu.M Trolox.

Non-naturally occurring truncated human elastin stimulates antioxidant genes in keratinocytes

Keratinocytes were incubated with medium alone or with 0.03% w/w polypeptide solution according to SEQ ID NO. 52 for 24 hours. RNA was extracted from the cells and analyzed for overall gene expression by Clariom microarray (ThermoFisher). FIG. 26 shows that a number of antioxidant genes (including SOD2, GPX4, GSTK1, GSTZ1, GSTA4, GSTM2, CCS, GPX1, GLRX, PRDX5, PRDX6 and PRDX 2) are up-regulated in keratinocytes treated with the polypeptide according to SEQ ID NO. 52 (FIG. 26, "B") relative to untreated cells (FIG. 26, "A").

Non-naturally occurring truncated human elastin reduces expression of apoptotic genes in keratinocytes

Keratinocytes were incubated with medium alone (untreated) or with 0.03% w/w polypeptide solution according to SEQ ID NO. 52 for 24 hours. RNA was extracted from the cells and analyzed for overall gene expression by Clariom microarray (ThermoFisher). FIG. 27 shows that a number of pro-apoptotic genes (including APAF1, BAK1, CASP7, CASP8, FADD, MCL1 and MET) were down-regulated in keratinocytes treated with the polypeptide according to SEQ ID NO. 52 (FIG. 27, "B") relative to untreated cells (FIG. 27, "A").

Non-naturally occurring truncated human elastin stimulates fibroblast cell production of elastin

Human primary fibroblast cultures were treated with medium alone (untreated) or with 0.03% w/w polypeptide solution having an amino acid sequence according to SEQ ID NO. 52 for 24 hours. The supernatant was collected and evaluated for elastin production using an ELISA kit. FIG. 28 shows that fibroblasts treated with a polypeptide having an amino acid sequence according to SEQ ID NO:52 (FIG. 28, "B") exhibited increased elastin production compared to fibroblasts treated with medium alone (FIG. 28, "A").

EXAMPLE 25 elastin polypeptide with hyaluronic acid, retinol, collagen polypeptide or truncations thereof, ascorbic acid, water Results of in vitro testing of combinations of salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid).

The combination of elastin polypeptides with ascorbic acid, retinol, collagen polypeptides or truncations thereof, hyaluronic acid, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) increases the viability and proliferation of keratinocytes and fibroblasts

In vitro studies, a (e.g., human) skin cell culture, (e.g., human) skin equivalent culture or ex vivo (e.g., human) skin is treated with an elastin polypeptide as provided herein (e.g., a (e.g., human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) alone or in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid). The viability and proliferation of keratinocytes and fibroblasts (e.g., as measured by, for example, an MTT viability assay) is increased (e.g., coordinated, additive) relative to treatment with an elastin polypeptide or truncate thereof, ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone, in combination with (e.g., a human) elastin polypeptide or truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52).

The combination of an elastin polypeptide with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) increases the production of extracellular matrix components and reduces the production of matrix degrading proteins.

In vitro studies, a (e.g., human) skin cell culture, (e.g., human) skin equivalent culture or ex vivo (e.g., human) skin is treated with an elastin polypeptide as provided herein (e.g., a (e.g., human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) alone or in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid). The treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergistically, accumulates) the production of extracellular matrix components (e.g., collagen, elastin, fibronectin, fibril, hyaluronic acid) relative to the treatment with (e.g., a human) elastin polypeptide or a truncate thereof, ascorbic acid, retinol, hyaluronic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone (e.g., in combination with the amino acid sequence of SEQ ID NO: 52). Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) the production of matrix degrading proteins (e.g., matrix metalloproteinases, proteases) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid), e.g., as measured by, e.g., transcriptome analysis, ELISA.

The combination of elastin polypeptides with ascorbic acid, retinol, hyaluronic acid, collagen polypeptides or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) enhances wound healing ability

In vitro studies, a (e.g., human) skin cell culture, (e.g., human) skin equivalent culture or ex vivo (e.g., human) skin is treated with an elastin polypeptide as provided herein (e.g., a (e.g., human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) alone or in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid). The wound healing capacity (e.g., as measured by, for example, a wound healing scratch assay) of skin cells (e.g., keratinocytes, fibroblasts) is increased (e.g., synergistically, additively) relative to treatment with (e.g., human) elastin polypeptide or a truncate thereof, ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or a truncate thereof, retinol, collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone, in combination with (e.g., human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52).

The combination of elastin polypeptides with ascorbic acid, retinol, hyaluronic acid, collagen polypeptides or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) has improved results following photodamage attack

In vitro studies, a (e.g., human) skin cell culture, a (e.g., human) skin equivalent culture, or ex vivo (e.g., human) skin is challenged with light injury (e.g., UV irradiation, blue light exposure), and then treated with an elastin polypeptide as provided herein (e.g., a (e.g., human) elastin polypeptide or truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) alone, or in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid). The treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone (e.g., a human) elastin polypeptide or a truncate thereof, ascorbic acid, retinol, hyaluronic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) as measured by, e.g., MTT viability assay. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) cytotoxicity and apoptosis (e.g., as measured by, e.g., fluorescence microscopy) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof. Compared with the single use For example, human) elastin polypeptide or truncations thereof, ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid), treating with (e.g., human) elastin polypeptide or truncations thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid) enhances (e.g., synergizes, accumulates) DNA repair of skin cells (e.g., keratinocytes, fibroblasts) as measured, for example, by ELISA assay of thymidine dimers. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) inflammation (e.g., as measured by, e.g., ELISA assays or Luminex detection of cytokines) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergistically, accumulates) reactive oxidative stress (e.g., as by, e.g., CM-H ₂ Measured by DCFDA detection).

The combination of elastin polypeptides with ascorbic acid, retinol, hyaluronic acid, collagen polypeptides or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) has improved results after contamination challenge

In vitro studies, a (e.g., human) skin cell culture, a (e.g., human) skin equivalent culture, or ex vivo (e.g., human) skin is challenged with a contaminant (e.g., urban dust exposure), and then treated with an elastin polypeptide as provided herein (e.g., a (e.g., human) elastin polypeptide or truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) alone or in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid). The treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone (e.g., a human) elastin polypeptide or a truncate thereof, ascorbic acid, retinol, hyaluronic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) as measured by, e.g., MTT viability assay. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) cytotoxicity and apoptosis (e.g., as measured by, e.g., fluorescence microscopy) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof. Relative to an elastin polypeptide alone (e.g., human) or a truncate thereof Treatment with a short, ascorbic, retinol, hyaluronic acid, collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) DNA repair of skin cells (e.g., keratinocytes, fibroblasts) in combination with (e.g., human) an elastin polypeptide or truncations thereof (e.g., having the amino acid sequence of SEQ ID NO: 52), ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid), as measured by, e.g., thymidine dimer ELISA assay. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) inflammation (e.g., as measured by, e.g., ELISA assays or Luminex detection of cytokines) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergistically, accumulates) reactive oxidative stress (e.g., as by, e.g., CM-H ₂ Measured by DCFDA detection).

The combination of elastin polypeptides with ascorbic acid, retinol, hyaluronic acid, collagen polypeptides or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acids (e.g., glycolic acid, lactic acid) has improved results after challenge with a highly irritating skin active substance

In an in vitro study, a skin cell culture (e.g., human), a skin equivalent culture (e.g., human), or ex vivo (e.g., human) skin is challenged with a skin active agent that is highly irritating (e.g., retinoic acid or retinol, benzoyl peroxide, salicylic acid), and then treated with an elastin polypeptide as provided herein (e.g., a (e.g., human) elastin polypeptide or truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) alone or in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). The treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) increases (e.g., synergizes, accumulates) the viability and proliferation of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone (e.g., a human) elastin polypeptide or a truncate thereof, ascorbic acid, retinol, hyaluronic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) as measured by, e.g., MTT viability assay. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) cytotoxicity and apoptosis (e.g., as measured by, e.g., fluorescence microscopy) of skin cells (e.g., keratinocytes, fibroblasts) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof. Phase (C) For treatment with (e.g., human) elastin polypeptide or truncations thereof alone, ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid), DNA repair of skin cells (e.g., keratinocytes, fibroblasts) is enhanced (e.g., as measured by, e.g., thymidine dimer ELISA assay) in combination with (e.g., human) elastin polypeptide or truncations thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid). Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergizes, accumulates) inflammation (e.g., as measured by, e.g., ELISA assays or Luminex detection of cytokines) relative to treatment with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof. Treatment with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) reduces (e.g., synergistically, accumulates) reactive oxidative stress (e.g., as by, e.g., CM-H ₂ Measured by DCFDA detection).

EXAMPLE 26 elastin polypeptide and hyaluronic acid, retinol, ascorbic acid, collagen polypeptide orTruncates, water Clinical test results of combinations of salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid)

Topical application of elastin polypeptides in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptides or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) improves skin appearance and quality.

In clinical studies, subjects use topical formulations of elastin polypeptides (e.g., a (e.g., human) elastin polypeptide or truncations thereof (e.g., having the amino acid sequence of SEQ ID NO: 52)) as provided herein alone, or in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptides or truncations thereof, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid). Subjects treated with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) exhibit reduced wrinkles and fine lines, reduced skin redness and pigmentation/hyperpigmentation, increased skin brightness, reduced pore size, reduced skin roughness and/or reduced acne (e.g., as measured using, for example, a CLARITY assay) relative to subjects treated with (e.g., a human) elastin polypeptide or a truncate thereof, ascorbic acid, retinol, hyaluronic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) alone.

The subject treated with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) exhibits increased skin elasticity, increased skin firmness, increased skin hydration, increased skin barrier function, increased skin collagen and elastin content and/or increased dermis density relative to a subject treated with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid).

Subjects treated with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) exhibit an improvement in the microflora of the skin (e.g., a decrease in the abundance of harmful microorganisms, an increase in the promotion of beneficial microorganisms and/or an increase in the diversity of microorganisms) relative to subjects treated with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof.

EXAMPLE 27 elastin polypeptide with hyaluronic acid, retinol, ascorbic acid, collagen polypeptide or truncations thereof, water Combinations of salicylic acid, benzoyl peroxide, niacinamide, or alpha hydroxy acids (e.g., glycolic acid, lactic acid) improve hair quality and appearance.

In clinical studies, subjects use a composition of an elastin polypeptide as provided herein, e.g., a (e.g., human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52), alone or in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide, or alpha hydroxy acid (e.g., glycolic acid, lactic acid), formulated for application to hair. Subjects treated with (e.g., a human) elastin polypeptide or a truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid) exhibit improved hair growth, thickened hair fiber diameter, increased combability, reduced hair loss, increased growth rate and/or increased tensile strength of hair relative to subjects treated with (e.g., a human) elastin polypeptide or a truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, a collagen polypeptide or a truncate thereof, benzoyl peroxide, nicotinamide or an alpha hydroxy acid (e.g., glycolic acid, lactic acid).

Subjects treated with (e.g., a human) elastin polypeptide or truncate thereof (e.g., having the amino acid sequence of SEQ ID NO: 52) in combination with ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncate thereof, salicylic acid, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid) exhibit improved hair fiber thickness and density, increased moisture, increased hydrophobicity, reduced hair tip bifurcation, reduced frizz/improved static control, fiber alignment/gloss, increased wet combability/dryability and/or greater resistance to hair breakage relative to subjects treated with (e.g., a human) elastin polypeptide or truncate thereof alone, ascorbic acid, retinol, hyaluronic acid, collagen polypeptide or truncate thereof, benzoyl peroxide, nicotinamide or alpha hydroxy acid (e.g., glycolic acid, lactic acid).

EXAMPLE 28 combination of collagen and hyaluronic acid as an ingestible nutraceutical

The non-naturally occurring collagen of the present invention in powder form is combined with hyaluronic acid in a ratio of 1:10, 2:5, 1:1, 5:2, 10:1, 50:1 and 100:1. The ratio and amount of the product are adjusted so that the uptake of the hyaluronic acid component is about 100mg to about 1g per day. Adding liquid, and making the composition into tea, tonic, broth or other liquid. The composition may also be added to a protein beverage, milk, nut milk, fruit shake or milk shake. The user consumes 1-2 parts of the beverage per day. Alternatively, the formulation may be formulated as a pill for daily administration. The health effects on the user were recorded daily for 1 month.

By using this method, the user observes that the skin is healthier, the skin is tighter, the skin is more moist and wrinkles are less.

EXAMPLE 29 combination of collagen and hyaluronic acid as injectable skin treatment agent

Injectable sterile combinations of the non-naturally occurring collagen and hyaluronic acid of the application in a sterile saline solution in a ratio of 1:10, 2:5, 1:1, 5:2, 10:1, 50:1 and 100:1. The composition is injected under the skin as a dermatological treatment for certain types of wound scars. The composition is also useful for injection into wrinkles or as a lip plumping agent.

By using this method, the treated individual sees faster wound scar healing, fuller lips, or less wrinkles after treatment.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Embodiments disclosed herein may be embodied in other specific forms without departing from the structures, methods, or other features as broadly described herein and claimed hereinafter. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Sequence listing

<110> Geltor Corp

<120> compositions comprising collagen or elastin polypeptides in combination with active ingredients and methods of use thereof

<130> 57607-714.601

<140>

<141>

<150> 63/091,799

<151> 2020-10-14

<150> 63/091,791

<151> 2020-10-14

<150> 63/085,857

<151> 2020-09-30

<150> 63/085,829

<151> 2020-09-30

<160> 55

<170> patent in version 3.5

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ggtgaaaaag gagaacaggg acgtacaggt gcagcaggta aacagggcag cccgggtgcc 240

gatggtgccc gtggcccgct gggtagcatt ggtcagcagg gtgcaagagg cgaaccgggc 300

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ggtccttcag gtcgtccagg tcaaccgggt gcaaatggtc tgccgggtgt taatggtcgt 420

ggcggtctgg aacgtggtct ggcaggaccg ccgggtcctg atggtcgccg cggtgaaacg 480

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ccagatggtg ttcgtggtat tccgggtaac gatggtcaga gcggtaaacc gggcattgat 660

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Polypeptides

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Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp His His His His His His His His

20 25 30

His Ser Gly Ser Ser Leu Val Pro Arg Gly Ser His Met Gly Pro Gln

35 40 45

Gly Val Val Gly Ala Asp Gly Lys Asp Gly Thr Pro Gly Glu Lys Gly

50 55 60

Glu Gln Gly Arg Thr Gly Ala Ala Gly Lys Gln Gly Ser Pro Gly Ala

65 70 75 80

Asp Gly Ala Arg Gly Pro Leu Gly Ser Ile Gly Gln Gln Gly Ala Arg

85 90 95

Gly Glu Pro Gly Asp Pro Gly Ser Pro Gly Leu Arg Gly Asp Thr Gly

100 105 110

Leu Ala Gly Val Lys Gly Val Ala Gly Pro Ser Gly Arg Pro Gly Gln

115 120 125

Pro Gly Ala Asn Gly Leu Pro Gly Val Asn Gly Arg Gly Gly Leu Glu

130 135 140

Arg Gly Leu Ala Gly Pro Pro Gly Pro Asp Gly Arg Arg Gly Glu Thr

145 150 155 160

Gly Ser Pro Gly Ile Ala Gly Ala Leu Gly Lys Pro Gly Leu Glu Gly

165 170 175

Pro Lys Gly Tyr Pro Gly Leu Arg Gly Arg Asp Gly Thr Asn Gly Lys

180 185 190

Arg Gly Glu Gln Gly Glu Thr Gly Pro Asp Gly Val Arg Gly Ile Pro

195 200 205

Gly Asn Asp Gly Gln Ser Gly Lys Pro Gly Ile Asp Gly Ile Asp Gly

210 215 220

Thr Asn Gly Gln Pro Gly Glu Ala Gly Tyr Gln Gly Gly Arg Gly Thr

225 230 235 240

Arg Gly Gln Leu Gly Glu Thr Gly Asp Val Gly Gln Asn Gly Asp Arg

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Gly Ala Pro Gly Pro Asp Gly Ser Lys Gly Ser Ala Gly Arg Pro Gly

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Leu Arg

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Polypeptides

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1 5 10 15

Glu Lys Gly Glu Gln Gly Arg Thr Gly Ala Ala Gly Lys Gln Gly Ser

20 25 30

Pro Gly Ala Asp Gly Ala Arg Gly Pro Leu Gly Ser Ile Gly Gln Gln

35 40 45

Gly Ala Arg Gly Glu Pro Gly Asp Pro Gly Ser Pro Gly Leu Arg Gly

50 55 60

Asp Thr Gly Leu Ala Gly Val Lys Gly Val Ala Gly Pro Ser Gly Arg

65 70 75 80

Pro Gly Gln Pro Gly Ala Asn Gly Leu Pro Gly Val Asn Gly Arg Gly

85 90 95

Gly Leu Glu Arg Gly Leu Ala Gly Pro Pro Gly Pro Asp Gly Arg Arg

100 105 110

Gly Glu Thr Gly Ser Pro Gly Ile Ala Gly Ala Leu Gly Lys Pro Gly

115 120 125

Leu Glu Gly Pro Lys Gly Tyr Pro Gly Leu Arg Gly Arg Asp Gly Thr

130 135 140

Asn Gly Lys Arg Gly Glu Gln Gly Glu Thr Gly Pro Asp Gly Val Arg

145 150 155 160

Gly Ile Pro Gly Asn Asp Gly Gln Ser Gly Lys Pro Gly Ile Asp Gly

165 170 175

Ile Asp Gly Thr Asn Gly Gln Pro Gly Glu Ala Gly Tyr Gln Gly Gly

180 185 190

Arg Gly Thr Arg Gly Gln Leu Gly Glu Thr Gly Asp Val Gly Gln Asn

195 200 205

Gly Asp Arg Gly Ala Pro Gly Pro Asp Gly Ser Lys Gly Ser Ala Gly

210 215 220

Arg Pro Gly Leu Arg

225

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<213> artificial sequence

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Polypeptides

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Gly Pro Gln Gly Val Val Gly Ala Asp Gly Lys Asp Gly Thr Pro Gly

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Asn Ala Gly Gln Lys Gly Pro Ser Gly Glu Pro Gly Ser Pro Gly Lys

20 25 30

Ala Gly Ser Ala Gly Glu Gln Gly Pro Pro Gly Lys Asp Gly Ser Asn

35 40 45

Gly Glu Pro Gly Ser Pro Gly Lys Glu Gly Glu Arg Gly Leu Ala Gly

50 55 60

Pro Pro Gly Pro Asp Gly Arg Arg Gly Glu Thr Gly Ser Pro Gly Ile

65 70 75 80

Ala Gly Ala Leu Gly Lys Pro Gly Leu Glu Gly Pro Lys Gly Tyr Pro

85 90 95

Gly Leu Arg Gly Arg Asp Gly Thr Asn Gly Lys Arg Gly Glu Gln Gly

100 105 110

Glu Thr Gly Pro Asp Gly Val Arg Gly Ile Pro Gly Asn Asp Gly Gln

115 120 125

Ser Gly Lys Pro Gly Ile Asp Gly Ile Asp Gly Thr Asn Gly Gln Pro

130 135 140

Gly Glu Ala Gly Tyr Gln Gly Gly Arg Gly Thr Arg Gly Gln Leu Gly

145 150 155 160

Glu Thr Gly Asp Val Gly Gln Asn Gly Asp Arg Gly Ala Pro Gly Pro

165 170 175

Asp Gly Ser Lys Gly Ser Ala Gly Arg Pro Gly Leu Arg

180 185

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atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60

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ggtcagctgg gtgaaacagg tgatgttggt cagaatggtg atcgcggcgc accgggtccg 600

gatggtagca aaggtagcgc cggtcgtccg ggtttacgtt aa 642

<210> 7

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 7

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp Gly Pro Gln Gly Val Val Gly Ala

20 25 30

Asp Gly Lys Asp Gly Thr Pro Gly Asn Ala Gly Gln Lys Gly Pro Ser

35 40 45

Gly Glu Pro Gly Ser Pro Gly Lys Ala Gly Ser Ala Gly Glu Gln Gly

50 55 60

Pro Pro Gly Lys Asp Gly Ser Asn Gly Glu Pro Gly Ser Pro Gly Lys

65 70 75 80

Glu Gly Glu Arg Gly Leu Ala Gly Pro Pro Gly Pro Asp Gly Arg Arg

85 90 95

Gly Glu Thr Gly Ser Pro Gly Ile Ala Gly Ala Leu Gly Lys Pro Gly

100 105 110

Leu Glu Gly Pro Lys Gly Tyr Pro Gly Leu Arg Gly Arg Asp Gly Thr

115 120 125

Asn Gly Lys Arg Gly Glu Gln Gly Glu Thr Gly Pro Asp Gly Val Arg

130 135 140

Gly Ile Pro Gly Asn Asp Gly Gln Ser Gly Lys Pro Gly Ile Asp Gly

145 150 155 160

Ile Asp Gly Thr Asn Gly Gln Pro Gly Glu Ala Gly Tyr Gln Gly Gly

165 170 175

Arg Gly Thr Arg Gly Gln Leu Gly Glu Thr Gly Asp Val Gly Gln Asn

180 185 190

Gly Asp Arg Gly Ala Pro Gly Pro Asp Gly Ser Lys Gly Ser Ala Gly

195 200 205

Arg Pro Gly Leu Arg

210

<210> 8

<211> 570

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 8

ggtccgcagg gtgttgttgg tgcagatggt aaagacggta ccccgggtaa tgcaggtcag 60

aaaggtccgt caggtgaacc tggcagccct ggtaaagcag gtagtgccgg tgagcagggt 120

ccgccgggca aagatggtag taatggtgag ccgggtagcc ctggcaaaga aggtgaacgt 180

ggtctggcag gaccgccggg tcctgatggt cgccgcggtg aaacgggttc accgggtatt 240

gccggtgccc tgggtaaacc aggtctggaa ggtccgaaag gttatcctgg tctgcgcggt 300

cgtgatggta ccaatggcaa acgtggcgaa cagggcgaaa ccggtccaga tggtgttcgt 360

ggtattccgg gtaacgatgg tcagagcggt aaaccgggca ttgatggtat tgatggcacc 420

aatggtcagc ctggcgaagc aggttatcag ggtggtcgcg gtacccgtgg tcagctgggt 480

gaaacaggtg atgttggtca gaatggtgat cgcggcgcac cgggtccgga tggtagcaaa 540

ggtagcgccg gtcgtccggg tttacgttaa 570

<210> 9

<211> 2232

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 9

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60

cagtatgaag atcaccatca ccaccaccac catcaccact ctggctcgag cctggtgccg 120

cgcggcagcc atatgtctgg ctcgagcagt aaaggtgaag aactgttcac cggtgttgtt 180

ccgatcctgg ttgaactgga tggtgatgtt aacggccaca aattctctgt tcgtggtgaa 240

ggtgaaggtg atgcaaccaa cggtaaactg accctgaaat tcatctgcac taccggtaaa 300

ctgccggttc catggccgac tctggtgact accctgacct atggtgttca gtgtttttct 360

cgttacccgg atcacatgaa gcagcatgat ttcttcaaat ctgcaatgcc ggaaggttat 420

gtacaggagc gcaccatttc tttcaaagac gatggcacct acaaaacccg tgcagaggtt 480

aaatttgaag gtgatactct ggtgaaccgt attgaactga aaggcattga tttcaaagag 540

gacggcaaca tcctgggcca caaactggaa tataacttca actcccataa cgtttacatc 600

accgcagaca aacagaagaa cggtatcaaa gctaacttca aaattcgcca taacgttgaa 660

gacggtagcg tacagctggc ggaccactac cagcagaaca ctccgatcgg tgatggtccg 720

gttctgctgc cggataacca ctacctgtcc acccagtcta aactgtccaa agacccgaac 780

gaaaagcgcg accacatggt gctgctggag ttcgttactg cagcaggtat cacgcacggc 840

atggatgaac tctacaaatc tggcgcgccg ggcggtccgc agggtgttgt tggtgcagat 900

ggtaaagacg gtaccccggg taatgcaggt cagaaaggtc cgtcaggtga acctggcagc 960

cctggtaaag caggtagtgc cggtgagcag ggtccgccgg gcaaagatgg tagtaatggt 1020

gagccgggta gccctggcaa agaaggtgaa cgtggtctgg caggaccgcc gggtcctgat 1080

ggtcgccgcg gtgaaacggg ttcaccgggt attgccggtg ccctgggtaa accaggtctg 1140

gaaggtccga aaggttatcc tggtctgcgc ggtcgtgatg gtaccaatgg caaacgtggc 1200

gaacagggcg aaaccggtcc agatggtgtt cgtggtattc cgggtaacga tggtcagagc 1260

ggtaaaccgg gcattgatgg tattgatggc accaatggtc agcctggcga agcaggttat 1320

cagggtggtc gcggtacccg tggtcagctg ggtgaaacag gtgatgttgg tcagaatggt 1380

gatcgcggcg caccgggtcc ggatggtagc aaaggtagcg ccggtcgtcc gggtttacgt 1440

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 1500

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 1560

tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 1620

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 1680

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 1740

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 1800

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 1860

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 1920

atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 1980

caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 2040

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 2100

attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 2160

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 2220

aagcattggt aa 2232

<210> 10

<211> 743

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 10

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp His His His His His His His His

20 25 30

His Ser Gly Ser Ser Leu Val Pro Arg Gly Ser His Met Ser Gly Ser

35 40 45

Ser Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

50 55 60

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu

65 70 75 80

Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys

85 90 95

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

100 105 110

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

115 120 125

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

130 135 140

Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val

145 150 155 160

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

165 170 175

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

180 185 190

Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly

195 200 205

Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val

210 215 220

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

225 230 235 240

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser

245 250 255

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

260 265 270

Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly

275 280 285

Ala Pro Gly Gly Pro Gln Gly Val Val Gly Ala Asp Gly Lys Asp Gly

290 295 300

Thr Pro Gly Asn Ala Gly Gln Lys Gly Pro Ser Gly Glu Pro Gly Ser

305 310 315 320

Pro Gly Lys Ala Gly Ser Ala Gly Glu Gln Gly Pro Pro Gly Lys Asp

325 330 335

Gly Ser Asn Gly Glu Pro Gly Ser Pro Gly Lys Glu Gly Glu Arg Gly

340 345 350

Leu Ala Gly Pro Pro Gly Pro Asp Gly Arg Arg Gly Glu Thr Gly Ser

355 360 365

Pro Gly Ile Ala Gly Ala Leu Gly Lys Pro Gly Leu Glu Gly Pro Lys

370 375 380

Gly Tyr Pro Gly Leu Arg Gly Arg Asp Gly Thr Asn Gly Lys Arg Gly

385 390 395 400

Glu Gln Gly Glu Thr Gly Pro Asp Gly Val Arg Gly Ile Pro Gly Asn

405 410 415

Asp Gly Gln Ser Gly Lys Pro Gly Ile Asp Gly Ile Asp Gly Thr Asn

420 425 430

Gly Gln Pro Gly Glu Ala Gly Tyr Gln Gly Gly Arg Gly Thr Arg Gly

435 440 445

Gln Leu Gly Glu Thr Gly Asp Val Gly Gln Asn Gly Asp Arg Gly Ala

450 455 460

Pro Gly Pro Asp Gly Ser Lys Gly Ser Ala Gly Arg Pro Gly Leu Arg

465 470 475 480

His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly

485 490 495

Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu

500 505 510

Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys

515 520 525

Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu

530 535 540

Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr

545 550 555 560

Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu

565 570 575

Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu

580 585 590

Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His

595 600 605

Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu

610 615 620

Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala

625 630 635 640

Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu

645 650 655

Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala

660 665 670

Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp

675 680 685

Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu

690 695 700

Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly

705 710 715 720

Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly

725 730 735

Ala Ser Leu Ile Lys His Trp

740

<210> 11

<211> 2232

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 11

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60

cagtatgaag atcaccatca ccaccaccac catcaccact ctggctcgag cctggtgccg 120

cgcggcagcc atatgtctgg ctcgagcagt aaaggtgaag aactgttcac cggtgttgtt 180

ccgatcctgg ttgaactgga tggtgatgtt aacggccaca aattctctgt tcgtggtgaa 240

ggtgaaggtg atgcaaccaa cggtaaactg accctgaaat tcatctgcac taccggtaaa 300

ctgccggttc catggccgac tctggtgact accctgacct atggtgttca gtgtttttct 360

cgttacccgg atcacatgaa gcagcatgat ttcttcaaat ctgcaatgcc ggaaggttat 420

gtacaggagc gcaccatttc tttcaaagac gatggcacct acaaaacccg tgcagaggtt 480

aaatttgaag gtgatactct ggtgaaccgt attgaactga aaggcattga tttcaaagag 540

gacggcaaca tcctgggcca caaactggaa tataacttca actcccataa cgtttacatc 600

accgcagaca aacagaagaa cggtatcaaa gctaacttca aaattcgcca taacgttgaa 660

gacggtagcg tacagctggc ggaccactac cagcagaaca ctccgatcgg tgatggtccg 720

gttctgctgc cggataacca ctacctgtcc acccagtcta aactgtccaa agacccgaac 780

gaaaagcgcg accacatggt gctgctggag ttcgttactg cagcaggtat cacgcacggc 840

atggatgaac tctacaaatc tggcgcgccg ggcggtccgc agggtgttgt tggtgcagat 900

ggtaaagacg gtaccccggg taatgcaggt cagaaaggtc cgtcaggtga acctggcagc 960

cctggtaaag caggtagtgc cggtgagcag ggtccgccgg gcaaagatgg tagtaatggt 1020

gagccgggta gccctggcaa agaaggtgaa cgtggtctgg caggaccgcc gggtcctgat 1080

ggtcgccgcg gtgaaacggg ttcaccgggt attgccggtg ccctgggtaa accaggtctg 1140

gaaggtccga aaggttatcc tggtctgcgc ggtcgtgatg gtaccaatgg caaacgtggc 1200

gaacagggcg aaaccggtcc agatggtgtt cgtggtattc cgggtaacga tggtcagagc 1260

ggtaaaccgg gcattgatgg tattgatggc accaatggtc agcctggcga agcaggttat 1320

cagggtggtc gcggtacccg tggtcagctg ggtgaaacag gtgatgttgg tcagaatggt 1380

gatcgcggcg caccgggtcc ggatggtagc aaaggtagcg ccggtcgtcc gggtttacgt 1440

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 1500

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 1560

tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 1620

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 1680

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 1740

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 1800

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 1860

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 1920

atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 1980

caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 2040

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 2100

attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 2160

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 2220

aagcattggt aa 2232

<210> 12

<211> 743

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 12

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp His His His His His His His His

20 25 30

His Ser Gly Ser Ser Leu Val Pro Arg Gly Ser His Met Ser Gly Ser

35 40 45

Ser Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

50 55 60

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu

65 70 75 80

Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys

85 90 95

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

100 105 110

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

115 120 125

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

130 135 140

Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val

145 150 155 160

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

165 170 175

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

180 185 190

Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly

195 200 205

Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val

210 215 220

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

225 230 235 240

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser

245 250 255

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

260 265 270

Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly

275 280 285

Ala Pro Gly Gly Pro Gln Gly Val Val Gly Ala Asp Gly Lys Asp Gly

290 295 300

Thr Pro Gly Asn Ala Gly Gln Lys Gly Pro Ser Gly Glu Pro Gly Ser

305 310 315 320

Pro Gly Lys Ala Gly Ser Ala Gly Glu Gln Gly Pro Pro Gly Lys Asp

325 330 335

Gly Ser Asn Gly Glu Pro Gly Ser Pro Gly Lys Glu Gly Glu Arg Gly

340 345 350

Leu Ala Gly Pro Pro Gly Pro Asp Gly Arg Arg Gly Glu Thr Gly Ser

355 360 365

Pro Gly Ile Ala Gly Ala Leu Gly Lys Pro Gly Leu Glu Gly Pro Lys

370 375 380

Gly Tyr Pro Gly Leu Arg Gly Arg Asp Gly Thr Asn Gly Lys Arg Gly

385 390 395 400

Glu Gln Gly Glu Thr Gly Pro Asp Gly Val Arg Gly Ile Pro Gly Asn

405 410 415

Asp Gly Gln Ser Gly Lys Pro Gly Ile Asp Gly Ile Asp Gly Thr Asn

420 425 430

Gly Gln Pro Gly Glu Ala Gly Tyr Gln Gly Gly Arg Gly Thr Arg Gly

435 440 445

Gln Leu Gly Glu Thr Gly Asp Val Gly Gln Asn Gly Asp Arg Gly Ala

450 455 460

Pro Gly Pro Asp Gly Ser Lys Gly Ser Ala Gly Arg Pro Gly Leu Arg

465 470 475 480

His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly

485 490 495

Ala Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu

500 505 510

Glu Ser Phe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys

515 520 525

Val Leu Leu Cys Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu

530 535 540

Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr

545 550 555 560

Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu

565 570 575

Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu

580 585 590

Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His

595 600 605

Asn Met Gly Asp His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu

610 615 620

Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala

625 630 635 640

Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu

645 650 655

Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala

660 665 670

Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp

675 680 685

Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala Ala Leu

690 695 700

Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly

705 710 715 720

Ser Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly

725 730 735

Ala Ser Leu Ile Lys His Trp

740

<210> 13

<211> 636

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 13

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60

cagtatgaag atgcaggttt tccgggtctg cctggtccgg caggcgaacc gggtcgtcat 120

ggtaaagatg gtctgatggg tagtccgggt tttaaaggtg aagcaggttc accgggtgca 180

cctggtcagg atggcacccg tggtgaaccg ggtattccgg gatttccggg taatcgtggc 240

ctgatgggtc agaaaggtga aattggtccg cctggtcagc agggtaaaaa aggcgcaccg 300

ggtatgccag gactgatggg ttcaaatggc agtccgggtc agccaggcac accgggttca 360

aaaggtagca aaggcgaacc tggtattcag ggtatgcctg gtgcaagcgg tctgaaaggc 420

gagccaggtg ccaccggttc tccgggtgaa ccaggttata tgggtctgcc aggtatccaa 480

ggcaaaaaag gtgataaagg taatcagggc gaaaaaggca ttcagggcca gaaaggcgaa 540

aatggccgtc agggtattcc aggccagcag ggcatccagg gtcatcatgg tgcaaaaggt 600

gaacgtggtg aaaagggcga accaggtgtt cgttta 636

<210> 14

<211> 211

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 14

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp Ala Gly Phe Pro Gly Leu Pro Gly

20 25 30

Pro Ala Gly Glu Pro Gly Arg His Gly Lys Asp Gly Leu Met Gly Ser

35 40 45

Pro Gly Phe Lys Gly Glu Ala Gly Ser Pro Gly Ala Pro Gly Gln Asp

50 55 60

Gly Thr Arg Gly Glu Pro Gly Ile Pro Gly Phe Pro Gly Asn Arg Gly

65 70 75 80

Leu Met Gly Gln Lys Gly Glu Ile Gly Pro Pro Gly Gln Gln Gly Lys

85 90 95

Lys Gly Ala Pro Gly Met Pro Gly Leu Met Gly Ser Asn Gly Ser Pro

100 105 110

Gly Gln Pro Gly Thr Pro Gly Ser Lys Gly Ser Lys Gly Glu Pro Gly

115 120 125

Ile Gln Gly Met Pro Gly Ala Ser Gly Leu Lys Gly Glu Pro Gly Ala

130 135 140

Thr Gly Ser Pro Gly Glu Pro Gly Tyr Met Gly Leu Pro Gly Ile Gln

145 150 155 160

Gly Lys Lys Gly Asp Lys Gly Asn Gln Gly Glu Lys Gly Ile Gln Gly

165 170 175

Gln Lys Gly Glu Asn Gly Arg Gln Gly Ile Pro Gly Gln Gln Gly Ile

180 185 190

Gln Gly His His Gly Ala Lys Gly Glu Arg Gly Glu Lys Gly Glu Pro

195 200 205

Gly Val Arg

210

<210> 15

<211> 565

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 15

tgcaggtttt ccgggtctgc ctggtccggc aggcgaaccg ggtcgtcatg gtaaagatgg 60

tctgatgggt agtccgggtt ttaaaggtga agcaggttca ccgggtgcac ctggtcagga 120

tggcacccgt ggtgaaccgg gtattccggg atttccgggt aatcgtggcc tgatgggtca 180

gaaaggtgaa attggtccgc ctggtcagca gggtaaaaaa ggcgcaccgg gtatgccagg 240

actgatgggt tcaaatggca gtccgggtca gccaggcaca ccgggttcaa aaggtagcaa 300

aggcgaacct ggtattcagg gtatgcctgg tgcaagcggt ctgaaaggcg agccaggtgc 360

caccggttct ccgggtgaac caggttatat gggtctgcca ggtatccaag gcaaaaaagg 420

tgataaaggt aatcagggcg aaaaaggcat tcagggccag aaaggcgaaa atggccgtca 480

gggtattcca ggccagcagg gcatccaggg tcatcatggt gcaaaaggtg aacgtggtga 540

aaagggcgaa ccaggtgttc gttaa 565

<210> 16

<211> 187

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 16

Ala Gly Phe Pro Gly Leu Pro Gly Pro Ala Gly Glu Pro Gly Arg His

1 5 10 15

Gly Lys Asp Gly Leu Met Gly Ser Pro Gly Phe Lys Gly Glu Ala Gly

20 25 30

Ser Pro Gly Ala Pro Gly Gln Asp Gly Thr Arg Gly Glu Pro Gly Ile

35 40 45

Pro Gly Phe Pro Gly Asn Arg Gly Leu Met Gly Gln Lys Gly Glu Ile

50 55 60

Gly Pro Pro Gly Gln Gln Gly Lys Lys Gly Ala Pro Gly Met Pro Gly

65 70 75 80

Leu Met Gly Ser Asn Gly Ser Pro Gly Gln Pro Gly Thr Pro Gly Ser

85 90 95

Lys Gly Ser Lys Gly Glu Pro Gly Ile Gln Gly Met Pro Gly Ala Ser

100 105 110

Gly Leu Lys Gly Glu Pro Gly Ala Thr Gly Ser Pro Gly Glu Pro Gly

115 120 125

Tyr Met Gly Leu Pro Gly Ile Gln Gly Lys Lys Gly Asp Lys Gly Asn

130 135 140

Gln Gly Glu Lys Gly Ile Gln Gly Gln Lys Gly Glu Asn Gly Arg Gln

145 150 155 160

Gly Ile Pro Gly Gln Gln Gly Ile Gln Gly His His Gly Ala Lys Gly

165 170 175

Glu Arg Gly Glu Lys Gly Glu Pro Gly Val Arg

180 185

<210> 17

<211> 639

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 17

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60

cagtatgaag atatgggtcc gcctggtagc cgtggtgcaa gtggtccggc aggcgttcgt 120

ggtccgaatg gtgatgcagg tcgtccgggt gaaccgggtc tgatgggtcc tcgtggtctg 180

cctggttcac cgggtaatat tggtcctgca ggtaaagaag gtccggttgg tctgccaggt 240

attgatggcc gtccgggtcc gattggtcca gccggtgcac gtggtgaacc tggcaatatt 300

ggttttccgg gtcctaaagg tccgaccggt gatccgggta aaaatggtga taaaggtcat 360

gcaggtctgg caggcgcacg cggtgcacct ggtccggatg gtaataatgg tgcacagggt 420

ccaccgggtc cgcagggtgt tcaaggtggt aaaggcgaac agggtcctgc cggtcctccg 480

ggttttcagg gactgcctgg tccgagcggt cctgcgggtg aagttggtaa acctggtgaa 540

cgcggtctgc atggtgaatt tggcctgcct gggcctgcag gtccgcgtgg cgaacgtggt 600

ccgccaggtg aaagcggtgc agcaggtccg acaggttaa 639

<210> 18

<211> 212

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 18

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp Met Gly Pro Pro Gly Ser Arg Gly

20 25 30

Ala Ser Gly Pro Ala Gly Val Arg Gly Pro Asn Gly Asp Ala Gly Arg

35 40 45

Pro Gly Glu Pro Gly Leu Met Gly Pro Arg Gly Leu Pro Gly Ser Pro

50 55 60

Gly Asn Ile Gly Pro Ala Gly Lys Glu Gly Pro Val Gly Leu Pro Gly

65 70 75 80

Ile Asp Gly Arg Pro Gly Pro Ile Gly Pro Ala Gly Ala Arg Gly Glu

85 90 95

Pro Gly Asn Ile Gly Phe Pro Gly Pro Lys Gly Pro Thr Gly Asp Pro

100 105 110

Gly Lys Asn Gly Asp Lys Gly His Ala Gly Leu Ala Gly Ala Arg Gly

115 120 125

Ala Pro Gly Pro Asp Gly Asn Asn Gly Ala Gln Gly Pro Pro Gly Pro

130 135 140

Gln Gly Val Gln Gly Gly Lys Gly Glu Gln Gly Pro Ala Gly Pro Pro

145 150 155 160

Gly Phe Gln Gly Leu Pro Gly Pro Ser Gly Pro Ala Gly Glu Val Gly

165 170 175

Lys Pro Gly Glu Arg Gly Leu His Gly Glu Phe Gly Leu Pro Gly Pro

180 185 190

Ala Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly Glu Ser Gly Ala Ala

195 200 205

Gly Pro Thr Gly

210

<210> 19

<211> 567

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 19

atgggtccgc ctggtagccg tggtgcaagt ggtccggcag gcgttcgtgg tccgaatggt 60

gatgcaggtc gtccgggtga accgggtctg atgggtcctc gtggtctgcc tggttcaccg 120

ggtaatattg gtcctgcagg taaagaaggt ccggttggtc tgccaggtat tgatggccgt 180

ccgggtccga ttggtccagc cggtgcacgt ggtgaacctg gcaatattgg ttttccgggt 240

cctaaaggtc cgaccggtga tccgggtaaa aatggtgata aaggtcatgc aggtctggca 300

ggcgcacgcg gtgcacctgg tccggatggt aataatggtg cacagggtcc accgggtccg 360

cagggtgttc aaggtggtaa aggcgaacag ggtcctgccg gtcctccggg ttttcaggga 420

ctgcctggtc cgagcggtcc tgcgggtgaa gttggtaaac ctggtgaacg cggtctgcat 480

ggtgaatttg gcctgcctgg gcctgcaggt ccgcgtggcg aacgtggtcc gccaggtgaa 540

agcggtgcag caggtccgac aggttaa 567

<210> 20

<211> 188

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 20

Met Gly Pro Pro Gly Ser Arg Gly Ala Ser Gly Pro Ala Gly Val Arg

1 5 10 15

Gly Pro Asn Gly Asp Ala Gly Arg Pro Gly Glu Pro Gly Leu Met Gly

20 25 30

Pro Arg Gly Leu Pro Gly Ser Pro Gly Asn Ile Gly Pro Ala Gly Lys

35 40 45

Glu Gly Pro Val Gly Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile

50 55 60

Gly Pro Ala Gly Ala Arg Gly Glu Pro Gly Asn Ile Gly Phe Pro Gly

65 70 75 80

Pro Lys Gly Pro Thr Gly Asp Pro Gly Lys Asn Gly Asp Lys Gly His

85 90 95

Ala Gly Leu Ala Gly Ala Arg Gly Ala Pro Gly Pro Asp Gly Asn Asn

100 105 110

Gly Ala Gln Gly Pro Pro Gly Pro Gln Gly Val Gln Gly Gly Lys Gly

115 120 125

Glu Gln Gly Pro Ala Gly Pro Pro Gly Phe Gln Gly Leu Pro Gly Pro

130 135 140

Ser Gly Pro Ala Gly Glu Val Gly Lys Pro Gly Glu Arg Gly Leu His

145 150 155 160

Gly Glu Phe Gly Leu Pro Gly Pro Ala Gly Pro Arg Gly Glu Arg Gly

165 170 175

Pro Pro Gly Glu Ser Gly Ala Ala Gly Pro Thr Gly

180 185

<210> 21

<211> 612

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 21

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcg 60

cagtatgaag atggttttca gggtcctgcc ggtgaaccgg gtgaacctgg tcagacaggt 120

ccggcaggcg cacgtggtcc tgcaggtcct cctggtaaag ccggtgaaga tggtcatccg 180

ggtaaaccgg gtcgtcctgg tgaacgtggt gttgttggtc cgcagggtgc ccgtggtttt 240

ccgggtactc cgggtctgcc aggttttaaa ggtattcgtg gtcataatgg tctggatggt 300

ctgaaaggtc agcctggtgc accgggtgtt aaaggtgaac caggtgctcc gggtgaaaat 360

ggcacaccgg gtcagaccgg tgcgcgtggt ctgcctggcg aacgcggtcg tgttggtgca 420

cctggtccag ccggtgcacg cggtagtgat ggtagcgttg gtccggttgg tccagcgggt 480

ccgattggta gcgcaggtcc accgggtttt ccaggcgcac cgggtccgaa aggtgaaatt 540

ggtgcagttg gtaatgcagg ccctgccggt ccagcaggac cgcgtggtga agttggcctg 600

cctggtctgt aa 612

<210> 22

<211> 203

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 22

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gln Tyr Glu Asp Gly Phe Gln Gly Pro Ala Gly Glu

20 25 30

Pro Gly Glu Pro Gly Gln Thr Gly Pro Ala Gly Ala Arg Gly Pro Ala

35 40 45

Gly Pro Pro Gly Lys Ala Gly Glu Asp Gly His Pro Gly Lys Pro Gly

50 55 60

Arg Pro Gly Glu Arg Gly Val Val Gly Pro Gln Gly Ala Arg Gly Phe

65 70 75 80

Pro Gly Thr Pro Gly Leu Pro Gly Phe Lys Gly Ile Arg Gly His Asn

85 90 95

Gly Leu Asp Gly Leu Lys Gly Gln Pro Gly Ala Pro Gly Val Lys Gly

100 105 110

Glu Pro Gly Ala Pro Gly Glu Asn Gly Thr Pro Gly Gln Thr Gly Ala

115 120 125

Arg Gly Leu Pro Gly Glu Arg Gly Arg Val Gly Ala Pro Gly Pro Ala

130 135 140

Gly Ala Arg Gly Ser Asp Gly Ser Val Gly Pro Val Gly Pro Ala Gly

145 150 155 160

Pro Ile Gly Ser Ala Gly Pro Pro Gly Phe Pro Gly Ala Pro Gly Pro

165 170 175

Lys Gly Glu Ile Gly Ala Val Gly Asn Ala Gly Pro Ala Gly Pro Ala

180 185 190

Gly Pro Arg Gly Glu Val Gly Leu Pro Gly Leu

195 200

<210> 23

<211> 540

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 23

ggttttcagg gtcctgccgg tgaaccgggt gaacctggtc agacaggtcc ggcaggcgca 60

cgtggtcctg caggtcctcc tggtaaagcc ggtgaagatg gtcatccggg taaaccgggt 120

cgtcctggtg aacgtggtgt tgttggtccg cagggtgccc gtggttttcc gggtactccg 180

ggtctgccag gttttaaagg tattcgtggt cataatggtc tggatggtct gaaaggtcag 240

cctggtgcac cgggtgttaa aggtgaacca ggtgctccgg gtgaaaatgg cacaccgggt 300

cagaccggtg cgcgtggtct gcctggcgaa cgcggtcgtg ttggtgcacc tggtccagcc 360

ggtgcacgcg gtagtgatgg tagcgttggt ccggttggtc cagcgggtcc gattggtagc 420

gcaggtccac cgggttttcc aggcgcaccg ggtccgaaag gtgaaattgg tgcagttggt 480

aatgcaggcc ctgccggtcc agcaggaccg cgtggtgaag ttggcctgcc tggtctgtaa 540

<210> 24

<211> 179

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 24

Gly Phe Gln Gly Pro Ala Gly Glu Pro Gly Glu Pro Gly Gln Thr Gly

1 5 10 15

Pro Ala Gly Ala Arg Gly Pro Ala Gly Pro Pro Gly Lys Ala Gly Glu

20 25 30

Asp Gly His Pro Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Val Val

35 40 45

Gly Pro Gln Gly Ala Arg Gly Phe Pro Gly Thr Pro Gly Leu Pro Gly

50 55 60

Phe Lys Gly Ile Arg Gly His Asn Gly Leu Asp Gly Leu Lys Gly Gln

65 70 75 80

Pro Gly Ala Pro Gly Val Lys Gly Glu Pro Gly Ala Pro Gly Glu Asn

85 90 95

Gly Thr Pro Gly Gln Thr Gly Ala Arg Gly Leu Pro Gly Glu Arg Gly

100 105 110

Arg Val Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Ser Asp Gly Ser

115 120 125

Val Gly Pro Val Gly Pro Ala Gly Pro Ile Gly Ser Ala Gly Pro Pro

130 135 140

Gly Phe Pro Gly Ala Pro Gly Pro Lys Gly Glu Ile Gly Ala Val Gly

145 150 155 160

Asn Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Glu Val Gly Leu

165 170 175

Pro Gly Leu

<210> 25

<211> 228

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 25

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Gly Asp Gln Gly Pro Val Gly Arg Thr Gly Glu Val Gly

20 25 30

Ala Val Gly Pro Pro Gly Phe Ala Gly Glu Lys Gly Pro Ser Gly Glu

35 40 45

Ala Gly Thr Ala Gly Pro Pro Gly Thr Pro Gly Pro Gln Gly Leu Leu

50 55 60

Gly Ala Pro Gly Ile Leu Gly Leu Pro Gly Ser Arg Gly Glu Arg Gly

65 70 75 80

Leu Pro Gly Val Ala Gly Ala Val Gly Glu Pro Gly Pro Leu Gly Ile

85 90 95

Ala Gly Pro Pro Gly Ala Arg Gly Pro Pro Gly Ala Val Gly Ser Pro

100 105 110

Gly Val Asn Gly Ala Pro Gly Glu Ala Gly Arg Asp Gly Asn Pro Gly

115 120 125

Asn Asp Gly Pro Pro Gly Arg Asp Gly Gln Pro Gly His Lys Gly Glu

130 135 140

Arg Gly Tyr Pro Gly Asn Ile Gly Pro Val Gly Ala Ala Gly Ala Pro

145 150 155 160

Gly Pro His Gly Pro Val Gly Pro Ala Gly Lys His Gly Asn Arg Gly

165 170 175

Glu Thr Gly Pro Ser Gly Pro Val Gly Pro Ala Gly Ala Val Gly Pro

180 185 190

Arg Gly Pro Ser Gly Pro Gln Gly Ile Arg Gly Asp Lys Gly Glu Pro

195 200 205

Gly Glu Lys Gly Pro Arg Gly Leu Pro Gly Leu Gly Asp Tyr Lys Asp

210 215 220

Asp Asp Asp Lys

225

<210> 26

<211> 687

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 26

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcgggt 60

gatcagggtc cggttggtcg taccggtgaa gttggtgcag tcgggccgcc gggttttgcg 120

ggtgaaaaag gcccgtcagg tgaagcaggc accgctggcc ctcctggcac gcctggccca 180

cagggtttac tgggcgcacc tggaattctg ggactgccgg gcagccgtgg agaacgcggt 240

ttaccaggtg ttgccggtgc cgttggtgaa cctggtccac tgggcattgc agggccgcct 300

ggcgcacggg gaccgcctgg tgctgttggt agtccgggtg tgaatggtgc tccgggtgaa 360

gccggtcgtg acggtaatcc gggaaatgac ggcccgccag gccgcgatgg tcagccgggt 420

cataaaggtg agcgtggtta cccaggtaat attggtccag tcggtgccgc cggtgcgccg 480

ggtcctcatg gccctgtcgg tccagccggt aaacatggta atcgcggtga gacaggtccg 540

tcaggaccag tgggccctgc tggcgcagtc ggtccgcgcg ggccgagtgg ccctcagggt 600

attcgtggcg ataaagggga accgggcgaa aaagggccgc ggggtctgcc aggcctgggt 660

gactacaaag acgacgacga caaataa 687

<210> 27

<211> 228

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 27

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Lys Gly His Asn Gly Leu Gln Gly Leu Pro Gly Ile Ala

20 25 30

Gly His His Gly Asp Gln Gly Ala Pro Gly Ser Val Gly Pro Ala Gly

35 40 45

Pro Arg Gly Pro Ala Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Arg

50 55 60

Thr Gly His Pro Gly Thr Val Gly Pro Ala Gly Ile Arg Gly Pro Gln

65 70 75 80

Gly His Gln Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly

85 90 95

Pro Pro Gly Val Ser Gly Gly Gly Tyr Asp Phe Gly Tyr Asp Gly Asp

100 105 110

Phe Tyr Arg Ala Asp Gln Pro Arg Ser Ala Pro Ser Leu Arg Pro Lys

115 120 125

Asp Tyr Glu Val Asp Ala Thr Leu Lys Ser Leu Asn Asn Gln Ile Glu

130 135 140

Thr Leu Leu Thr Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys

145 150 155 160

Arg Asp Leu Arg Leu Ser His Pro Glu Trp Ser Ser Gly Tyr Tyr Trp

165 170 175

Ile Asp Pro Asn Gln Gly Cys Thr Met Asp Ala Ile Lys Val Tyr Cys

180 185 190

Asp Phe Ser Thr Gly Glu Thr Cys Ile Arg Ala Gln Pro Glu Asn Ile

195 200 205

Pro Ala Lys Asn Trp Tyr Arg Ser Ser Lys Asp Gly Asp Tyr Lys Asp

210 215 220

Asp Asp Asp Lys

225

<210> 28

<211> 687

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 28

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcgaaa 60

ggtcacaatg gactgcaagg cctgccaggt attgcaggtc atcatggtga tcaaggtgcc 120

ccgggaagcg ttggtccggc ggggccgaga ggccctgcgg gaccttcagg tccggcaggc 180

aaagatggtc ggacaggcca tccgggcacc gttggccctg caggaattcg tggaccgcag 240

ggtcatcagg gacctgctgg tccgccaggt cccccgggcc ctccgggacc accgggtgtt 300

agtggtggtg gttatgattt tggctatgat ggtgattttt atcgtgcaga tcagccgcgt 360

agcgcaccga gcctgcgtcc taaagattat gaagttgatg caaccctgaa aagcctgaat 420

aatcagattg aaacactgct gacaccggaa ggtagccgta aaaatccggc ccgtacctgt 480

cgtgatctgc gtctgagcca cccggaatgg agcagcggtt attattggat tgatccgaat 540

caaggttgta ccatggatgc aattaaagtt tattgtgatt ttagcacagg tgaaacatgt 600

atccgtgcac agccggaaaa tattccggcc aaaaattggt atcgtagtag caaagatggt 660

gactacaaag acgacgacga caaataa 687

<210> 29

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 29

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Tyr Glu Val Asp Ala Thr Leu Lys Ser Leu Asn Asn Gln

20 25 30

Ile Glu Thr Leu Leu Thr Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg

35 40 45

Thr Cys Arg Asp Leu Arg Leu Ser His Pro Glu Trp Ser Ser Gly Tyr

50 55 60

Tyr Trp Ile Asp Pro Asn Gln Gly Cys Thr Met Asp Ala Ile Lys Val

65 70 75 80

Tyr Cys Asp Phe Ser Thr Gly Glu Thr Cys Ile Arg Ala Gln Pro Glu

85 90 95

Asn Ile Pro Ala Lys Asn Trp Tyr Arg Ser Ser Lys Asp Lys Lys His

100 105 110

Val Trp Leu Gly Glu Thr Ile Asn Ala Gly Ser Gln Phe Glu Tyr Asn

115 120 125

Val Glu Gly Val Thr Ser Lys Glu Met Ala Thr Gln Leu Ala Phe Met

130 135 140

Arg Leu Leu Ala Asn Tyr Ala Ser Gln Asn Ile Thr Tyr His Cys Lys

145 150 155 160

Asn Ser Ile Ala Tyr Met Asp Glu Glu Thr Gly Asn Leu Lys Lys Ala

165 170 175

Val Ile Leu Gln Gly Ser Asn Asp Val Glu Leu Val Ala Glu Gly Asn

180 185 190

Ser Arg Phe Thr Tyr Thr Val Leu Val Asp Gly Cys Ser Lys Lys Thr

195 200 205

Asn Glu Trp Gly Lys Thr Ile Ile Glu Tyr Lys Thr Asn Lys Pro Ser

210 215 220

Arg Leu Pro Phe Leu Asp Ile Ala Pro Leu Asp Ile Gly Gly Ala Asp

225 230 235 240

Gln Glu Phe Phe Val Asp Ile Gly Pro Val Cys Phe Lys Gly Asp Tyr

245 250 255

Lys Asp Asp Asp Asp Lys

260

<210> 30

<211> 788

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 30

tgaaaaagat ttggctggcg ctggctggtt tagttttagc gtttagcgca tcggcgtatg 60

aagttgatgc aaccctgaaa agcctgaata atcagattga aacactgctg acaccggaag 120

gtagccgtaa aaatccggcc cgtacctgtc gtgatctgcg tctgagccac ccggaatgga 180

gcagcggtta ttattggatt gatccgaatc aaggttgtac catggatgca attaaagttt 240

attgtgattt tagcacaggt gaaacatgta tccgtgcaca gccggaaaat attccggcca 300

aaaattggta tcgtagtagc aaagataaaa aacatgtgtg gctgggtgaa accattaatg 360

caggtagcca gtttgaatac aatgttgaag gtgttaccag caaagaaatg gcaacacagc 420

tggcatttat gcgtctgctg gcaaattatg caagccagaa tattacatat cattgtaaaa 480

atagcattgc atatatggat gaagaaaccg gtaatctgaa aaaagcagtt attctgcagg 540

gtagcaatga tgttgaactg gttgccgaag gtaatagccg ttttacatat accgttctgg 600

ttgatggttg tagcaaaaaa accaatgaat ggggtaaaac catcattgaa tataaaacca 660

acaaaccgag ccgtctgccg tttctggata tcgctccgct ggatattggt ggtgccgatc 720

aggaattttt tgtcgatatc ggtcctgtgt gttttaaagg tgactacaaa gacgacgacg 780

acaaataa 788

<210> 31

<211> 957

<212> PRT

<213> Chile person

<400> 31

Met Ala His Tyr Ile Thr Phe Leu Cys Met Val Leu Val Leu Leu Leu

1 5 10 15

Gln Asn Ser Val Leu Ala Glu Asp Gly Glu Val Arg Ser Ser Cys Arg

20 25 30

Thr Ala Pro Thr Asp Leu Val Phe Ile Leu Asp Gly Ser Tyr Ser Val

35 40 45

Gly Pro Glu Asn Phe Glu Ile Val Lys Lys Trp Leu Val Asn Ile Thr

50 55 60

Lys Asn Phe Asp Ile Gly Pro Lys Phe Ile Gln Val Gly Val Val Gln

65 70 75 80

Tyr Ser Asp Tyr Pro Val Leu Glu Ile Pro Leu Gly Ser Tyr Asp Ser

85 90 95

Gly Glu His Leu Thr Ala Ala Val Glu Ser Ile Leu Tyr Leu Gly Gly

100 105 110

Asn Thr Lys Thr Gly Lys Ala Ile Gln Phe Ala Leu Asp Tyr Leu Phe

115 120 125

Ala Lys Ser Ser Arg Phe Leu Thr Lys Ile Ala Val Val Leu Thr Asp

130 135 140

Gly Lys Ser Gln Asp Asp Val Lys Asp Ala Ala Gln Ala Ala Arg Asp

145 150 155 160

Ser Lys Ile Thr Leu Phe Ala Ile Gly Val Gly Ser Glu Thr Glu Asp

165 170 175

Ala Glu Leu Arg Ala Ile Ala Asn Lys Pro Ser Ser Thr Tyr Val Phe

180 185 190

Tyr Val Glu Asp Tyr Ile Ala Ile Ser Lys Ile Arg Glu Val Met Lys

195 200 205

Gln Lys Leu Cys Glu Glu Ser Val Cys Pro Thr Arg Ile Pro Val Ala

210 215 220

Ala Arg Asp Glu Arg Gly Phe Asp Ile Leu Leu Gly Leu Asp Val Asn

225 230 235 240

Lys Lys Val Lys Lys Arg Ile Gln Leu Ser Pro Lys Lys Ile Lys Gly

245 250 255

Tyr Glu Val Thr Ser Lys Val Asp Leu Ser Glu Leu Thr Ser Asn Val

260 265 270

Phe Pro Glu Gly Leu Pro Pro Ser Tyr Val Phe Val Ser Thr Gln Arg

275 280 285

Phe Lys Val Lys Lys Ile Trp Asp Leu Trp Arg Ile Leu Thr Ile Asp

290 295 300

Gly Arg Pro Gln Ile Ala Val Thr Leu Asn Gly Val Asp Lys Ile Leu

305 310 315 320

Leu Phe Thr Thr Thr Ser Val Ile Asn Gly Ser Gln Val Val Thr Phe

325 330 335

Ala Asn Pro Gln Val Lys Thr Leu Phe Asp Glu Gly Trp His Gln Ile

340 345 350

Arg Leu Leu Val Thr Glu Gln Asp Val Thr Leu Tyr Ile Asp Asp Gln

355 360 365

Gln Ile Glu Asn Lys Pro Leu His Pro Val Leu Gly Ile Leu Ile Asn

370 375 380

Gly Gln Thr Gln Ile Gly Lys Tyr Ser Gly Lys Glu Glu Thr Val Gln

385 390 395 400

Phe Asp Val Gln Lys Leu Arg Ile Tyr Cys Asp Pro Glu Gln Asn Asn

405 410 415

Arg Glu Thr Ala Cys Glu Ile Pro Gly Phe Asn Gly Glu Cys Leu Asn

420 425 430

Gly Pro Ser Asp Val Gly Ser Thr Pro Ala Pro Cys Ile Cys Pro Pro

435 440 445

Gly Lys Pro Gly Leu Gln Gly Pro Lys Gly Asp Pro Gly Leu Pro Gly

450 455 460

Asn Pro Gly Tyr Pro Gly Gln Pro Gly Gln Asp Gly Lys Pro Gly Tyr

465 470 475 480

Gln Gly Ile Ala Gly Thr Pro Gly Val Pro Gly Ser Pro Gly Ile Gln

485 490 495

Gly Ala Arg Gly Leu Pro Gly Tyr Lys Gly Glu Pro Gly Arg Asp Gly

500 505 510

Asp Lys Gly Asp Arg Gly Leu Pro Gly Phe Pro Gly Leu His Gly Met

515 520 525

Pro Gly Ser Lys Gly Glu Met Gly Ala Lys Gly Asp Lys Gly Ser Pro

530 535 540

Gly Phe Tyr Gly Lys Lys Gly Ala Lys Gly Glu Lys Gly Asn Ala Gly

545 550 555 560

Phe Pro Gly Leu Pro Gly Pro Ala Gly Glu Pro Gly Arg His Gly Lys

565 570 575

Asp Gly Leu Met Gly Ser Pro Gly Phe Lys Gly Glu Ala Gly Ser Pro

580 585 590

Gly Ala Pro Gly Gln Asp Gly Thr Arg Gly Glu Pro Gly Ile Pro Gly

595 600 605

Phe Pro Gly Asn Arg Gly Leu Met Gly Gln Lys Gly Glu Ile Gly Pro

610 615 620

Pro Gly Gln Gln Gly Lys Lys Gly Ala Pro Gly Met Pro Gly Leu Met

625 630 635 640

Gly Ser Asn Gly Ser Pro Gly Gln Pro Gly Thr Pro Gly Ser Lys Gly

645 650 655

Ser Lys Gly Glu Pro Gly Ile Gln Gly Met Pro Gly Ala Ser Gly Leu

660 665 670

Lys Gly Glu Pro Gly Ala Thr Gly Ser Pro Gly Glu Pro Gly Tyr Met

675 680 685

Gly Leu Pro Gly Ile Gln Gly Lys Lys Gly Asp Lys Gly Asn Gln Gly

690 695 700

Glu Lys Gly Ile Gln Gly Gln Lys Gly Glu Asn Gly Arg Gln Gly Ile

705 710 715 720

Pro Gly Gln Gln Gly Ile Gln Gly His His Gly Ala Lys Gly Glu Arg

725 730 735

Gly Glu Lys Gly Glu Pro Gly Val Arg Gly Ala Ile Gly Ser Lys Gly

740 745 750

Glu Ser Gly Val Asp Gly Leu Met Gly Pro Ala Gly Pro Lys Gly Gln

755 760 765

Pro Gly Asp Pro Gly Pro Gln Gly Pro Pro Gly Leu Asp Gly Lys Pro

770 775 780

Gly Arg Glu Phe Ser Glu Gln Phe Ile Arg Gln Val Cys Thr Asp Val

785 790 795 800

Ile Arg Ala Gln Leu Pro Val Leu Leu Gln Ser Gly Arg Ile Arg Asn

805 810 815

Cys Asp His Cys Leu Ser Gln His Gly Ser Pro Gly Ile Pro Gly Pro

820 825 830

Pro Gly Pro Ile Gly Pro Glu Gly Pro Arg Gly Leu Pro Gly Leu Pro

835 840 845

Gly Arg Asp Gly Val Pro Gly Leu Val Gly Val Pro Gly Arg Pro Gly

850 855 860

Val Arg Gly Leu Lys Gly Leu Pro Gly Arg Asn Gly Glu Lys Gly Ser

865 870 875 880

Gln Gly Phe Gly Tyr Pro Gly Glu Gln Gly Pro Pro Gly Pro Pro Gly

885 890 895

Pro Glu Gly Pro Pro Gly Ile Ser Lys Glu Gly Pro Pro Gly Asp Pro

900 905 910

Gly Leu Pro Gly Lys Asp Gly Asp His Gly Lys Pro Gly Ile Gln Gly

915 920 925

Gln Pro Gly Pro Pro Gly Ile Cys Asp Pro Ser Leu Cys Phe Ser Val

930 935 940

Ile Ala Arg Arg Asp Pro Phe Arg Lys Gly Pro Asn Tyr

945 950 955

<210> 32

<211> 1366

<212> PRT

<213> Chile person

<400> 32

Met Leu Ser Phe Val Asp Thr Arg Thr Leu Leu Leu Leu Ala Val Thr

1 5 10 15

Leu Cys Leu Ala Thr Cys Gln Ser Leu Gln Glu Glu Thr Val Arg Lys

20 25 30

Gly Pro Ala Gly Asp Arg Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly

35 40 45

Pro Pro Gly Arg Asp Gly Glu Asp Gly Pro Thr Gly Pro Pro Gly Pro

50 55 60

Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Ala Gln

65 70 75 80

Tyr Asp Gly Lys Gly Val Gly Leu Gly Pro Gly Pro Met Gly Leu Met

85 90 95

Gly Pro Arg Gly Pro Pro Gly Ala Ala Gly Ala Pro Gly Pro Gln Gly

100 105 110

Phe Gln Gly Pro Ala Gly Glu Pro Gly Glu Pro Gly Gln Thr Gly Pro

115 120 125

Ala Gly Ala Arg Gly Pro Ala Gly Pro Pro Gly Lys Ala Gly Glu Asp

130 135 140

Gly His Pro Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Val Val Gly

145 150 155 160

Pro Gln Gly Ala Arg Gly Phe Pro Gly Thr Pro Gly Leu Pro Gly Phe

165 170 175

Lys Gly Ile Arg Gly His Asn Gly Leu Asp Gly Leu Lys Gly Gln Pro

180 185 190

Gly Ala Pro Gly Val Lys Gly Glu Pro Gly Ala Pro Gly Glu Asn Gly

195 200 205

Thr Pro Gly Gln Thr Gly Ala Arg Gly Leu Pro Gly Glu Arg Gly Arg

210 215 220

Val Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Ser Asp Gly Ser Val

225 230 235 240

Gly Pro Val Gly Pro Ala Gly Pro Ile Gly Ser Ala Gly Pro Pro Gly

245 250 255

Phe Pro Gly Ala Pro Gly Pro Lys Gly Glu Ile Gly Ala Val Gly Asn

260 265 270

Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Glu Val Gly Leu Pro

275 280 285

Gly Leu Ser Gly Pro Val Gly Pro Pro Gly Asn Pro Gly Ala Asn Gly

290 295 300

Leu Thr Gly Ala Lys Gly Ala Ala Gly Leu Pro Gly Val Ala Gly Ala

305 310 315 320

Pro Gly Leu Pro Gly Pro Arg Gly Ile Pro Gly Pro Val Gly Ala Ala

325 330 335

Gly Ala Thr Gly Ala Arg Gly Leu Val Gly Glu Pro Gly Pro Ala Gly

340 345 350

Ser Lys Gly Glu Ser Gly Asn Lys Gly Glu Pro Gly Ser Ala Gly Pro

355 360 365

Gln Gly Pro Pro Gly Pro Ser Gly Glu Glu Gly Lys Arg Gly Pro Asn

370 375 380

Gly Glu Ala Gly Ser Ala Gly Pro Pro Gly Pro Pro Gly Leu Arg Gly

385 390 395 400

Ser Pro Gly Ser Arg Gly Leu Pro Gly Ala Asp Gly Arg Ala Gly Val

405 410 415

Met Gly Pro Pro Gly Ser Arg Gly Ala Ser Gly Pro Ala Gly Val Arg

420 425 430

Gly Pro Asn Gly Asp Ala Gly Arg Pro Gly Glu Pro Gly Leu Met Gly

435 440 445

Pro Arg Gly Leu Pro Gly Ser Pro Gly Asn Ile Gly Pro Ala Gly Lys

450 455 460

Glu Gly Pro Val Gly Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile

465 470 475 480

Gly Pro Ala Gly Ala Arg Gly Glu Pro Gly Asn Ile Gly Phe Pro Gly

485 490 495

Pro Lys Gly Pro Thr Gly Asp Pro Gly Lys Asn Gly Asp Lys Gly His

500 505 510

Ala Gly Leu Ala Gly Ala Arg Gly Ala Pro Gly Pro Asp Gly Asn Asn

515 520 525

Gly Ala Gln Gly Pro Pro Gly Pro Gln Gly Val Gln Gly Gly Lys Gly

530 535 540

Glu Gln Gly Pro Pro Gly Pro Pro Gly Phe Gln Gly Leu Pro Gly Pro

545 550 555 560

Ser Gly Pro Ala Gly Glu Val Gly Lys Pro Gly Glu Arg Gly Leu His

565 570 575

Gly Glu Phe Gly Leu Pro Gly Pro Ala Gly Pro Arg Gly Glu Arg Gly

580 585 590

Pro Pro Gly Glu Ser Gly Ala Ala Gly Pro Thr Gly Pro Ile Gly Ser

595 600 605

Arg Gly Pro Ser Gly Pro Pro Gly Pro Asp Gly Asn Lys Gly Glu Pro

610 615 620

Gly Val Val Gly Ala Val Gly Thr Ala Gly Pro Ser Gly Pro Ser Gly

625 630 635 640

Leu Pro Gly Glu Arg Gly Ala Ala Gly Ile Pro Gly Gly Lys Gly Glu

645 650 655

Lys Gly Glu Pro Gly Leu Arg Gly Glu Ile Gly Asn Pro Gly Arg Asp

660 665 670

Gly Ala Arg Gly Ala Pro Gly Ala Val Gly Ala Pro Gly Pro Ala Gly

675 680 685

Ala Thr Gly Asp Arg Gly Glu Ala Gly Ala Ala Gly Pro Ala Gly Pro

690 695 700

Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Val Gly Pro Ala

705 710 715 720

Gly Pro Asn Gly Phe Ala Gly Pro Ala Gly Ala Ala Gly Gln Pro Gly

725 730 735

Ala Lys Gly Glu Arg Gly Ala Lys Gly Pro Lys Gly Glu Asn Gly Val

740 745 750

Val Gly Pro Thr Gly Pro Val Gly Ala Ala Gly Pro Ala Gly Pro Asn

755 760 765

Gly Pro Pro Gly Pro Ala Gly Ser Arg Gly Asp Gly Gly Pro Pro Gly

770 775 780

Met Thr Gly Phe Pro Gly Ala Ala Gly Arg Thr Gly Pro Pro Gly Pro

785 790 795 800

Ser Gly Ile Ser Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu

805 810 815

Gly Leu Arg Gly Pro Arg Gly Asp Gln Gly Pro Val Gly Arg Thr Gly

820 825 830

Glu Val Gly Ala Val Gly Pro Pro Gly Phe Ala Gly Glu Lys Gly Pro

835 840 845

Ser Gly Glu Ala Gly Thr Ala Gly Pro Pro Gly Thr Pro Gly Pro Gln

850 855 860

Gly Leu Leu Gly Ala Pro Gly Ile Leu Gly Leu Pro Gly Ser Arg Gly

865 870 875 880

Glu Arg Gly Leu Pro Gly Val Ala Gly Ala Val Gly Glu Pro Gly Pro

885 890 895

Leu Gly Ile Ala Gly Pro Pro Gly Ala Arg Gly Pro Pro Gly Ala Val

900 905 910

Gly Ser Pro Gly Val Asn Gly Ala Pro Gly Glu Ala Gly Arg Asp Gly

915 920 925

Asn Pro Gly Asn Asp Gly Pro Pro Gly Arg Asp Gly Gln Pro Gly His

930 935 940

Lys Gly Glu Arg Gly Tyr Pro Gly Asn Ile Gly Pro Val Gly Ala Ala

945 950 955 960

Gly Ala Pro Gly Pro His Gly Pro Val Gly Pro Ala Gly Lys His Gly

965 970 975

Asn Arg Gly Glu Thr Gly Pro Ser Gly Pro Val Gly Pro Ala Gly Ala

980 985 990

Val Gly Pro Arg Gly Pro Ser Gly Pro Gln Gly Ile Arg Gly Asp Lys

995 1000 1005

Gly Glu Pro Gly Glu Lys Gly Pro Arg Gly Leu Pro Gly Leu Lys

1010 1015 1020

Gly His Asn Gly Leu Gln Gly Leu Pro Gly Ile Ala Gly His His

1025 1030 1035

Gly Asp Gln Gly Ala Pro Gly Ser Val Gly Pro Ala Gly Pro Arg

1040 1045 1050

Gly Pro Ala Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Arg Thr

1055 1060 1065

Gly His Pro Gly Thr Val Gly Pro Ala Gly Ile Arg Gly Pro Gln

1070 1075 1080

Gly His Gln Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro

1085 1090 1095

Gly Pro Pro Gly Val Ser Gly Gly Gly Tyr Asp Phe Gly Tyr Asp

1100 1105 1110

Gly Asp Phe Tyr Arg Ala Asp Gln Pro Arg Ser Ala Pro Ser Leu

1115 1120 1125

Arg Pro Lys Asp Tyr Glu Val Asp Ala Thr Leu Lys Ser Leu Asn

1130 1135 1140

Asn Gln Ile Glu Thr Leu Leu Thr Pro Glu Gly Ser Arg Lys Asn

1145 1150 1155

Pro Ala Arg Thr Cys Arg Asp Leu Arg Leu Ser His Pro Glu Trp

1160 1165 1170

Ser Ser Gly Tyr Tyr Trp Ile Asp Pro Asn Gln Gly Cys Thr Met

1175 1180 1185

Asp Ala Ile Lys Val Tyr Cys Asp Phe Ser Thr Gly Glu Thr Cys

1190 1195 1200

Ile Arg Ala Gln Pro Glu Asn Ile Pro Ala Lys Asn Trp Tyr Arg

1205 1210 1215

Ser Ser Lys Asp Lys Lys His Val Trp Leu Gly Glu Thr Ile Asn

1220 1225 1230

Ala Gly Ser Gln Phe Glu Tyr Asn Val Glu Gly Val Thr Ser Lys

1235 1240 1245

Glu Met Ala Thr Gln Leu Ala Phe Met Arg Leu Leu Ala Asn Tyr

1250 1255 1260

Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr

1265 1270 1275

Met Asp Glu Glu Thr Gly Asn Leu Lys Lys Ala Val Ile Leu Gln

1280 1285 1290

Gly Ser Asn Asp Val Glu Leu Val Ala Glu Gly Asn Ser Arg Phe

1295 1300 1305

Thr Tyr Thr Val Leu Val Asp Gly Cys Ser Lys Lys Thr Asn Glu

1310 1315 1320

Trp Gly Lys Thr Ile Ile Glu Tyr Lys Thr Asn Lys Pro Ser Arg

1325 1330 1335

Leu Pro Phe Leu Asp Ile Ala Pro Leu Asp Ile Gly Gly Ala Asp

1340 1345 1350

Gln Glu Phe Phe Val Asp Ile Gly Pro Val Cys Phe Lys

1355 1360 1365

<210> 33

<211> 429

<212> PRT

<213> Hydractinia carnea

<400> 33

Gly Pro Gln Gly Val Val Gly Ala Asp Gly Lys Asp Gly Thr Pro Gly

1 5 10 15

Glu Lys Gly Glu Gln Gly Arg Thr Gly Ala Ala Gly Lys Gln Gly Ser

20 25 30

Pro Gly Ala Asp Gly Ala Arg Gly Pro Leu Gly Ser Ile Gly Gln Gln

35 40 45

Gly Ala Arg Gly Glu Pro Gly Asp Pro Gly Ser Pro Gly Leu Arg Gly

50 55 60

Asp Thr Gly Leu Ala Gly Val Lys Gly Val Ala Gly Pro Ser Gly Arg

65 70 75 80

Pro Gly Gln Pro Gly Ala Asn Gly Leu Pro Gly Val Asn Gly Arg Gly

85 90 95

Gly Leu Arg Gly Lys Pro Gly Ala Lys Gly Ile Ala Gly Ser Asp Gly

100 105 110

Glu Ala Gly Glu Ser Gly Ala Pro Gly Gln Ser Gly Pro Thr Gly Pro

115 120 125

Arg Gly Gln Arg Gly Pro Ser Gly Glu Asp Gly Asn Pro Gly Leu Gln

130 135 140

Gly Leu Pro Gly Ser Asp Gly Glu Pro Gly Glu Glu Gly Gln Pro Gly

145 150 155 160

Arg Ser Gly Gln Pro Gly Gln Gln Gly Pro Arg Gly Ser Pro Gly Glu

165 170 175

Val Gly Pro Arg Gly Ser Lys Gly Pro Ser Gly Asp Arg Gly Asp Arg

180 185 190

Gly Glu Arg Gly Val Pro Gly Gln Thr Gly Ser Ala Gly Asn Val Gly

195 200 205

Glu Asp Gly Glu Gln Gly Gly Lys Gly Val Asp Gly Ala Ser Gly Pro

210 215 220

Ser Gly Ala Leu Gly Ala Arg Gly Pro Pro Gly Ser Arg Gly Asp Thr

225 230 235 240

Gly Ala Val Gly Pro Pro Gly Pro Thr Gly Arg Ser Gly Leu Pro Gly

245 250 255

Asn Ala Gly Gln Lys Gly Pro Ser Gly Glu Pro Gly Ser Pro Gly Lys

260 265 270

Ala Gly Ser Ala Gly Glu Gln Gly Pro Pro Gly Lys Asp Gly Ser Asn

275 280 285

Gly Glu Pro Gly Ser Pro Gly Lys Glu Gly Glu Arg Gly Leu Ala Gly

290 295 300

Pro Pro Gly Pro Asp Gly Arg Arg Gly Glu Thr Gly Ser Pro Gly Ile

305 310 315 320

Ala Gly Ala Leu Gly Lys Pro Gly Leu Glu Gly Pro Lys Gly Tyr Pro

325 330 335

Gly Leu Arg Gly Arg Asp Gly Thr Asn Gly Lys Arg Gly Glu Gln Gly

340 345 350

Glu Thr Gly Pro Asp Gly Val Arg Gly Ile Pro Gly Asn Asp Gly Gln

355 360 365

Ser Gly Lys Pro Gly Ile Asp Gly Ile Asp Gly Thr Asn Gly Gln Pro

370 375 380

Gly Glu Ala Gly Tyr Gln Gly Gly Arg Gly Thr Arg Gly Gln Leu Gly

385 390 395 400

Glu Thr Gly Asp Val Gly Gln Asn Gly Asp Arg Gly Ala Pro Gly Pro

405 410 415

Asp Gly Ser Lys Gly Ser Ala Gly Arg Pro Gly Leu Arg

420 425

<210> 34

<211> 6

<212> PRT

<213> Chile person

<400> 34

Val Gly Val Ala Pro Gly

1 5

<210> 35

<211> 12

<212> PRT

<213> Chile person

<400> 35

Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly

1 5 10

<210> 36

<211> 18

<212> PRT

<213> Chile person

<400> 36

Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala

1 5 10 15

Pro Gly

<210> 37

<211> 930

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 37

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcgggt 60

ccgcaacctg gggttccgtt aggttatccg attaaagcac cgaaactgcc cggcggttat 120

ggtctgccgt acacaaccgg taaactgccg tatggttatg gcccgggtgg agttgcgggt 180

gcagcaggta aagcgggtta tcctaccgga accggtgtag gtccgcaggc cgctgctgcc 240

gccgccgcaa aagcagcggc taaatttggc gccggagcag cgggtgttct gcctggagtt 300

ggtggtgcgg gcgtgccagg ggtacctggt gcaattccgg gtattggtgg tattgccggt 360

gtcggcaccc cggccgcggc agctgcggca gcggcggctg ccaaagctgc taaatacggt 420

gccgcggcgg gtctggtgcc aggaggtccg ggttttggtc cgggagtggt tggcgtgcct 480

ggcgcaggcg ttcctggtgt gggcgttcca ggtgcaggga ttcctgttgt gcctggtgcc 540

ggtattcccg gcgcggccgt tccgggggtg gttagcccgg aagccgcagc gaaggctgcg 600

gcaaaggcag caaagtatgg cgcacgccca ggagtcggcg tgggtggtat cccgacctat 660

ggggtgggcg cagggggttt tcctggtttc ggcgtaggtg taggaggtat accgggcgtg 720

gccggtgtac caggggttgg tggcgtccct ggtgttggcg gtgtgccagg tgttggtatt 780

tcaccggaag cacaggcagc agccgcagct aaggcagcga aatatggtgc cgccggcgca 840

ggagttttag gtgggctggt tccgggcccg caggcagctg tgccgggggt tccaggcacc 900

ggtggtgtcc ctggagtcgg tacgccgtaa 930

<210> 38

<211> 309

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 38

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Gly Pro Gln Pro Gly Val Pro Leu Gly Tyr Pro Ile Lys

20 25 30

Ala Pro Lys Leu Pro Gly Gly Tyr Gly Leu Pro Tyr Thr Thr Gly Lys

35 40 45

Leu Pro Tyr Gly Tyr Gly Pro Gly Gly Val Ala Gly Ala Ala Gly Lys

50 55 60

Ala Gly Tyr Pro Thr Gly Thr Gly Val Gly Pro Gln Ala Ala Ala Ala

65 70 75 80

Ala Ala Ala Lys Ala Ala Ala Lys Phe Gly Ala Gly Ala Ala Gly Val

85 90 95

Leu Pro Gly Val Gly Gly Ala Gly Val Pro Gly Val Pro Gly Ala Ile

100 105 110

Pro Gly Ile Gly Gly Ile Ala Gly Val Gly Thr Pro Ala Ala Ala Ala

115 120 125

Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly

130 135 140

Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro

145 150 155 160

Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val

165 170 175

Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser

180 185 190

Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala

195 200 205

Arg Pro Gly Val Gly Val Gly Gly Ile Pro Thr Tyr Gly Val Gly Ala

210 215 220

Gly Gly Phe Pro Gly Phe Gly Val Gly Val Gly Gly Ile Pro Gly Val

225 230 235 240

Ala Gly Val Pro Gly Val Gly Gly Val Pro Gly Val Gly Gly Val Pro

245 250 255

Gly Val Gly Ile Ser Pro Glu Ala Gln Ala Ala Ala Ala Ala Lys Ala

260 265 270

Ala Lys Tyr Gly Ala Ala Gly Ala Gly Val Leu Gly Gly Leu Val Pro

275 280 285

Gly Pro Gln Ala Ala Val Pro Gly Val Pro Gly Thr Gly Gly Val Pro

290 295 300

Gly Val Gly Thr Pro

305

<210> 39

<211> 873

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 39

ggtccgcaac ctggggttcc gttaggttat ccgattaaag caccgaaact gcccggcggt 60

tatggtctgc cgtacacaac cggtaaactg ccgtatggtt atggcccggg tggagttgcg 120

ggtgcagcag gtaaagcggg ttatcctacc ggaaccggtg taggtccgca ggccgctgct 180

gccgccgccg caaaagcagc ggctaaattt ggcgccggag cagcgggtgt tctgcctgga 240

gttggtggtg cgggcgtgcc aggggtacct ggtgcaattc cgggtattgg tggtattgcc 300

ggtgtcggca ccccggccgc ggcagctgcg gcagcggcgg ctgccaaagc tgctaaatac 360

ggtgccgcgg cgggtctggt gccaggaggt ccgggttttg gtccgggagt ggttggcgtg 420

cctggcgcag gcgttcctgg tgtgggcgtt ccaggtgcag ggattcctgt tgtgcctggt 480

gccggtattc ccggcgcggc cgttccgggg gtggttagcc cggaagccgc agcgaaggct 540

gcggcaaagg cagcaaagta tggcgcacgc ccaggagtcg gcgtgggtgg tatcccgacc 600

tatggggtgg gcgcaggggg ttttcctggt ttcggcgtag gtgtaggagg tataccgggc 660

gtggccggtg taccaggggt tggtggcgtc cctggtgttg gcggtgtgcc aggtgttggt 720

atttcaccgg aagcacaggc agcagccgca gctaaggcag cgaaatatgg tgccgccggc 780

gcaggagttt taggtgggct ggttccgggc ccgcaggcag ctgtgccggg ggttccaggc 840

accggtggtg tccctggagt cggtacgccg taa 873

<210> 40

<211> 290

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 40

Gly Pro Gln Pro Gly Val Pro Leu Gly Tyr Pro Ile Lys Ala Pro Lys

1 5 10 15

Leu Pro Gly Gly Tyr Gly Leu Pro Tyr Thr Thr Gly Lys Leu Pro Tyr

20 25 30

Gly Tyr Gly Pro Gly Gly Val Ala Gly Ala Ala Gly Lys Ala Gly Tyr

35 40 45

Pro Thr Gly Thr Gly Val Gly Pro Gln Ala Ala Ala Ala Ala Ala Ala

50 55 60

Lys Ala Ala Ala Lys Phe Gly Ala Gly Ala Ala Gly Val Leu Pro Gly

65 70 75 80

Val Gly Gly Ala Gly Val Pro Gly Val Pro Gly Ala Ile Pro Gly Ile

85 90 95

Gly Gly Ile Ala Gly Val Gly Thr Pro Ala Ala Ala Ala Ala Ala Ala

100 105 110

Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro

115 120 125

Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val Pro Gly Ala Gly

130 135 140

Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro Val Val Pro Gly

145 150 155 160

Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val Ser Pro Glu Ala

165 170 175

Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Arg Pro Gly

180 185 190

Val Gly Val Gly Gly Ile Pro Thr Tyr Gly Val Gly Ala Gly Gly Phe

195 200 205

Pro Gly Phe Gly Val Gly Val Gly Gly Ile Pro Gly Val Ala Gly Val

210 215 220

Pro Gly Val Gly Gly Val Pro Gly Val Gly Gly Val Pro Gly Val Gly

225 230 235 240

Ile Ser Pro Glu Ala Gln Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr

245 250 255

Gly Ala Ala Gly Ala Gly Val Leu Gly Gly Leu Val Pro Gly Pro Gln

260 265 270

Ala Ala Val Pro Gly Val Pro Gly Thr Gly Gly Val Pro Gly Val Gly

275 280 285

Thr Pro

290

<210> 41

<211> 238

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 41

Gly Pro Gly Gly Val Ala Ala Ala Ala Lys Ser Ala Ala Lys Val Ala

1 5 10 15

Ala Lys Ala Gln Leu Arg Ala Ala Ala Gly Leu Gly Ala Gly Ile Pro

20 25 30

Gly Leu Gly Val Gly Val Gly Val Pro Gly Leu Gly Val Gly Ala Gly

35 40 45

Val Pro Gly Leu Gly Val Gly Ala Gly Val Pro Gly Phe Gly Ala Gly

50 55 60

Ala Asp Glu Gly Val Arg Arg Ser Leu Ser Pro Glu Leu Arg Glu Gly

65 70 75 80

Asp Pro Ser Ser Ser Gln His Leu Pro Ser Thr Pro Ser Ser Pro Arg

85 90 95

Val Pro Gly Ala Leu Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala

100 105 110

Val Pro Gly Val Leu Gly Gly Leu Gly Ala Leu Gly Gly Val Gly Ile

115 120 125

Pro Gly Gly Val Val Gly Ala Gly Pro Ala Ala Ala Ala Ala Ala Ala

130 135 140

Lys Ala Ala Ala Lys Ala Ala Gln Phe Gly Leu Val Gly Ala Ala Gly

145 150 155 160

Leu Gly Gly Leu Gly Val Gly Gly Leu Gly Val Pro Gly Val Gly Gly

165 170 175

Leu Gly Gly Ile Pro Pro Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly

180 185 190

Ala Ala Gly Leu Gly Gly Val Leu Gly Gly Ala Gly Gln Phe Pro Leu

195 200 205

Gly Gly Val Ala Ala Arg Pro Gly Phe Gly Leu Ser Pro Ile Phe Pro

210 215 220

Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly Arg Lys Arg Lys

225 230 235

<210> 42

<211> 717

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 42

ggtccgggcg gtgtcgcagc agcagctaaa agcgcggcga aagttgcggc caaagcccaa 60

ctgcgcgccg ccgcgggcct cggtgcaggt attccggggc tgggtgtcgg agttggagtc 120

ccgggtttgg gcgtgggcgc gggagttccg ggactgggag tgggtgccgg agttcctggc 180

tttggtgcag gcgcagatga aggtgttcgt cgtagcctga gtccggaact gcgtgaaggt 240

gatccgagta gcagccagca tctgccgagc accccgagca gcccgcgtgt tccgggtgca 300

ttagctgcag caaaagccgc caagtatggt gcagccgtgc cgggcgtctt aggtggtctg 360

ggcgccctgg gtggtgtagg cattccggga ggtgttgtgg gtgcaggacc ggccgccgca 420

gctgcggccg ccaaagcagc tgcaaaagcg gcccagtttg gtttagtggg cgccgcaggt 480

ttaggcggtt taggtgtggg tggactgggt gtacctggcg taggcggtct gggtggaatt 540

ccgcccgcag cggccgcgaa agcggcaaaa tatggcgcgg caggcctggg cggcgtgctg 600

ggtggggcag gtcagtttcc gctgggcggg gttgccgcac gtccgggatt tggtctgagc 660

ccgattttcc ctggcggcgc atgtctgggt aaagcatgtg gtcgtaaacg taaataa 717

<210> 43

<211> 693

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 43

Gly Gly Val Pro Gly Ala Ile Pro Gly Gly Val Pro Gly Gly Val Phe

1 5 10 15

Tyr Pro Gly Ala Gly Leu Gly Ala Leu Gly Gly Gly Ala Leu Gly Pro

20 25 30

Gly Gly Lys Pro Leu Lys Pro Val Pro Gly Gly Leu Ala Gly Ala Gly

35 40 45

Leu Gly Ala Gly Leu Gly Ala Phe Pro Ala Val Thr Phe Pro Gly Ala

50 55 60

Leu Val Pro Gly Gly Val Ala Asp Ala Ala Ala Ala Tyr Lys Ala Ala

65 70 75 80

Lys Ala Gly Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly

85 90 95

Val Ser Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro

100 105 110

Gly Lys Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val

115 120 125

Leu Pro Gly Ala Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro

130 135 140

Thr Gly Ala Gly Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe

145 150 155 160

Ala Gly Ile Pro Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val

165 170 175

Pro Leu Gly Tyr Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly

180 185 190

Leu Pro Tyr Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly

195 200 205

Val Ala Gly Ala Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val

210 215 220

Gly Pro Gln Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe

225 230 235 240

Gly Ala Gly Ala Ala Gly Val Leu Pro Gly Val Gly Gly Ala Gly Val

245 250 255

Pro Gly Val Pro Gly Ala Ile Pro Gly Ile Gly Gly Ile Ala Gly Val

260 265 270

Gly Thr Pro Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala

275 280 285

Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly

290 295 300

Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val

305 310 315 320

Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala

325 330 335

Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala

340 345 350

Lys Ala Ala Lys Tyr Gly Ala Arg Pro Gly Val Gly Val Gly Gly Ile

355 360 365

Pro Thr Tyr Gly Val Gly Ala Gly Gly Phe Pro Gly Phe Gly Val Gly

370 375 380

Val Gly Gly Ile Pro Gly Val Ala Gly Val Pro Gly Val Gly Gly Val

385 390 395 400

Pro Gly Val Gly Gly Val Pro Gly Val Gly Ile Ser Pro Glu Ala Gln

405 410 415

Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Gly Ala Gly

420 425 430

Val Leu Gly Gly Leu Val Pro Gly Pro Gln Ala Ala Val Pro Gly Val

435 440 445

Pro Gly Thr Gly Gly Val Pro Gly Val Gly Thr Pro Ala Ala Ala Ala

450 455 460

Lys Ser Ala Ala Lys Val Ala Ala Lys Ala Gln Leu Arg Ala Ala Ala

465 470 475 480

Gly Leu Gly Ala Gly Ile Pro Gly Leu Gly Val Gly Val Gly Val Pro

485 490 495

Gly Leu Gly Val Gly Ala Gly Val Pro Gly Leu Gly Val Gly Ala Gly

500 505 510

Val Pro Gly Phe Gly Ala Gly Ala Asp Glu Gly Val Arg Arg Ser Leu

515 520 525

Ser Pro Glu Leu Arg Glu Gly Asp Pro Ser Ser Ser Gln His Leu Pro

530 535 540

Ser Thr Pro Ser Ser Pro Arg Val Pro Gly Ala Leu Ala Ala Ala Lys

545 550 555 560

Ala Ala Lys Tyr Gly Ala Ala Val Pro Gly Val Leu Gly Gly Leu Gly

565 570 575

Ala Leu Gly Gly Val Gly Ile Pro Gly Gly Val Val Gly Ala Gly Pro

580 585 590

Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Gln Phe

595 600 605

Gly Leu Val Gly Ala Ala Gly Leu Gly Gly Leu Gly Val Gly Gly Leu

610 615 620

Gly Val Pro Gly Val Gly Gly Leu Gly Gly Ile Pro Pro Ala Ala Ala

625 630 635 640

Ala Lys Ala Ala Lys Tyr Gly Ala Ala Gly Leu Gly Gly Val Leu Gly

645 650 655

Gly Ala Gly Gln Phe Pro Leu Gly Gly Val Ala Ala Arg Pro Gly Phe

660 665 670

Gly Leu Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly Lys Ala Cys

675 680 685

Gly Arg Lys Arg Lys

690

<210> 44

<211> 2082

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 44

ggtggcgtac caggcgcaat tcctgggggt gtcccaggcg gtgtttttta tccgggcgcc 60

ggtcttggcg cactgggtgg cggtgcactg ggcccgggcg gcaaaccgct gaaaccggta 120

ccaggtggtt tagcaggcgc cggcttaggc gcaggtctgg gagcatttcc ggcagttacc 180

tttccagggg cactggttcc tggaggtgtg gccgatgcag ccgcggcata taaagccgct 240

aaagccggtg cgggtttagg aggcgtccca ggtgtcggtg gcctgggtgt tagcgccggt 300

gcagttgttc cgcagccggg agcaggggtt aaacctggta aagtgccggg agtaggtctg 360

ccaggcgttt atcctggtgg tgttttgccg ggtgcccgtt ttccgggcgt tggtgttctt 420

ccaggcgtgc cgaccggagc cggtgttaaa ccgaaagccc ccggtgttgg aggtgcattt 480

gcaggcatcc cgggagttgg cccgtttggt ggtccgcaac ctggggttcc gttaggttat 540

ccgattaaag caccgaaact gcccggcggt tatggtctgc cgtacacaac cggtaaactg 600

ccgtatggtt atggcccggg tggagttgcg ggtgcagcag gtaaagcggg ttatcctacc 660

ggaaccggtg taggtccgca ggccgctgct gccgccgccg caaaagcagc ggctaaattt 720

ggcgccggag cagcgggtgt tctgcctgga gttggtggtg cgggcgtgcc aggggtacct 780

ggtgcaattc cgggtattgg tggtattgcc ggtgtcggca ccccggccgc ggcagctgcg 840

gcagcggcgg ctgccaaagc tgctaaatac ggtgccgcgg cgggtctggt gccaggaggt 900

ccgggttttg gtccgggagt ggttggcgtg cctggcgcag gcgttcctgg tgtgggcgtt 960

ccaggtgcag ggattcctgt tgtgcctggt gccggtattc ccggcgcggc cgttccgggg 1020

gtggttagcc cggaagccgc agcgaaggct gcggcaaagg cagcaaagta tggcgcacgc 1080

ccaggagtcg gcgtgggtgg tatcccgacc tatggggtgg gcgcaggggg ttttcctggt 1140

ttcggcgtag gtgtaggagg tataccgggc gtggccggtg taccaggggt tggtggcgtc 1200

cctggtgttg gcggtgtgcc aggtgttggt atttcaccgg aagcacaggc agcagccgca 1260

gctaaggcag cgaaatatgg tgccgccggc gcaggagttt taggtgggct ggttccgggc 1320

ccgcaggcag ctgtgccggg ggttccaggc accggtggtg tccctggagt cggtacgccg 1380

gcagcagcag ctaaaagcgc ggcgaaagtt gcggccaaag cccaactgcg cgccgccgcg 1440

ggcctcggtg caggtattcc ggggctgggt gtcggagttg gagtcccggg tttgggcgtg 1500

ggcgcgggag ttccgggact gggagtgggt gccggagttc ctggctttgg tgcaggcgca 1560

gatgaaggtg ttcgtcgtag cctgagtccg gaactgcgtg aaggtgatcc gagtagcagc 1620

cagcatctgc cgagcacccc gagcagcccg cgtgttccgg gtgcattagc tgcagcaaaa 1680

gccgccaagt atggtgcagc cgtgccgggc gtcttaggtg gtctgggcgc cctgggtggt 1740

gtaggcattc cgggaggtgt tgtgggtgca ggaccggccg ccgcagctgc ggccgccaaa 1800

gcagctgcaa aagcggccca gtttggttta gtgggcgccg caggtttagg cggtttaggt 1860

gtgggtggac tgggtgtacc tggcgtaggc ggtctgggtg gaattccgcc cgcagcggcc 1920

gcgaaagcgg caaaatatgg cgcggcaggc ctgggcggcg tgctgggtgg ggcaggtcag 1980

tttccgctgg gcggggttgc cgcacgtccg ggatttggtc tgagcccgat tttccctggc 2040

ggcgcatgtc tgggtaaagc atgtggtcgt aaacgtaaat aa 2082

<210> 45

<211> 846

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 45

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcc 60

ggtctgggtg gcgtgcctgg cgttggtggc ctgggcgtta gcgccggtgc agttgttccg 120

cagcctggtg cgggtgttaa accgggtaaa gttcctggtg ttggtctgcc tggtgtttat 180

ccaggcggtg ttctgccagg tgcgcgtttt cctggcgtgg gtgttctgcc gggtgttccg 240

accggtgcag gcgtgaaacc gaaagcacca ggtgttggtg gtgcatttgc aggtattcca 300

ggtgtgggtc cgtttggtgg tccgcagcca ggcgttccgc tgggttatcc gattaaagca 360

ccgaaactgc ctggcggtta tggtctgccg tataccaccg gtaaactgcc gtatggttat 420

ggccctggcg gtgttgccgg tgcggcaggt aaagcaggct atccgaccgg taccggtgta 480

ggtccgcagg cagcagccgc agcagcggca aaagcagcag cgaaatttgg tgcgggtgca 540

gccggtgtgc tgccaggcgt aggtggcgct ggtgtacctg gtgtccctgg cgcaattcca 600

ggtattggcg gtattgcagg cgttggtact ccggcagctg cagccgctgc cgcagccgca 660

gctaaagcag ccaaatatgg tgcagcggca ggcctggttc ctggcggtcc aggttttggt 720

ccgggtgttg ttggcgtccc aggtgccggt gtgcctggtg tgggtgttcc aggtgcgggt 780

attccggttg ttcctggcgc aggcattccg ggtgccgcag ttccgggtgt agttagcccg 840

gaataa 846

<210> 46

<211> 281

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 46

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly

20 25 30

Val Ser Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro

35 40 45

Gly Lys Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val

50 55 60

Leu Pro Gly Ala Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro

65 70 75 80

Thr Gly Ala Gly Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe

85 90 95

Ala Gly Ile Pro Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val

100 105 110

Pro Leu Gly Tyr Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly

115 120 125

Leu Pro Tyr Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly

130 135 140

Val Ala Gly Ala Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val

145 150 155 160

Gly Pro Gln Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe

165 170 175

Gly Ala Gly Ala Ala Gly Val Leu Pro Gly Val Gly Gly Ala Gly Val

180 185 190

Pro Gly Val Pro Gly Ala Ile Pro Gly Ile Gly Gly Ile Ala Gly Val

195 200 205

Gly Thr Pro Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala

210 215 220

Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly

225 230 235 240

Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val

245 250 255

Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala

260 265 270

Ala Val Pro Gly Val Val Ser Pro Glu

275 280

<210> 47

<211> 789

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 47

gccggtctgg gtggcgtgcc tggcgttggt ggcctgggcg ttagcgccgg tgcagttgtt 60

ccgcagcctg gtgcgggtgt taaaccgggt aaagttcctg gtgttggtct gcctggtgtt 120

tatccaggcg gtgttctgcc aggtgcgcgt tttcctggcg tgggtgttct gccgggtgtt 180

ccgaccggtg caggcgtgaa accgaaagca ccaggtgttg gtggtgcatt tgcaggtatt 240

ccaggtgtgg gtccgtttgg tggtccgcag ccaggcgttc cgctgggtta tccgattaaa 300

gcaccgaaac tgcctggcgg ttatggtctg ccgtatacca ccggtaaact gccgtatggt 360

tatggccctg gcggtgttgc cggtgcggca ggtaaagcag gctatccgac cggtaccggt 420

gtaggtccgc aggcagcagc cgcagcagcg gcaaaagcag cagcgaaatt tggtgcgggt 480

gcagccggtg tgctgccagg cgtaggtggc gctggtgtac ctggtgtccc tggcgcaatt 540

ccaggtattg gcggtattgc aggcgttggt actccggcag ctgcagccgc tgccgcagcc 600

gcagctaaag cagccaaata tggtgcagcg gcaggcctgg ttcctggcgg tccaggtttt 660

ggtccgggtg ttgttggcgt cccaggtgcc ggtgtgcctg gtgtgggtgt tccaggtgcg 720

ggtattccgg ttgttcctgg cgcaggcatt ccgggtgccg cagttccggg tgtagttagc 780

ccggaataa 789

<210> 48

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 48

Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly Val Ser Ala

1 5 10 15

Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val

20 25 30

Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly

35 40 45

Ala Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro Thr Gly Ala

50 55 60

Gly Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe Ala Gly Ile

65 70 75 80

Pro Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val Pro Leu Gly

85 90 95

Tyr Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly Leu Pro Tyr

100 105 110

Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly Val Ala Gly

115 120 125

Ala Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val Gly Pro Gln

130 135 140

Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe Gly Ala Gly

145 150 155 160

Ala Ala Gly Val Leu Pro Gly Val Gly Gly Ala Gly Val Pro Gly Val

165 170 175

Pro Gly Ala Ile Pro Gly Ile Gly Gly Ile Ala Gly Val Gly Thr Pro

180 185 190

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly

195 200 205

Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val

210 215 220

Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala

225 230 235 240

Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro

245 250 255

Gly Val Val Ser Pro Glu

260

<210> 49

<211> 492

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 49

atgaaaaaga tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcggcc 60

ggtctgggtg gcgtgcctgg cgttggtggc ctgggcgtta gcgccggtgc agttgttccg 120

cagcctggtg cgggtgttaa accgggtaaa gttcctggtg ttggtctgcc tggtgtttat 180

ccaggcggtg ttctgccagg tgcgcgtttt cctggcgtgg gtgttctgcc gggtgttccg 240

accggtgcag gcgtgaaacc gaaagcacca ggtgttggtg gtgcatttgc aggtattcca 300

ggtgtgggtc cgtttggtgg tccgcagcca ggcgttccgc tgggttatcc gattaaagca 360

ccgaaactgc ctggcggtta tggtctgccg tataccaccg gtaaactgcc gtatggttat 420

ggccctggcg gtgttgccgg tgcggcaggt aaagcaggct atccgaccgg taccggtgta 480

ggtccgcagt aa 492

<210> 50

<211> 163

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 50

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly

20 25 30

Val Ser Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro

35 40 45

Gly Lys Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val

50 55 60

Leu Pro Gly Ala Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro

65 70 75 80

Thr Gly Ala Gly Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe

85 90 95

Ala Gly Ile Pro Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val

100 105 110

Pro Leu Gly Tyr Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly

115 120 125

Leu Pro Tyr Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly

130 135 140

Val Ala Gly Ala Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val

145 150 155 160

Gly Pro Gln

<210> 51

<211> 435

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polynucleotide

<400> 51

gccggtctgg gtggcgtgcc tggcgttggt ggcctgggcg ttagcgccgg tgcagttgtt 60

ccgcagcctg gtgcgggtgt taaaccgggt aaagttcctg gtgttggtct gcctggtgtt 120

tatccaggcg gtgttctgcc aggtgcgcgt tttcctggcg tgggtgttct gccgggtgtt 180

ccgaccggtg caggcgtgaa accgaaagca ccaggtgttg gtggtgcatt tgcaggtatt 240

ccaggtgtgg gtccgtttgg tggtccgcag ccaggcgttc cgctgggtta tccgattaaa 300

gcaccgaaac tgcctggcgg ttatggtctg ccgtatacca ccggtaaact gccgtatggt 360

tatggccctg gcggtgttgc cggtgcggca ggtaaagcag gctatccgac cggtaccggt 420

gtaggtccgc agtaa 435

<210> 52

<211> 144

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

Polypeptides

<400> 52

Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly Val Ser Ala

1 5 10 15

Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val

20 25 30

Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly

35 40 45

Ala Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro Thr Gly Ala

50 55 60

Gly Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe Ala Gly Ile

65 70 75 80

Pro Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val Pro Leu Gly

85 90 95

Tyr Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly Leu Pro Tyr

100 105 110

Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly Val Ala Gly

115 120 125

Ala Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val Gly Pro Gln

130 135 140

<210> 53

<211> 760

<212> PRT

<213> Chile person

<400> 53

Gly Gly Val Pro Gly Ala Ile Pro Gly Gly Val Pro Gly Gly Val Phe

1 5 10 15

Tyr Pro Gly Ala Gly Leu Gly Ala Leu Gly Gly Gly Ala Leu Gly Pro

20 25 30

Gly Gly Lys Pro Leu Lys Pro Val Pro Gly Gly Leu Ala Gly Ala Gly

35 40 45

Leu Gly Ala Gly Leu Gly Ala Phe Pro Ala Val Thr Phe Pro Gly Ala

50 55 60

Leu Val Pro Gly Gly Val Ala Asp Ala Ala Ala Ala Tyr Lys Ala Ala

65 70 75 80

Lys Ala Gly Ala Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly

85 90 95

Val Ser Ala Gly Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro

100 105 110

Gly Lys Val Pro Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val

115 120 125

Leu Pro Gly Ala Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro

130 135 140

Thr Gly Ala Gly Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe

145 150 155 160

Ala Gly Ile Pro Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val

165 170 175

Pro Leu Gly Tyr Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly

180 185 190

Leu Pro Tyr Thr Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly

195 200 205

Val Ala Gly Ala Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val

210 215 220

Gly Pro Gln Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe

225 230 235 240

Gly Ala Gly Ala Ala Gly Val Leu Pro Gly Val Gly Gly Ala Gly Val

245 250 255

Pro Gly Val Pro Gly Ala Ile Pro Gly Ile Gly Gly Ile Ala Gly Val

260 265 270

Gly Thr Pro Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala

275 280 285

Lys Tyr Gly Ala Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly

290 295 300

Pro Gly Val Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val

305 310 315 320

Pro Gly Ala Gly Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala

325 330 335

Ala Val Pro Gly Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala

340 345 350

Lys Ala Ala Lys Tyr Gly Ala Arg Pro Gly Val Gly Val Gly Gly Ile

355 360 365

Pro Thr Tyr Gly Val Gly Ala Gly Gly Phe Pro Gly Phe Gly Val Gly

370 375 380

Val Gly Gly Ile Pro Gly Val Ala Gly Val Pro Gly Val Gly Gly Val

385 390 395 400

Pro Gly Val Gly Gly Val Pro Gly Val Gly Ile Ser Pro Glu Ala Gln

405 410 415

Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Gly Ala Gly

420 425 430

Val Leu Gly Gly Leu Val Pro Gly Pro Gln Ala Ala Val Pro Gly Val

435 440 445

Pro Gly Thr Gly Gly Val Pro Gly Val Gly Thr Pro Ala Ala Ala Ala

450 455 460

Ala Lys Ala Ala Ala Lys Ala Ala Gln Phe Gly Leu Val Pro Gly Val

465 470 475 480

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro

485 490 495

Gly Val Gly Leu Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

500 505 510

Ala Pro Gly Val Gly Val Ala Pro Gly Ile Gly Pro Gly Gly Val Ala

515 520 525

Ala Ala Ala Lys Ser Ala Ala Lys Val Ala Ala Lys Ala Gln Leu Arg

530 535 540

Ala Ala Ala Gly Leu Gly Ala Gly Ile Pro Gly Leu Gly Val Gly Val

545 550 555 560

Gly Val Pro Gly Leu Gly Val Gly Ala Gly Val Pro Gly Leu Gly Val

565 570 575

Gly Ala Gly Val Pro Gly Phe Gly Ala Gly Ala Asp Glu Gly Val Arg

580 585 590

Arg Ser Leu Ser Pro Glu Leu Arg Glu Gly Asp Pro Ser Ser Ser Gln

595 600 605

His Leu Pro Ser Thr Pro Ser Ser Pro Arg Val Pro Gly Ala Leu Ala

610 615 620

Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Val Pro Gly Val Leu Gly

625 630 635 640

Gly Leu Gly Ala Leu Gly Gly Val Gly Ile Pro Gly Gly Val Val Gly

645 650 655

Ala Gly Pro Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala

660 665 670

Ala Gln Phe Gly Leu Val Gly Ala Ala Gly Leu Gly Gly Leu Gly Val

675 680 685

Gly Gly Leu Gly Val Pro Gly Val Gly Gly Leu Gly Gly Ile Pro Pro

690 695 700

Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Gly Leu Gly Gly

705 710 715 720

Val Leu Gly Gly Ala Gly Gln Phe Pro Leu Gly Gly Val Ala Ala Arg

725 730 735

Pro Gly Phe Gly Leu Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly

740 745 750

Lys Ala Cys Gly Arg Lys Arg Lys

755 760

<210> 54

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

His tag

<220>

<221> SITE

<222> (1)..(30)

<223> the sequence may comprise 2-30 residues

<400> 54

His His His His His His His His His His His His His His His His

1 5 10 15

His His His His His His His His His His His His His His

20 25 30

<210> 55

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> description of artificial sequence synthetic

His tag

<220>

<221> SITE

<222> (1)..(20)

<223> the sequence may comprise 2-20 residues

<220>

<223> for a detailed description of alternative and preferred embodiments, please refer to the submitted description.

<400> 55

His His His His His His His His His His His His His His His His

1 5 10 15

His His His His

20

Claims

1. A composition comprising a non-naturally occurring recombinant polypeptide selected from the group consisting of human collagen or a truncate thereof, jellyfish collagen or a truncate thereof, and elastin or a truncate thereof, wherein the composition further comprises one or more active ingredients, wherein the non-naturally occurring recombinant polypeptide and the one or more active ingredients provide a benefit to the skin and/or hair.

2. The composition of claim 1, wherein the one or more active ingredients are selected from the group consisting of retinoids or vitamin a compounds, vitamin C compounds, alpha hydroxy acids, hyaluronic acid, coenzyme Q10, tea extract, grape seed extract, niacinamide, antibacterial agents, or acne-preventing agents, and any combination of two or more thereof.

3. The composition of claim 1 or 2, wherein the benefit to the skin is: (i) Improving skin firmness, elasticity, brightness, hydration, tactile texture, visual texture, collagen content or elastin content; (ii) Reducing skin damage, lines or wrinkles present on the skin, or erythema or redness of the skin; (iii) preventing or treating ultraviolet radiation damage to the skin; (iv) promoting repair of damaged skin; (v) Protecting skin cells from exposure to urban dust; (vi) Stimulating collagen production or elastin production in the skin; or (vii) any combination of two or more of (i) - (vi).

4. The composition of any one of claims 1-3, wherein the non-naturally occurring recombinant polypeptide is a truncated collagen polypeptide or a truncated elastin polypeptide that is truncated at the C-terminus, N-terminus, internal truncation, or both the C-terminus and N-terminus relative to a naturally occurring collagen or elastin.

5. The composition of any one of claims 1-4, wherein the non-naturally occurring recombinant polypeptide is truncated by 50 to 800 amino acids at the C-terminus, 50 to 800 amino acids at the N-terminus, or both.

6. The composition of any one of claims 1-5, wherein the non-naturally occurring recombinant polypeptide is internally truncated by 50 to 800 amino acids.

7. The composition of claim 4, wherein the non-naturally occurring recombinant polypeptide is human collagen or jellyfish collagen.

8. The composition of claim 7, wherein the non-naturally occurring recombinant polypeptide is 50 to 900 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 in length.

9. The composition of claim 7, wherein the non-naturally occurring recombinant polypeptide is a polypeptide having an N-terminal truncation of 50 to 650 amino acids and a C-terminal truncation of 50 to 250 amino acids.

10. The composition of claim 7, wherein the non-naturally occurring recombinant polypeptide is human type 21 collagen.

11. The composition of any one of claims 7-10, wherein the non-naturally occurring recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity relative to SEQ ID No. 31.

12. The composition of any one of claims 7-11, wherein the non-naturally occurring recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID No. 14 and SEQ ID No. 16.

13. The composition of claim 12, wherein the non-naturally occurring recombinant polypeptide comprises SEQ ID No. 16.

14. The composition of claim 13, wherein the non-naturally occurring recombinant polypeptide consists of SEQ ID No. 16.

15. The composition of claim 7, wherein the non-naturally occurring recombinant polypeptide is human type 1 collagen.

16. The composition of claim 15, wherein the non-naturally occurring recombinant polypeptide is a polypeptide comprising an amino acid sequence having at least 80% sequence identity relative to SEQ ID No. 32.

17. The composition of claim 15, wherein the non-naturally occurring recombinant polypeptide comprises an amino acid sequence having at least about 80% sequence identity to an amino acid sequence selected from the group consisting of 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.

18. The composition of claim 17, wherein the non-naturally occurring recombinant polypeptide comprises an amino acid sequence selected from the group consisting of 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.

19. The composition of claim 7, wherein the non-naturally occurring recombinant polypeptide is jellyfish collagen.

20. The composition of claim 19, wherein the non-naturally occurring recombinant polypeptide is a polypeptide comprising an amino acid sequence having at least 80% sequence identity relative to SEQ ID No. 33.

21. The composition of claim 19, wherein the non-naturally occurring recombinant polypeptide is a polypeptide comprising an amino acid sequence having at least 80% sequence identity relative to an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 10, and SEQ ID No. 12.

22. The composition of claim 21, wherein the non-naturally occurring recombinant polypeptide is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 10, and SEQ ID No. 12.

23. The composition of claim 21, wherein the non-naturally occurring recombinant polypeptide is a polypeptide having at least 80% sequence identity to SEQ ID No. 5.

24. The composition of claim 4, wherein the non-naturally occurring recombinant polypeptide is human elastin.

25. The composition of claim 24, wherein the non-naturally occurring recombinant polypeptide is 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 in length.

26. The composition of claim 24, wherein the non-naturally occurring recombinant polypeptide comprises a C-terminal truncation, an N-terminal truncation, an internal truncation, or any combination thereof, relative to a full-length elastin polypeptide.

27. The composition of claim 24, wherein the non-naturally occurring recombinant polypeptide is a polypeptide having an N-terminal truncation of 50 to 200 amino acids and a C-terminal truncation of 50 to 600 amino acids.

28. The composition of claim 24, wherein the non-naturally occurring recombinant polypeptide is a polypeptide having at least 80% sequence identity relative to SEQ ID No. 53.

29. The composition of claim 24, wherein the non-naturally occurring recombinant polypeptide is a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 50 or SEQ ID No. 52.

30. The composition of claim 29, wherein the non-naturally occurring recombinant polypeptide is a polypeptide having at least 80% sequence identity to SEQ ID No. 52.

31. The composition of claim 29, wherein the non-naturally occurring recombinant polypeptide is a polypeptide comprising the amino acid sequence of any one of SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 43, SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 50, or SEQ ID No. 52.

32. A topical formulation comprising the composition of any one of the preceding claims and one or more additional ingredients or carriers.

33. The topical formulation of claim 32, wherein the one or more additional ingredients or carriers are selected from the group consisting of: water, oil glyceryl polyether-8 esters, glycerin, coconut alkanes, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, pentanediol, disodium EDTA, octanoyl glycol, chlorophenyl glyoxyl, phenoxyethanol, vegetable oils, and any combination thereof.

34. The topical formulation of claim 32, wherein the topical formulation is a personal care product formulated for application to the skin and/or hair of a subject.

35. The topical formulation of claim 34, wherein the one or more carriers is a topical carrier.

36. The topical formulation of claim 35, wherein the topical carrier is selected from the group consisting of: liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, water, and any combination thereof.

37. The topical formulation of any one of claims 34-36, further comprising a preservative.

38. The topical formulation of any one of claims 34-37, wherein the non-naturally occurring recombinant polypeptide or truncate thereof is present in the topical formulation in an amount of about 0.001% (w/w) to about 30% (w/w).

39. The topical formulation of any one of claims 34-38, wherein the non-naturally occurring recombinant polypeptide or truncate thereof is present in the topical formulation in an amount of about 0.001% (w/w) to about 10% (w/w).

40. The topical formulation of any one of claims 34-39, wherein the hyaluronic acid is present in an amount of about 0.001% (w/w) to about 10% (w/w).

41. The topical formulation of any one of claims 34-40, wherein retinol is present in an amount of about 0.001% (w/w) to about 5% (w/w).

42. The topical formulation according to any one of claims 34-41, wherein the ascorbic acid is present in an amount of about 0.01% (w/w) to about 50% (w/w).

43. The topical formulation according to any one of claims 34-42, wherein salicylic acid is present in an amount of about 0.1% (w/w) to about 50% (w/w).

44. The topical formulation according to any one of claims 34-43, wherein benzoyl peroxide is present in an amount of about 0.1% (w/w) to about 20% (w/w).

45. The topical formulation according to any one of claims 34-44, wherein the nicotinamide, when present, is present in an amount of about 0.1% (w/w) to about 50% (w/w).

46. The topical formulation according to any one of claims 34-45, wherein the alpha hydroxy acid, when present, is present in an amount of about 0.1% (w/w) to about 50% (w/w).

47. The topical formulation according to any one of claims 34-46, wherein the personal care product is a cosmetic.

48. The topical formulation according to any one of claims 34-47, wherein the personal care product is selected from the group consisting of: face masks, skin cleansers, cleansing creams, cleansing lotions, facial cleansers, cleansing milks, cleansing pads, cleansing milks, face creams, body creams, facial moisturizers, body moisturizers, facial essences, facial masks, body films, lotions, facial sprays, eye creams, eye care products, exfoliating formulations, lipsticks, shampoos, hair conditioners, body washes, hair care essences, scalp essences, hair sprays, hair gels, eye shadows, concealers, mascaras, and color cosmetics.

49. The topical formulation according to any one of claims 34-48, wherein the personal care product is configured to form a film.

50. A method of providing a benefit to the skin of a subject, the method comprising: applying the topical formulation of any one of claims 34-49 to the subject's skin, thereby providing a benefit to the subject's skin.

51. The method of claim 50, wherein the benefit to the skin is: (i) Improving skin firmness, elasticity, brightness, hydration, tactile texture, visual texture, collagen content or elastin content; (ii) Reducing skin damage, lines or wrinkles present on the skin, or erythema or redness of the skin; (iii) preventing or treating ultraviolet radiation damage to the skin; (iv) promoting repair of damaged skin; (v) Protecting skin cells from exposure to urban dust; (vi) Stimulating collagen production or elastin production in the skin; or (vii) any combination of two or more of (i) - (vi).

52. A method of providing benefits to hair of a subject, the method comprising: applying the topical formulation of any one of claims 34-49 to the subject's hair, thereby providing a benefit to the subject's hair.

53. The method of claim 52 wherein the benefit to the hair is: (i) improving the appearance and/or quality of hair; (ii) improving hair growth or thickness; (iii) reducing hair loss; (iv) Improving hair tensile strength, hair moisture, hydrophobicity, static control, fiber alignment, gloss, wet comb, dry comb, hair breakage resistance; (v) reduced bifurcation and curling of the hair tips; or (vi) any combination of two or more of (i) - (v).

54. A composition comprising a non-naturally occurring recombinant elastin polypeptide or a truncate thereof and a non-naturally occurring recombinant collagen polypeptide or a truncate thereof.

55. The composition of claim 54, further comprising one or more active ingredients.

56. The composition of claim 55, wherein the one or more active ingredients are selected from the group consisting of retinoids or vitamin a compounds, vitamin C compounds, alpha hydroxy acids, hyaluronic acid, coenzyme Q10, tea extract, grape seed extract, niacinamide, antibacterial agents, or acne-preventing agents, and any combination of two or more thereof.