Recombinant elastin and its production

Abstract

The present disclosure provides non-naturally occurring truncated elastin molecules. The non-naturally occurring truncated elastin molecules can improve the firmness, elasticity, brightness, hydration, tactile texture and / or visual texture of the skin. The non-naturally occurring truncated elastin molecules can reduce the degradation of the extracellular matrix.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 833,415, filed on April 12, 2019, and 62 / 889,397, filed on August 20, 2019, which are incorporated herein by reference in their entirety. Background Art

[0003] Elastin is an elastic protein that is essential for the normal function of arteries, lungs, tendons, ligaments, skin and other tissues. Elastin provides tissues with the ability to stretch and return to their original shape. The protein tropoelastin is a component of elastin. Unlike other structural proteins (such as collagen) that contain a series of genes, there is only one tropoelastin gene in humans. When the gene encoding tropoelastin is expressed, the single tropoelastin gene is spliced ​​to produce different forms of tropoelastin. Multiple tropoelastin molecules combine to form elastin.

[0004] The structure of native elastin is similar to collagen, with an amino acid sequence rich in glycine and proline. A single polypeptide chain consists of a repeating triplet amino acid sequence designated GLY-XY, interspersed with strings of alanine to provide flexibility. X and Y can be any amino acids, and the first amino acid is glycine. The amino acids proline and hydroxyproline are found in high concentrations in elastin. Elastin is often covalently cross-linked with 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 tissues.

[0005] Certain domains of elastin can function as signaling domains for intracellular communication. In addition, certain elastin domains can function as binding domains for peptides and other biomolecules. Degradation products of elastin have been shown to activate cellular processes, such as phagocytosis. These cell-interaction domains are interspersed with alanine stretches that create the stretchy, elastic structure that is characteristic of elastin. Alanine stretches are also rich in lysine, which in tissues is converted to aldehyde lysine by lysyl oxidase and cross-linked into a fishing net-like structure, making it very difficult to extract elastin from animal tissues, which requires breaking down the elastin molecules for their release. Due to this difficult extraction process, commercially available elastin is only 3-5 kDa in size.

[0006] In human native elastin, the molecule has two parts, each containing three consecutive VGVAPG repeats, namely VGVAPGVGVAPGVGVAPG. The amino acid sequence VGVAPG has been reported to promote extracellular matrix degradation. Summary of the invention

[0007] In one aspect, a non-naturally occurring polypeptide is provided, which consists of: an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids); and optionally one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the group consisting of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the group consisting of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site.In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the group consisting of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the group consisting of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and one or more selected from the group consisting of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19; and one or more selected from the group consisting of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site.In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids). In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19. In some cases, the secretion tag is DsbA. In some cases, the non-naturally occurring polypeptide is a recombinant polypeptide.

[0008] In another aspect, a composition is provided comprising 0.001 to 30% w / w of a non-naturally occurring polypeptide of any of the foregoing. In some cases, the composition comprises 0.001% to 1% w / w of a non-naturally occurring polypeptide, 0.001% to 0.05% w / w of a non-naturally occurring polypeptide, 0.001% to 0.03% w / w of a non-naturally occurring polypeptide, 0.001% to 0.02% w / w of a non-naturally occurring polypeptide, 0.001% to 0.01% w / w of a non-naturally occurring polypeptide, 0.005% to 0.1% w / w of a non-naturally occurring polypeptide, 0.005% to 0.03% w / w of a non-naturally occurring polypeptide, 0.005% to 0.02% w / w of a non-naturally occurring polypeptide, or 0.005% to 0.01% w / w of a non-naturally occurring polypeptide. In some cases, the composition can stimulate the growth of fibroblasts, stimulate the synthesis of tropoelastin, reduce the formation of thymine-thymine (TT) dimers, or any combination thereof. In some cases, the composition is formulated for topical application. In some cases, the composition comprises one or more topical carriers and a preservative. In some cases, the topical carrier is selected from water, glycereth-8 ester, glycerin, coconut alkanes, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, pentylene glycol, disodium EDTA, caprylyl glycol, chlorphenesin and phenoxyethanol, liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifier, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone and cyclopentasiloxane. In some cases, the preservative is selected from: tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, cis-isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract and EDTA. In some cases, the composition is a cosmetic.

[0009] On the other hand, a method for treating the skin of a subject is provided, the method comprising applying any of the foregoing compositions to the skin of the subject, thereby treating the skin of the subject. In some cases, the treatment comprises reducing skin damage, promoting damaged skin repair, protecting skin from ultraviolet damage, increasing the vitality of skin cells, protecting skin cells from the impact of urban dust exposure, or any combination thereof. In some cases, the vitality of fibroblasts, keratinocytes or both present in the subject's skin increases. In some cases, the fibroblasts present in the subject's skin increase the synthesis of procollagen. In some cases, the keratinocytes present in the subject's skin increase the expression of one or more antioxidant genes. In some cases, the one or more antioxidant genes are selected from: SOD2, GPX2, GPX4, GSTK1, GSTZ1, GSTA4, GSTM2, CCS, GPX1, GLRX, PRDX5, PRDX6 and PRDX2. In some cases, the keratinocytes present in the subject's skin reduce the expression of one or more pro-apoptotic genes. In some cases, the one or more pro-apoptotic genes are selected from: APAF1, BAK1, CASP7, CASP8, FADD, MCL1 and MET. In some cases, the keratinocytes present in the subject's skin reduce the production of inflammatory cytokines. In some cases, the inflammatory cytokine is IL-1α. In some cases, the viability of keratinocytes present in the subject's skin is increased.

[0010] On the other hand, a polynucleotide encoding a non-naturally occurring polypeptide according to any of the foregoing is provided. In some cases, the polynucleotide is contained in a vector. In some cases, the polynucleotide comprises a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18. In some cases, the polynucleotide comprises a nucleic acid sequence having at least 85% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18. In some cases, the polynucleotide comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18. In some cases, the polynucleotide comprises a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18. In some cases, the polynucleotide comprises a nucleic acid sequence having at least 98% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18. In some cases, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18. In some cases, the polynucleotide further comprises a nucleic acid sequence encoding one or more of a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the nucleic acid sequence is codon-optimized for expression in a host cell.

[0011] On the other hand, a recombinant cell is provided, which comprises at least one copy of a heterologous nucleic acid sequence encoding a non-naturally occurring polypeptide of any of the foregoing. In some cases, the recombinant cell is a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is an Escherichia coli species. In some cases, the heterologous nucleic acid sequence has at least 95% sequence identity with a nucleic acid sequence selected from the following: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18. In some cases, the heterologous nucleic acid sequence comprises a nucleic acid sequence selected from the following: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18. In some cases, the heterologous nucleic acid sequence is codon optimized to be expressed in a cell. In some cases, the recombinant cell is capable of secreting the non-naturally occurring polypeptide extracellularly.

[0012] In another aspect, a composition is provided, comprising a recombinant cell of any of the foregoing and a culture medium comprising any of the foregoing non-naturally occurring polypeptides.

[0013] In another aspect, a method for producing a non-naturally occurring polypeptide is provided, the method comprising: a) cultivating a recombinant cell of any of the preceding items in a culture medium, wherein the recombinant cell secretes the recombinant polypeptide into the culture medium; b) collecting the culture medium containing the recombinant polypeptide secreted therein; and c) purifying the recombinant polypeptide from the culture medium.

[0014] In another aspect, a non-naturally occurring polypeptide is provided, which consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 19, or a truncate thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 19, or a truncate thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19, or a truncation thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 19, or a truncate thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and optionally one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site. In some cases, the non-naturally occurring polypeptide consists of an amino acid sequence according to SEQ ID NO: 19, or a truncate thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids); and one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein, and a protease cleavage site.In some cases, the non-naturally occurring polypeptide consists of the amino acid sequence according to SEQ ID NO: 19, or a truncate thereof (e.g., a polypeptide having at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids). In some cases, the non-naturally occurring polypeptide consists of the amino acid sequence according to SEQ ID NO: 19. In some cases, the secretion tag is DsbA. In some cases, the non-naturally occurring polypeptide is a recombinant polypeptide. In another aspect, a composition is provided comprising 0.001% to 30% w / w of a non-naturally occurring polypeptide. In some cases, the composition comprises 0.001 to 1% w / w of the non-naturally occurring polypeptide, 0.001 to 0.05% w / w of the non-naturally occurring polypeptide, 0.001 to 0.03% w / w of the non-naturally occurring polypeptide, 0.001 to 0.02% w / w of the non-naturally occurring polypeptide, 0.001 to 0.01% w / w of the non-naturally occurring polypeptide, 0.005 to 0.1% w / w of the non-naturally occurring polypeptide, 0.005 to 0.03% w / w of the non-naturally occurring polypeptide, 0.005 to 0.02% w / w of the non-naturally occurring polypeptide, or 0.005 to 0.01% w / w of the non-naturally occurring polypeptide. In some cases, the composition can achieve one or more selected from the following: stimulate the growth of fibroblasts, increase the vigor of fibroblasts or keratinocytes, stimulate the synthesis of tropoelastin, stimulate the synthesis of tropoelastin, stimulate the expression of one or more antioxidant genes, reduce the expression of one or more pro-apoptotic genes, reduce the generation of one or more inflammatory cytokines and reduce the formation of thymine-thymine (TT) dimers. In some cases, the composition is formulated for topical application. In some cases, the composition comprises one or more local carriers and preservatives. In some cases, the topical carrier is selected from water, glyceryl polyether-8 oil, glycerin, coconut alkanes, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, pentylene glycol, disodium EDTA, caprylyl glycol, chlorphenesin and phenoxyethanol, liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusted powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone and cyclopentasiloxane.In some cases, the preservative is selected from: tocopherol, diiodomethyl-p-tolyl sulfone, 2-bromo-2-nitropropane-1,3-diol, cis-isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azonia adamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclic oxazolidine, methyl paraben, sorbic acid, Germaben II, rosemary extract and EDTA. In some cases, the composition is a cosmetic. On the other hand, a recombinant cell is provided, which comprises at least one copy of a heterologous nucleic acid sequence encoding a non-naturally occurring polypeptide. In some cases, the recombinant cell is an Escherichia coli species.

[0015] In certain embodiments, various polypeptides, compositions comprising such polypeptides, and methods of using such polypeptides and / or compositions thereof are described herein. In certain embodiments, such polypeptides include non-natural and / or recombinant polypeptides, for example, comprising one or more amino acid sequences (e.g., full-length proteins) that are truncated relative to native proteins, such as native proteins described herein. In some cases, such polypeptides are described herein as "truncated proteins." In specific embodiments, the polypeptide includes one or more (e.g., two or more) truncated amino acid sequences of native human proteins.

[0016] On the one hand, non-naturally occurring full length or non-naturally occurring truncated human elastin molecules are provided. In one embodiment, the truncated human elastin molecules are produced by host cells. The non-naturally occurring elastin can be human elastin. In one embodiment, the non-naturally occurring elastin can be full length or truncated elastin (e.g., relative to naturally occurring and / or full length elastin).

[0017] In one embodiment, a non-naturally occurring polypeptide (e.g., truncated human elastin) is provided, wherein the non-naturally occurring polypeptide does not comprise one or more consecutive amino acid sequences, and wherein each of the one or more consecutive amino acid sequences has a VGVAPG sequence (SEQ ID NO: 1). In another embodiment, the non-naturally occurring polypeptide (e.g., truncated human elastin) does not comprise two or more consecutive amino acid sequences VGVAPG. For example, in one embodiment, the non-naturally occurring polypeptide (e.g., truncated human elastin) does not comprise the amino acid sequence VGVAPGVGVAPG (SEQ ID NO: 2) or the non-naturally occurring polypeptide (e.g., truncated human elastin) does not comprise the amino acid sequence VGVAPGVGVAPGVGVAPG (SEQ ID NO: 3). In some cases, the non-naturally occurring polypeptides described herein (e.g., truncated human elastin) reduce extracellular matrix degradation, or do not promote or cause extracellular matrix degradation.

[0018] In various aspects, the non-naturally occurring polypeptides disclosed herein (e.g., truncated human elastin) can have a molecular weight of 1 kDa to 60 kDa, 5 kDa to 55 kDa, 5 kDa to 50 kDa, 5 kDa to 45 kDa, 5 kDa to 40 kDa, 5 kDa to 35 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, 5 kDa to 15 kDa, 5 kDa to 10 kDa, 10 kDa to 40 kDa, 10 kDa to 35 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, or 10 kDa to 20 kDa.

[0019] In yet another embodiment, a non-naturally occurring polypeptide (e.g., truncated human elastin) is provided, wherein the non-naturally occurring polypeptide is truncated at the C-terminus relative to naturally occurring and / or full-length human elastin, truncated at the N-terminus relative to naturally occurring and / or full-length human elastin, internally truncated relative to naturally occurring and / or full-length human elastin, or truncated at both the C-terminus and the N-terminus relative to naturally occurring and / or full-length human elastin. In some cases, the non-naturally occurring polypeptide described herein comprises a truncation relative to SEQ ID NO: 20 (e.g., at the N-terminus, at the C-terminus and / or internally truncated).

[0020] The length of the non-naturally occurring polypeptides described herein (e.g., truncated human elastin) can 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 non-naturally occurring polypeptides described herein (e.g., truncated human elastin) can be molecules that are at least 1, 5, 10, 20, 50, 100 or more amino acids shorter than full-length human tropoelastin or full-length human elastin.

[0021] In specific embodiments, the non-naturally occurring polypeptides herein comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and homologues thereof (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity thereto). In more specific embodiments, the non-naturally occurring polypeptides herein consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and homologues thereof (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity thereto).

[0022] In another aspect, the non-naturally occurring polypeptide described herein further comprises one or more amino acid sequences selected from the group consisting of a secretion tag, a histidine tag, a green fluorescent protein tag, and a protease cleavage site. In one aspect, the secretion tag is 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 a specific embodiment, the secretion tag is DsbA.

[0023] In certain embodiments, compositions are provided herein. In some embodiments, such compositions include any polypeptide described herein (e.g., truncated or non-naturally occurring elastin). On the one hand, compositions are provided herein comprising any suitable amount (e.g., 0.001% w / w to 30% w / w) of any polypeptide provided herein (e.g., truncated and / or non-naturally occurring elastin). In some cases, the composition comprises 0.001% w / w to 1% w / w, 0.001% w / w to 0.05% w / w, 0.001% w / w to 0.03% w / w, 0.001% w / w to 0.02% w / w, 0.001% w / w to 0.01% w / w, 0.005% w / w to 0.1% w / w, 0.005% w / w to 0.03% w / w, 0.005% w / w to 0.02% w / w, or 0.005% w / w to 0.01% w / w of any polypeptide provided herein (e.g., truncated and / or non-naturally occurring elastin).

[0024] In certain embodiments, the compositions provided herein are topical compositions, such as compositions formulated and / or suitable for topical application or use. The topical compositions provided herein may include any polypeptide described herein (e.g., truncated and / or non-naturally occurring elastin, e.g., in any suitable amount) and at least one other component. In one aspect, a method is provided herein, such as to provide (e.g., as described herein) benefits to the skin of a subject, the method comprising applying the topical composition to the subject's skin topically. In a specific embodiment, the topical composition may be used to reduce skin damage, promote damaged skin repair, stimulate skin cells to produce collagen, improve skin health and moisturizing, improve skin appearance. In certain embodiments, the topical composition may be used to increase, promote, stimulate or otherwise increase the method for producing elastin in the skin. In certain embodiments, the topical composition may be used to stimulate fibroblast growth and / or stimulate tropoelastin synthesis and / or reduce the method for the formation of thymine-thymine (TT) dimers in cells. The topical composition may also include at least one other component, including a topical carrier or a preservative.

[0025] In one embodiment, the topical composition comprises one or more topical carriers selected from the group consisting of water, glyceryl ether-8 oil, glycerin, coconut alkanes, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, pentylene glycol, disodium EDTA, caprylyl glycol, chlorphenesin and phenoxyethanol, liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusted powders, biodegradable polymers, mineral oil, triglyceride oils, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone and cyclopentasiloxane.

[0026] In another embodiment, the topical composition comprises one or more preservatives 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-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA.

[0027] In some embodiments, a method for reducing skin damage and / or promoting damaged skin repair is provided. The method may include applying a composition (e.g., a topical composition) comprising any non-naturally occurring polypeptide described herein (e.g., truncated and / or non-naturally occurring human elastin) to the skin of a subject. In some cases, the method increases the vigor of fibroblasts or keratinocytes of the subject's skin. In some cases, the application of the composition (e.g., a topical composition) increases the synthesis of tropoelastin by fibroblasts of the subject's skin. In some cases, the topical application of the composition (e.g., a topical composition) protects skin or keratinocytes from ultraviolet damage. In some cases, the application of the composition (e.g., a topical composition) to the subject's skin reduces the formation of thymine-thymine (TT) dimers.

[0028] On the other hand, the non-naturally occurring polypeptide (e.g., truncated human elastin) is a recombinant polypeptide (e.g., produced by a host cell, e.g., by recombinant expression). The recombinant polypeptide can be a recombinant human elastin (e.g., a non-naturally occurring and / or truncated human elastin). In one embodiment, the recombinant elastin is a full-length or truncated elastin. In one embodiment, the recombinant polypeptide (e.g., truncated human elastin) is truncated at the C-terminus relative to natural and / or full-length elastin, truncated at the N-terminus relative to natural and / or full-length elastin, truncated internally relative to natural and / or full-length elastin, or truncated at both the C-terminus and the N-terminus of natural and / or full-length elastin.

[0029] In some aspects, the recombinant polypeptides described herein (e.g., non-naturally occurring and / or truncated human elastin) can be 10 to 700 amino acids, 10 to 600, 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 length.

[0030] In another embodiment, the molecular weight of a recombinant polypeptide described herein (e.g., non-naturally occurring and / or truncated human elastin) may be from 1 kDa to 60 kDa, 5 kDa to 55 kDa, 5 kDa to 50 kDa, 5 kDa to 45 kDa, 5 kDa to 40 kDa, 5 kDa to 35 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, 5 kDa to 15 kDa, 5 kDa to 10 kDa, 10 kDa to 40 kDa, 10 kDa to 35 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, or 10 kDa to 20 kDa.

[0031] In various aspects, the recombinant host cell may comprise at least one copy of a heterologous nucleic acid sequence encoding a recombinant polypeptide described herein (e.g., a non-naturally occurring and / or truncated elastin protein). In some cases, the nucleic acid sequence encoding a recombinant polypeptide (e.g., a non-naturally occurring and / or truncated human elastin protein) may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and homologs thereof (e.g., having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity thereto). In another embodiment, the nucleic acid sequence encoding a recombinant polypeptide (e.g., non-naturally occurring and / or truncated human elastin) may consist of a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, and homologs thereof (e.g., having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity thereto).

[0032] In another embodiment, the recombinant polypeptide described herein may include one or more selected from the following: a secretion tag, a histidine tag, a fluorescent protein tag, and a protease cleavage site. In some cases, the secretion tag is 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 a specific embodiment, the secretion tag is DsbA.

[0033] Polynucleotides encoding the polypeptides described herein (e.g., non-naturally occurring and / or truncated elastin) and vectors comprising the polynucleotides can be used to transform host cells, which can then express the recombinant polypeptide (e.g., non-naturally occurring and / or truncated elastin).

[0034] In another embodiment, a host cell (e.g., a recombinant host cell) that has been engineered to express a disclosed recombinant polypeptide (e.g., a non-naturally occurring and / or truncated elastin) is provided. The host cell can be any host cell, including bacterial cells, yeast cells, fungal cells, insect cells, mammalian cells, plant cells, and any other cells for expressing exogenous polynucleotides. In a specific embodiment, the host cell is Escherichia coli. In a specific embodiment, the host cell is capable of secreting the recombinant polypeptide extracellularly (e.g., secreted into the culture medium).

[0035] In another embodiment, a method for producing a polypeptide as described herein (e.g., a recombinant polypeptide, such as a non-naturally occurring elastin and / or a truncated elastin) is provided. The method comprises the following steps: inoculating a culture medium with a recombinant host cell comprising at least one copy of a heterologous nucleic acid sequence encoding a polypeptide as described herein (e.g., a non-naturally occurring and / or truncated elastin), culturing the host cell (e.g., culturing the host cell in a culture medium), whereby the host cell secretes the recombinant polypeptide extracellularly (e.g., secretes into the culture medium), and isolating the recombinant polypeptide (e.g., from the host cell and / or the culture medium).

[0036] On the other hand, provided herein is a method for reducing the production of inflammatory cytokines in skin cells. In one embodiment, the skin cell is a keratinocyte or a fibroblast. The method includes applying a polypeptide as described herein (e.g., non-naturally occurring and / or truncated elastin, such as in a topical formulation) to a subject's skin cell or skin. In some cases, the production of inflammatory cytokines (e.g., TNFα, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18, and IL-1RA) is reduced.

[0037] On the other hand, a method for reducing skin damage or promoting damaged skin repair is provided. The method may include applying a composition (e.g., a composition formulated for topical application) comprising a non-natural polypeptide as described herein (e.g., truncated elastin) to the skin of a subject. In some cases, the method increases the vigor of fibroblasts or keratinocytes of the subject's skin. In some cases, the application of the composition increases the synthesis of tropoelastin by fibroblasts or keratinocytes of the subject's skin. In some cases, the topical application of the composition protects skin, fibroblasts or keratinocytes from ultraviolet damage. In some cases, the formation of thymine-thymine (TT) dimers is reduced by topical application of the composition provided herein.

[0038] On the other hand, provided herein is a method for protecting skin cells from exposure to urban dust. In some cases, the method includes applying a non-naturally occurring polypeptide as described herein (e.g., a truncated elastin) to a skin cell or skin of a subject. In some cases, exposure of the skin cell or skin of the subject to a non-naturally occurring polypeptide increases the vitality of the skin cell after exposure to urban dust. The skin cell can be a keratinocyte or a fibroblast. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the present invention are utilized, in which:

[0040] Figure 1 Shown is a photo of a protein gel. Lane 1 is a size marker. Lanes 2 and 3 show the 23 kDa truncated human elastin of Example 10, which diffuses on the gel with an expected molecular weight. Lane 4 is blank and does not load any protein. Lanes 5 and 6 show the 13 kDa truncated human elastin of Example 11, which diffuses on the gel with an expected molecular weight.

[0041] Figure 2 Depicted is an SDS-PAGE protein gel showing the high purity and homogeneity of the recombinantly produced polypeptides described herein (comprising non-naturally occurring truncated elastin amino acid sequences).

[0042] Figure 3 Depicted are the secretion levels of procollagen type I C-peptide following treatment of fibroblasts with exemplary polypeptides provided herein (comprising a non-naturally occurring truncated elastin amino acid sequence).

[0043] Figure 4The antioxidant capacity of exemplary polypeptides provided herein (comprising non-naturally occurring truncated elastin amino acid sequences) is depicted.

[0044] Figure 5 Depicted are the expression levels of mRNA for various antioxidant genes following treatment of keratinocytes with exemplary polypeptides provided herein (comprising non-naturally occurring truncated elastin amino acid sequences).

[0045] Figure 6 Depicted are the expression levels of mRNA for various pro-apoptotic genes following treatment of keratinocytes with exemplary polypeptides provided herein (comprising a non-naturally occurring truncated elastin amino acid sequence). DETAILED DESCRIPTION

[0046] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure can be practiced without these details.

[0047] As used herein, the term "about" generally refers to ± 10%.

[0048] The term “consisting of” means “including and limited to.” Generally, disclosures of “comprising” include disclosures of “consisting of.”

[0049] The term "consisting essentially of means that the composition, method, or structure may include additional ingredients, steps, and / or parts, but only if the additional ingredients, steps, and / or parts do not materially change the basic and novel characteristics of the claimed composition, method, or structure. Generally, disclosures of "comprising" include disclosures of "consisting essentially of."

[0050] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0051] Throughout the document, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be construed as a strict limitation of the scope of the present disclosure. Therefore, the description of the range should be considered as specifically disclosing all possible subranges and each numerical value within the range. For example, the description of a range such as from 1 to 6 should be considered as explicitly disclosing subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and each numeral within the range, such as 1, 2, 3, 4, 5 and 6. No matter how wide the scope is, this applies.

[0052] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrase "range between" a first indicated numeral and a second indicated numeral and the phrase "range from" a first indicated numeral to" a second indicated numeral are used interchangeably herein and are intended to include the first indicated numeral and the second indicated numeral and all fractions and integers therebetween.

[0053] As used herein, the term "elastin" or "elastin-like" refers to such (e.g., monomeric) polypeptides in some cases, which may be associated with one or more elastin or elastin-like polypeptides, and / or may be combined with another polypeptide, nucleic acid, polysaccharide, lipid, or other molecule. Typically, the term "elastin" or "elastin-like" refers to a polypeptide with one or more functions associated with naturally occurring elastin. When non-naturally occurring polypeptides provided herein (e.g., truncated elastin) have one or more functions associated with naturally occurring elastin, they may be referred to as "elastin" or "elastin-like". As used herein, the term "tropoelastin" generally refers to a polypeptide that may be further processed (e.g., by cutting, splicing, etc.) to produce an elastin polypeptide. A non-limiting example of elastin is a naturally occurring human elastin with SEQ ID NO:20 amino acid sequence.

[0054] As used herein, the term "expression vector" or "vector" generally refers to a nucleic acid assembly capable of directing the expression of an exogenous gene. An expression vector may include a promoter operably linked to an exogenous gene, a restriction endonuclease site, a nucleic acid encoding one or more selection markers, and other nucleic acids that can be used in the practice of recombinant technology.

[0055] As used herein, the term "extracellular matrix" generally refers to a network of extracellular macromolecules (e.g., elastin, collagen, enzymes, and glycoproteins) that provide a scaffold for cells in multicellular organisms. The extracellular matrix can provide structural components that mediate cell adhesion, intercellular communication, and other functions.

[0056] As used herein, the term "fibroblast" generally refers to cells that synthesize tropoelastin, tropoelastin, and other structural proteins. Fibroblasts are widely distributed in the body and are present in the skin, connective tissue, and other tissues.

[0057] The term "fluorescent protein" generally refers to a protein that can be used as a reporter molecule for the expression of exogenous polynucleotides in genetic engineering technology. The protein fluoresces and emits bright visible light when exposed to ultraviolet or blue light. Proteins that emit green light include green fluorescent protein (GFP), and proteins that emit red light include red fluorescent protein (RFP).

[0058] As used herein, the term "gene" generally refers to a polynucleotide encoding a specific protein, and 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.

[0059] The term "histidine tag" generally refers to a series of 2-30 consecutive histidine residues on a recombinant polypeptide.

[0060] The term "host cell" generally refers to a cell that has been engineered to express an introduced exogenous polynucleotide.

[0061] The term "keratinocyte" generally refers to cells that produce keratin, tropoelastin, and other cellular components found in the epidermal layer of the skin.

[0062] As used herein, the term "non-naturally occurring" refers to genes, polypeptides or proteins that are not usually found in nature, such as elastin. Non-naturally occurring elastin can be recombinantly prepared (e.g., by being expressed by a recombinant host cell). Non-naturally occurring elastin can be a recombinant elastin (e.g., by being expressed by a recombinant host cell). Non-naturally occurring elastin is a truncated elastin. Other non-naturally occurring elastin polypeptides include chimeric elastin. Chimeric elastin can be a polypeptide in which a portion of an elastin polypeptide is adjacent to a portion of a second elastin polypeptide. For example, a molecule comprising a portion of human elastin adjacent to a portion of another human polypeptide can be a chimeric elastin. In another embodiment, the non-naturally occurring elastin includes 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.

[0063] Generally, the disclosure of elastin or truncated elastin (e.g., with a specific amino acid sequence) provided herein includes a polypeptide having or comprising this precise amino acid sequence and its homologue. In some cases, the homologue of the amino acid sequence provided herein can have a longer or shorter sequence, and can have the displacement of one or more amino acid residues of the amino acid sequence. Such homologue has a specific sequence identity (e.g., with a sequence identity amount provided herein) with the sequence. Sequence identity (e.g., for the purpose of assessing percent identity) can be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including the default parameters. In some cases, the non-naturally occurring elastin protein may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the sequences disclosed herein.

[0064] The term "protease cleavage site" generally refers to an amino acid sequence that is cleaved by a specific protease.

[0065] The term "secretion tag" or "signal peptide" generally refers to an amino acid sequence that recruits the cellular machinery of the host cell to transport the expressed protein to a specific location or organelle of the host cell.

[0066] The term "truncated elastin" generally refers to a monomeric polypeptide that is 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, truncated at the N-terminus relative to the full-length elastin, truncated by removing one or more internal portions of the full-length elastin polypeptide (e.g., internal truncations), or truncated at both the C-terminus and the N-terminus relative to the full-length elastin. In non-limiting embodiments, the truncated human elastin may comprise an amino acid sequence according to SEQ ID NO: 19 or a homolog thereof. Generally, the truncated elastin provided herein may have similar or substantially similar functions and / or provide similar or substantially similar benefits (e.g., as provided herein) to natural or full-length elastin. In some cases, the truncated elastin provided herein may have improved or increased functions and / or benefits (e.g., as provided herein) compared to natural or full-length elastin.

[0067] When used to refer to an amino acid position, "truncation" includes the amino acid position. For example, an N-terminal truncation at amino acid position 100 of a full-length protein means that 100 amino acids are truncated from the N-terminus of the full-length protein (i.e., the truncated protein lacks amino acids 1 to 100 of the full-length protein). Similarly, a C-terminal truncation of a full-length protein (assuming a full-length protein of 1000 amino acids) at amino acid position 901 means that 100 amino acids are truncated from the C-terminus (i.e., the truncated protein lacks amino acids 901 to 1000 of the full-length protein). Similarly, internal truncations at amino acid positions 101 and 200 refer to internal truncations 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).

[0068] In some embodiments, the cell culture may further include 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.

[0069] The host bacterial cells can be cultured continuously or discontinuously; in batch, fed-batch or repeated fed-batch processes.

[0070] On the one hand, a non-naturally occurring elastin protein is provided. In some cases, the non-naturally occurring elastin protein is a recombinant polypeptide produced by a host cell (e.g., a recombinant cell). In some cases, the non-naturally occurring elastin protein can be human elastin protein. In some cases, the non-naturally occurring elastin protein can be a truncated elastin protein. The truncated elastin protein can be truncated relative to the full-length elastin protein (e.g., with SEQ ID NO: 20 amino acid sequence). Truncation can be an internal truncation relative to the full-length elastin protein, a truncation relative to the full-length elastin protein at the N-terminal portion, a truncation relative to the full-length elastin protein at the C-terminal portion, or a truncation relative to the full-length elastin protein at the C-terminal and N-terminal portions.

[0071] The truncated 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 length relative to the full-length elastin. In another embodiment, the truncated elastin may be truncated by 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700 or 750 amino acids. The truncated elastin may be encoded by a portion of the polynucleotide sequence disclosed herein or the entire polynucleotide sequence.

[0072] In some embodiments, the truncated elastin (e.g., an amino acid sequence thereof) (e.g., of a polypeptide provided herein) can be truncated at the C-terminus (relative to the 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, and the like. In some cases, the truncated elastin may be truncated at the C-terminus by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210

[0073] In some embodiments, the truncated elastin (e.g., an amino acid sequence thereof) (e.g., of a polypeptide provided herein) can be truncated at the N-terminus (relative to the 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 at the N-terminus (relative to the full-length elastin) by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210

[0074] In some embodiments, the truncated elastin (e.g., its amino acid sequence) (e.g., polypeptide provided herein) can be truncated at both the N-terminus and the C-terminus relative to the full-length elastin. In some cases, the truncated elastin can be truncated at the N-terminus (relative to the full-length elastin) by any suitable number of amino acid residues, for example 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 1 00 and so on; and can be truncated at the C-terminus (relative to full-length elastin) by any suitable number of amino acid residues, for example, 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 and so on.In some cases, the truncated elastin may be truncated at the N-terminus (relative to the full-length elastin) by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370 , 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more , 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200

[0075] In some embodiments, the truncated elastin (e.g., an amino acid sequence thereof) (e.g., of a polypeptide provided herein) can be internally truncated (relative to the 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, and the like. In some cases, the truncated elastin may be internally truncated (relative to the full-length elastin) by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200

[0076] The truncated elastin disclosed herein may include a truncation relative to the full-length elastin. In some embodiments, the truncated elastin disclosed herein may include a truncation relative to the full-length human elastin. In some cases, the full-length human elastin has the amino acid sequence of SEQ ID NO:20 provided in Table 1 below.

[0077] Table 1. Full-length elastin amino acid sequence

[0078]

[0079]

[0080] In some cases, a truncated elastin protein as described herein may comprise an N-terminal truncation at any amino acid position between amino acid positions 1 and 68, between amino acid positions 1 and 73, between amino acid positions 1 and 78, between amino acid positions 1 and 83, between amino acid positions 1 and 88, between amino acid positions 1 and 93, or between amino acid positions 1 and 98 of SEQ ID NO: 20. In some cases, a truncated elastin protein as described herein may comprise a C-terminal truncation at any amino acid position between amino acid positions 213 and 760, between amino acid positions 218 and 760, between amino acid positions 223 and 760, between amino acid positions 228 and 760, between amino acid positions 233 and 760, between amino acid positions 238 and 760, or between 243 and 760 of SEQ ID NO: 20. In some cases, a truncated elastin protein as described herein may comprise both an N-terminal truncation and a C-terminal truncation. For example, the truncated elastin protein as described herein can comprise an N-terminal truncation at any amino acid position between amino acid positions 1 and 68, between amino acid positions 1 and 73, between amino acid positions 1 and 78, between amino acid positions 1 and 83, between amino acid positions 1 and 88, between amino acid positions 1 and 93, or between amino acid positions 1 and 98 of SEQ ID NO: 20; and a C-terminal truncation at any amino acid position between amino acid positions 213 and 760, between amino acid positions 218 and 760, between amino acid positions 223 and 760, between amino acid positions 228 and 760, between amino acid positions 233 and 760, between amino acid positions 238 and 760, or between 243 and 760 of SEQ ID NO: 20. In a specific embodiment, the truncated elastin protein disclosed herein can comprise an N-terminal truncation at amino acid position 83 of SEQ ID NO: 20; and a C-terminal truncation at amino acid position 228 of SEQ ID NO: 20.

[0081] In some cases, a truncated elastin protein as described herein may comprise an N-terminal truncation at any amino acid position between amino acid positions 1 and 68, between amino acid positions 1 and 73, between amino acid positions 1 and 78, between amino acid positions 1 and 83, between amino acid positions 1 and 88, between amino acid positions 1 and 93, or between amino acid positions 1 and 98 of SEQ ID NO: 20. In some cases, a truncated elastin protein as described herein may comprise a C-terminal truncation at any amino acid position between amino acid positions 331 and 760, between amino acid positions 336 and 760, between amino acid positions 341 and 760, between amino acid positions 346 and 760, between amino acid positions 351 and 760, between amino acid positions 356 and 760, or between amino acid positions 361 and 760 of SEQ ID NO: 20. In some cases, a truncated elastin protein as described herein may comprise an N-terminal truncation and a C-terminal truncation. For example, a truncated elastin protein as described herein may comprise an N-terminal truncation at any amino acid position between amino acid positions 1 and 68, between amino acid positions 1 and 73, between amino acid positions 1 and 78, between amino acid positions 1 and 83, between amino acid positions 1 and 88, between amino acid positions 1 and 93, or between amino acid positions 1 and 98 of SEQ ID NO: 20; and may comprise a C-terminal truncation at any amino acid position between amino acid positions 331 and 760, between amino acid positions 336 and 760, between amino acid positions 341 and 760, between amino acid positions 346 and 760, between amino acid positions 351 and 760, between amino acid positions 356 and 760, or between amino acid positions 361 and 760 of SEQ ID NO: 20. In a specific embodiment, a truncated elastin protein as described herein may comprise an N-terminal truncation at amino acid position 83 of SEQ ID NO: 20; and a C-terminal truncation at amino acid position 346 of SEQ ID NO: 20.

[0082] In some cases, the truncated elastin protein may comprise any of the amino acid sequences provided in Table 2 below. In some cases, the truncated elastin protein may consist of any of the amino acid sequences provided in Table 2 below. In some cases, the truncated elastin protein may consist essentially of any of the amino acid sequences provided in Table 2 below. In specific embodiments, the non-naturally occurring elastin protein is or comprises any of the following amino acid sequences: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19. In some embodiments, the truncated elastin protein 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 of: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

[0083] In some embodiments, the truncated elastin can be a truncation (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 any of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19; or can be a truncation (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: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19. NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. 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: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; or an N-terminal truncation, a C-terminal truncation and / or an internal truncation relative to 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: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.

[0084] Table 2. Non-limiting examples of truncated elastin proteins

[0085]

[0086]

[0087]

[0088]

[0089] In various aspects, the truncated human elastin protein does not comprise two or more consecutive amino acid sequences of VGVAPG (SEQ ID NO: 1). In some cases, the truncated human elastin protein does not comprise VGVAPGVGVAPG (SEQ ID NO: 2). In some cases, the truncated human elastin protein does not comprise VGVAPGVGVAPGVGVAPG (SEQ ID NO: 3). In some cases, the non-naturally occurring truncated human elastin protein reduces or does not cause extracellular matrix degradation.

[0090] In some cases, the length of the truncated elastin protein can 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 250 amino acids, 160 to 220 amino acids, 170 to 200 amino acids, 180 to 190 amino acids, or 185 to 190 amino acids.

[0091] In some aspects, the molecular weight of the truncated human elastin may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or greater than 50 kDa. In some embodiments, the molecular weight is less than about 10, 15, 20, 25, 30, 35, 40, 45 or 50 kDa. In some embodiments, the molecular weight is 1 kDa to 60 kDa, 5 kDa to 55 kDa, 5 kDa to 50 kDa, 5 kDa to 45 kDa, 5 kDa to 40 kDa, 5 kDa to 35 kDa, 5 kDa to 30 kDa, 5 kDa to 25 kDa, 5 kDa to 20 kDa, 5 kDa to 15 kDa, 5 kDa to 10 kDa, 10 kDa to 40 kDa, 10 kDa to 35 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa or 10 kDa to 20 kDa.

[0092] In some embodiments, the non-naturally occurring elastin may comprise a signal sequence. Typically, the signal sequence may be a component of an expression vector, or may be a part of an exogenous gene inserted into a vector. The selected signal sequence may be a signal 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 natural signal sequence of an exogenous gene, the signal sequence may be replaced by any commonly known bacterial signal sequence. In some embodiments, the DsbA signal sequence may be used to target the recombinantly produced polypeptide to the periplasmic space. Dinh and Bernhardt, J Bacteriol, September 2011, 4984-4987.

[0093] In some embodiments, the non-naturally occurring elastin may further include an amino acid sequence comprising a secretion tag. The secretion tag may direct the elastin to the periplasmic space of the host cell. In a specific embodiment, the signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, DegP or Hy1A, or a hybrid secretion tag comprising a portion of a secretion tag fused to a portion of a second secretion tag. On the one hand, the secretion tag is attached to the non-naturally occurring elastin. On the other hand, the secretion tag may be cut from the non-naturally occurring elastin.

[0094] In some embodiments, the elastin protein that non-naturally occurs further comprises a histidine tag. The histidine tag or polyhistidine tag can be a sequence of 2 to 20 histidine residues attached to the elastin protein. The histidine tag can comprise 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues or 7 to 10 histidine residues. The histidine tag can be used for protein purification by adopting a chromatographic method based on a nickel-based chromatographic medium. On the one hand, the histidine tag can be attached to the elastin protein that non-naturally occurs. On the other hand, the histidine tag can be cut from the elastin protein that non-naturally occurs.

[0095] In some embodiments, the non-naturally occurring elastin protein also 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 elastin protein comprises GFP and / or RFP. In one embodiment, a super-folded GFP can be fused with the non-naturally occurring elastin protein. A super-folded GFP can be a GFP that can be correctly folded even when fused with a poorly folded polypeptide.

[0096] In some embodiments, the non-naturally occurring elastin protein also comprises a protease cleavage site. The protease cleavage site can be used to cut the recombinantly produced elastin protein to remove one or more parts of the polypeptide. The polypeptide portion that can be removed includes a secretion tag, a histidine tag, a fluorescent protein tag and / or a protease cleavage site. Proteases can include endoproteinases, exoproteinases, serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases and metalloproteinases. Exemplary protease cleavage sites include amino acids that are cut by thrombin, TEV protease, factor Xa, enteropeptidase and rhinovirus 3C protease. On the one hand, the cleavage tag can be attached to the non-naturally occurring elastin protein. On the other hand, the cleavage tag is removed from the non-naturally occurring elastin protein by an appropriate protease.

[0097] Provided in certain embodiments herein is a composition or preparation (e.g., local) comprising one or more polypeptides provided herein. In some embodiments, compositions, for example, provide any suitable amount of polypeptides provided herein with any suitable amount (e.g., when given or applied to an individual or cell, the amount suitable for providing a benefit). In some specific embodiments, compositions include when (e.g., topically) applied to subject's skin, the amount suitable for providing a beneficial effect to subject's skin. In specific embodiments, compositions include 0.001% to 30% w / w of, for example, polypeptides (or non-naturally occurring elastin) provided herein. In more specific embodiments, the composition comprises 0.001% to 20% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin), 0.001% to 10% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin), 0.001% to 5% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin), 0.001% to 2% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin), 0.001% to 1% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin), 0.001% to 0.5% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin), and 0.001% to 0.2% w / w of a polypeptide, e.g., as provided herein (or a non-naturally occurring elastin).

[0098] On the one hand, the composition comprising non-naturally occurring human elastin is a personal care product (e.g., cosmetics). In some embodiments, the composition is formulated for topical application. The composition may include other cosmetic ingredients suitable for human use. Personal care products can be used to prevent or treat damage to human skin or hair caused by ultraviolet radiation. Personal care products can be used to increase the firmness, elasticity, brightness, hydration, tactile texture or visual texture of the skin, and / or stimulate collagen production. Personal care products can be applied to skin or hair. Compositions include, for example, facial masks, skin cleansers (such as soap, cleansing cream, cleansing lotion, facial cleanser, cleansing milk, cleansing pads, facial cleansers), face and body creams and moisturizers, facial essences, facial masks and body masks, toners and facial sprays, eye creams and eye care agents, exfoliating formulas, lip balms and lipsticks, shampoos, conditioners and shower gels, hair and scalp essences, hair mists and sprays, eye shadows, concealers, mascara and other color cosmetics.

[0099] The composition comprising non-naturally occurring elastin may further comprise at least one additional ingredient, including a topical carrier or preservative. Topical carriers include topical carriers selected from liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusted powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerine, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and water. Preservatives include preservatives selected from tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride cis isomer, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, phenoxyethanol, butylene glycol, 1,2-hexanediol, methylparaben, sorbic acid, Germaben II, rosemary extract and EDTA.

[0100] Also provided herein is a method for reducing skin damage, promoting damaged skin repair, protecting skin from ultraviolet damage and / or protecting skin cells from the effects of urban dust exposure. In another embodiment, a method for increasing the firmness, elasticity, brightness, hydration, tactile texture or visual texture of the skin and / or stimulating elastin or collagen production is provided. The method includes applying a composition comprising non-natural elastin to the skin of a subject. Without being bound by a particular theory or mechanism, when applied to the skin, the non-natural elastin in the composition reduces skin damage by being protected from ultraviolet damage, and / or promotes damaged skin repair by increasing cell viability and / or increasing tropoelastin or tropoelastin synthesis, and / or promotes the viability of skin cells. On the one hand, non-naturally occurring truncated human elastin reduces the formation of thymine-thymine (TT) dimers.

[0101] The methods provided herein include, for example, using the compositions for the treatments indicated in the methods by the steps provided herein. In embodiments, the disclosure provides uses of the compositions provided herein (e.g., truncated elastin or a preparation comprising truncated elastin) in the methods provided herein.

[0102] In some embodiments, the administration of the composition stimulates the growth of fibroblasts, and / or stimulates the synthesis of tropoelastin, and / or reduces the formation of thymine-thymine (TT) dimers. In some embodiments, the administration of the composition stimulates the growth of fibroblasts by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to skin or skin cells to which the composition is not applied. In some embodiments, the administration of the composition stimulates the synthesis of tropoelastin by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to skin or skin cells to which the composition is not applied. In some embodiments, administration of the composition reduces the formation of TT dimers by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% compared to skin or skin cells to which the composition has not been applied. In some embodiments, the formation of TT dimers is measured using an ELISA-based assay.

[0103] In some embodiments, disclosed herein is a method for promoting the repair of damaged skin, protecting the skin from ultraviolet damage, or increasing the vitality of skin cells, the method comprising the step of applying a composition to the skin or skin cells of a subject. In some embodiments, when the composition is applied, the damaged skin can be repaired by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, compared to skin or skin cells to which the composition is not applied. In some embodiments, when the composition is applied, the skin can be protected from ultraviolet damage by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, compared to skin or skin cells to which the composition is not applied. In some embodiments, the viability of the skin cells is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% compared to skin or skin cells to which the composition is not applied. In some embodiments, the skin cells can be keratinocytes. In some embodiments, the skin cells can be fibroblasts. In some embodiments, the viability of fibroblasts can be measured using an MTT assay.

[0104] In some embodiments, disclosed herein is a method for increasing the synthesis of procollagen by fibroblasts by applying a composition to the skin or skin cells of a subject. In some embodiments, when the composition is applied, the synthesis of procollagen by fibroblasts present in the subject's skin may increase by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%, compared to skin or skin cells to which the composition is not applied. In some embodiments, the synthesis of procollagen by fibroblasts present in the subject's skin may increase by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%, as measured by an ELISA-based assay. In some embodiments, the level of procollagen-type IC peptides is measured based on an ELISA-based assay.

[0105] In some embodiments, disclosed herein is a method for reducing the production of inflammatory cytokines by skin cells, the method comprising applying a non-naturally occurring elastin or a composition comprising non-naturally occurring elastin to the skin or skin cells of a subject. In some embodiments, cytokines include TNFα, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18, IL-1RA, or a combination thereof. In some embodiments, inflammatory cytokines may be interleukins, such as IL-1, IL-6, or IL-8. In some embodiments, inflammatory cytokines may be IL-1a. In some embodiments, when the composition is applied, the inflammatory cytokines produced by skin cells may be reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, compared to skin or skin cells to which the composition is not applied. In some embodiments, skin cells may be keratinocytes. In some embodiments, skin cells may be fibroblasts. In some embodiments, cytokine production can be measured using ELISA-based assays.

[0106] In some embodiments, the truncated elastin provided herein can stimulate the expression of antioxidant genes in the skin cells or skin of the experimenter. In some cases, the truncated elastin provided herein can stimulate the expression of one or more antioxidant genes selected from the group consisting of SOD2, GPX2, GPX4, GSTK1, GSTZ1, GSTA4, GSTM2, CCS, GPX1, GLRX, PRDX5, PRDX6 and PRDX2. In some cases, mRNA expression (e.g., by microarray, RNA sequencing, etc.) can be measured.

[0107] In some embodiments, the truncated elastin provided herein can reduce the expression of pro-apoptotic genes in the skin cells or skin of the subject. In some cases, the truncated elastin provided herein can reduce the expression of one or more pro-apoptotic genes selected from the group consisting of APAF1, BAK1, CASP7, CASP8, FADD, MCL1 and MET. In some cases, mRNA expression can be measured (e.g., by microarray, RNA sequencing, etc.).

[0108] In some embodiments, disclosed herein is a method for increasing the viability of skin cells, the method comprising applying a non-naturally occurring elastin or a composition comprising non-naturally occurring elastin to the skin or skin cells of a subject. In some embodiments, when the composition is applied, the viability of the skin cells may be increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100% compared to skin or skin cells to which the composition is not applied. In some embodiments, the skin cells may be keratinocytes. In some embodiments, the skin cells may be fibroblasts.

[0109] In some embodiments, disclosed herein is a method for protecting skin cells from exposure to urban dust, the method comprising applying a non-naturally occurring elastin or a composition comprising a non-naturally occurring elastin to the skin or skin cells of a subject, wherein the viability of the skin cells is increased. In some embodiments, the viability of the skin cells may be increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater than 100% compared to untreated cells. In some embodiments, the skin cells may be keratinocytes. In some embodiments, the skin cells may be fibroblasts. In some embodiments, the MTT assay may be used to measure viability.

[0110] In some embodiments, the composition has antioxidant capacity. In some embodiments, the antioxidant capacity can be measured using the oxygen radical absorbance capacity (ORAC) assay. In some embodiments, the antioxidant capacity can be measured in Trolox equivalent units. In some embodiments, the antioxidant capacity of the composition can be at least 50 μM, 100 μM, 150 μM, 200 μM, 250 μM or greater than 250 μM Trolox equivalent units.

[0111] 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 include a polynucleotide according to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or a homolog thereof (e.g., having at least 80%, 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).

[0112] In one aspect, the polynucleotide can be contained in a vector (e.g., to transform a host cell). The polynucleotide can further comprise a nucleic acid encoding an enzyme that allows the host organism to grow in the presence of a selection agent. The selection agent can include certain sugars, including galactose-containing sugars, or antibiotics, including ampicillin, hygromycin, G418, etc. The enzyme used to confer resistance to the selection agent can include beta-galactosidase.

[0113] In one aspect, a host cell expressing the disclosed polynucleotides (and polypeptides encoded by the polynucleotides) is provided. In some cases, the host cell may contain at least one copy of a heterologous nucleic acid sequence (e.g., encoding a polypeptide as described herein). The host cell may be any host cell, including gram-negative bacterial cells, gram-positive bacterial cells, yeast cells, insect cells, mammalian cells, plant cells, or any other cell for expressing exogenous polynucleotides. In a specific embodiment, the host cell is Escherichia coli. In various aspects, the host cell may be able to secrete non-naturally occurring elastin outside the cell (e.g., secreted into the culture medium).

[0114] In addition to carbon, nitrogen and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations, introduced alone or as a mixture with another supplement or culture (such as a complex nitrogen source). In certain embodiments, the culture medium further comprises one or more components selected from the group consisting of ammonium chloride, ammonium sulfate, calcium chloride, casamino acids, iron (II) sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate and yeast extract.

[0115] Another embodiment provides a method for producing non-natural elastin. The method includes inoculating a culture medium with a recombinant host cell comprising at least one copy of a heterologous nucleic acid sequence, wherein the heterologous nucleic acid sequence encodes a non-natural elastin, cultivating the host cell (e.g., cultivating the host cell in a culture medium) and isolating the non-natural elastin from the host cell. In some cases, the method includes cultivating a recombinant host cell in a culture medium, wherein the recombinant host cell secretes the non-natural elastin into the culture medium, collecting the culture medium comprising the non-natural elastin secreted therein, and purifying and / or separating (e.g., by centrifugation, filtration, etc.) the non-natural elastin from the culture medium.

[0116] In another aspect, a method for preparing protein by fermentation is provided. The method comprises the following steps:

[0117] a) culturing a recombinant bacterial cell in a medium comprising a magnesium salt, wherein the concentration of magnesium ions in the medium is at least about 6 mM, and wherein the bacterial cell comprises an exogenous gene encoding the protein; and

[0118] b) harvesting the protein from the culture medium.

[0119] Bacteria can be cultured continuously (as described in, for example, WO 05 / 021772) or discontinuously in a batch process (batch culture) or in a fed batch or repeated fed batch process for the production of target protein. In some embodiments, protein production is carried out on a large scale. Various large-scale fermentation procedures can be used to produce recombinant proteins. Large-scale fermentation has a capacity of at least 1,000 liters, preferably a capacity of about 1,000 to 100,000 liters. In some cases, the fermentor uses an agitator impeller to distribute oxygen and nutrients, especially glucose (preferred carbon source / energy source). Small-scale fermentation generally refers to fermentation in a fermentor, and the capacity of the fermentor is no more than about 20 liters.

[0120] In order to accumulate the target protein, the host cells are cultured under conditions sufficient to accumulate the target protein. Such conditions include, for example, temperature, nutrients, and cell density conditions that allow the cells to express and accumulate the protein. In addition, as known to those skilled in the art, such conditions may refer to conditions under which the cells can perform the following basic cellular functions for secreted proteins: transcription, translation, and transfer of proteins from one cell compartment to another.

[0121] Bacterial cells are cultured at a suitable temperature. For E. coli growth, for example, a typical temperature range is from about 20°C to about 39°C. In one embodiment, the temperature is from about 20°C to about 37°C. In another embodiment, the temperature is about 30°C. In one embodiment, host cells can be cultured at a temperature in a non-switched state or a switched state, and switched to different temperatures to induce protein production. Host cells can be cultured at a temperature to propagate cells, and then cultured at a lower temperature to induce protein production. The first temperature can be 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C. The second temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C. Culturing at the second temperature can be carried out for 1 hour to 100 hours, 5 hours to 90 hours, 5 hours to 80 hours, 5 hours to 80 hours, 5 hours to 70 hours, 10 hours to 70 hours, 15 hours to 70 hours, 15 hours to 65 hours, 15 hours to 60 hours, 20 hours to 60 hours, 20 hours to 55 hours, 20 hours to 50 hours, 24 hours to 50 hours, 24 hours to 48 hours, 30 hours to 50 hours, 30 hours to 45 hours or 30 hours to 40 hours.

[0122] The pH of the culture medium can be any pH from about 5-9, depending primarily on the host organism. For E. coli, the pH can be from about 6.0 to about 7.4, from about 6.2 to about 7.2, from about 6.2 to about 7.0, from about 6.2 to about 6.8, from about 6.2 to about 6.6, about 6.4, or about 6.5.

[0123] In order to induce gene expression, cells can generally be cultured until a certain optical density is reached, for example, OD 600 is about 1.1, at which point induction is initiated (e.g., by adding an inducer, by exhausting a repressor, inhibitor or medium component, etc.) to induce the expression of an exogenous gene encoding a target protein. In some embodiments, the expression of an exogenous gene can be induced by an inducer selected from, for example, isopropyl-β-d-1-thiogalactopyranoside, lactose, arabinose, maltose, tetracycline, anhydrotetracycline, vavlycin, xylose, copper, zinc, etc. Induction of gene expression can also be achieved by reducing the dissolved oxygen level during fermentation. The dissolved oxygen level of fermentation during cell propagation can be between 10% and 30%. In order to induce gene expression, the dissolved oxygen level can be reduced to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%. In a host cell, whether in a physiological state or in a conversion state, protein production can be induced by reducing the fermentation temperature as disclosed herein.

[0124] Example

[0125] Example 1. 25 kDa truncated human elastin

[0126] The following discloses a non-naturally occurring truncated human elastin protein without a His tag, a linker and / or a thrombin cleavage site. The non-naturally occurring truncated human elastin protein is truncated relative to the full-length human elastin protein (SEQ ID NO: 20). The following discloses a codon-optimized nucleotide sequence and an amino acid sequence encoding the elastin protein. In SEQ ID NO: 4, the DsbA secretion tag is encoded by nucleotides 1-57 and encodes amino acids 1-19 of SEQ ID NO: 5. In SEQ ID NO: 4, the non-naturally occurring truncated human elastin protein sequence is encoded by nucleotides 58-927 and encodes amino acids 20-309 of SEQ ID NO: 5.

[0127] The codon-optimized nucleotide sequence encoding the non-naturally occurring truncated elastin is provided in SEQ ID NO:4.

[0128] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGGTCCGCAACCTGGGGTTCCGTTAGGTTATCCGATTAAAGCACCGAAACTGCCCGGCGGTTATGGTCTGCCGTACACAACCGGTAAACTGCCGTATGGTTATGGCCCGGGTGGAGTTGCGGGTGCAGCAGGTAAAGCGGGTTATCCTACCGGAACCGGTGTAGGTCCGCAGGCCGCTGCTGCCGCCGCCGCAAAAGCAGCGGCTAAATTTGGCGCCGGAGCAGCGGGTGTTCTGCCTGGAGTTGGTGGTGCGGGCGTGCCAGGGGTACCTGGTGCAATTCCGGGTATTGGTGGTATTGCCGGTGTCGGCACCCCGGCCGCGGCAGCTGCGGCAGCGGCGGCTGCCAAAGCTGCTAAATACGGTGCCGCGGCGGGTCTGGTGCCAGGAGGTCCGGGTTTTGGTCCGGGAGTGGTTGGCGTGCCTGGCGCAGGCGTTCCTGGTGTGGGCGTTCCAGGTGCAGGGATTCCTGTTGTGCCTGGTGCCGGTATTCCCGGCGCGGCCGTTCCGGGGGTGGTTAGCCCGGAAGCCGCAGCGAAGGCTGCGGCAAAGGCAGCAAAGTATGGCGCACGCCCAGGAGTCGGCGTGGGTGGTATCCCGACCTATGGGGTGGGCGCAGGGGGTTTTCCTGGTTTCGGCGTAGGTGTAGGAGGTATACCGGGCGTGGCCGGTGTACCAGGGGTTGGTGGCGTCCCTGGTGTTGGCGGTGTGCCAGGTGTTGGTATTTCACCGGAAGCACAGGCAGCAGCCGCAGCTAAGGCAGCGAAATATGGTGCCGCCGGCGCAGGAGTTTTAGGTGGGCTGGTTCCGGGCCCGCAGGCAGCTGTGCCGGGGGTTCCAGGCACCGGTGGTGTCCCTGGAGTCGGTACGCCGTAA(SEQ ID NO:4)

[0129] The amino acid sequence of the 25 kDa non-naturally occurring truncated human elastin sequence including the DsbA secretion signal is disclosed in SEQ ID NO:5.

[0130] MKKIWLALAGLVLAFSASAGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGV VGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGPQAAVPGVPGTGGVPGVGTP(SEQ ID NO:5)

[0131] The codon-optimized nucleotide sequence encoding a non-naturally occurring truncated human elastin protein without the DsbA secretion tag is provided in SEQ ID NO:6.

[0132] GGTCCGCAACCTGGGGTTCCGTTAGGTTATCCGATTAAAGCACCGAAACTGCCCGGCGGTTATGGTCTGCCGTACACAACCGGTAAACTGCCGTATGGTTATGGCCCGGGTGGAGTTGCGGGTGCAGCAGGTAAAGCGGGTTATCCTACCGGAACCGGTGTAGGTCCGCAGGCCGCTGCTGCCGCCGCCGCAAAAGCAGCGGCTAAATTTGGCGCCGGAGCAGCGGGTGTTCTGCCTGGAGTTGGTGGTGCGGGCGTGCCAGGGGTACCTGGTGCAATTCCGGGTATTGGTGGTATTGCCGGTGTCGGCACCCCGGCCGCGGCAGCTGCGGCAGCGGCGGCTGCCAAAGCTGCTAAATACGGTGCCGCGGCGGGTCTGGTGCCAGGAGGTCCGGGTTTTGGTCCGGGAGTGGTTGGCGTGCCTGGCGCAGGCGTTCCTGGTGTGGGCGTTCCAGGTGCAGGGATTCCTGTTGTGCCTGGTGCCGGTATTCCCGGCGCGGCCGTTCCGGGGGTGGTTAGCCCGGAAGCCGCAGCGAAGGCTGCGGCAAAGGCAGCAAAGTATGGCGCACGCCCAGGAGTCGGCGTGGGTGGTATCCCGACCTATGGGGTGGGCGCAGGGGGTTTTCCTGGTTTCGGCGTAGGTGTAGGAGGTATACCGGGCGTGGCCGGTGTACCAGGGGTTGGTGGCGTCCCTGGTGTTGGCGGTGTGCCAGGTGTTGGTATTTCACCGGAAGCACAGGCAGCAGCCGCAGCTAAGGCAGCGAAATATGGTGCCGCCGGCGCAGGAGTTTTAGGTGGGCTGGTTCCGGGCCCGCAGGCAGCTGTGCCGGGGGTTCCAGGCACCGGTGGTGTCCCTGGAGTCGGTACGCCGTAA(SEQ ID NO:6)

[0133] The amino acid sequence of a non-naturally occurring truncated human elastin without the DsbA secretion tag is disclosed in SEQ ID NO:7.

[0134] GPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPG VGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGPQAAVPGVPGTGGVPGVGTP(SEQ IDNO:7)

[0135] The polynucleotide of SEQ ID NO: 4 was synthesized in-house by Gen9 DNA (now Ginkgo Bioworks). The pET28 vector was designed with overlapping sequences between SEQ ID NO: 4 and SEQ ID NO: 6 to be 20 to 30 bp long and PCR was performed by enzyme GXL polymerase (www.takarabio.com / products / pcr / gc-rich-pcr / primestar-gxl-dna-polymerase) was added. The opened pET28a vector and the insert DNA (SEQ ID NO: 4) were then assembled into the final plasmid using In-Fusion cloning (www.takarabio.com / products / cloning / in-fusion-cloning). The plasmid sequence was then verified by Sanger sequencing using Genewiz (www.genewiz.com / en).

[0136] Transformed cells were grown in minimal medium and frozen in 1.5 ml aliquots with vegetable glycerol at a 50:50 ratio of cells:glycerol. A vial of this frozen culture was revived overnight in 50 ml minimal medium at 37°C, 200 rpm. Cells were transferred to 300 ml minimal medium and grown for 6-9 hours to reach an OD600 of 5-10.

[0137] The minimal medium used in this example and throughout this disclosure was prepared as follows:

[0138] 1) Autoclave 5 L of 550 g / kg glucose syrup (VWR, product number 97061-170) in DI water.

[0139] 2) Autoclave the following in 3946 mL of DI water:

[0140] 20 g (NH4)2HPO4 (VWR, product number 97061-932).

[0141] 66.5 g KH2PO4 (VWR, product number 97062-348).

[0142] 22.5g H3C6H5O7 (VWR, product number BDH9228-2.5KG).

[0143] 8.85 g MgSO4.7H2O (VWR, product number 97062-134).

[0144] 10 mL of 1000x Trace Metals Preparation (Table 3).

[0145] After autoclaving, add

[0146] 118g (1) to (2)

[0147] 5mL of 25mg / mL Kanamycin Sulfate (VWR-V0408)

[0148] 28% NH4OH (VWR, product number BDH3022) was used to adjust the pH to 6.1.

[0149] Table 3. Trace metal preparations

[0150] 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

[0151] Fermentations were performed at different temperatures ranging from 25°C to 28°C. For some fermentations, the fermentation temperature was maintained at a constant temperature. For other fermentations, the fermentation temperature was maintained for a desired period of time, and when the cell density OD600 reached 10-20, the temperature was reduced to induce protein production. Typically, the temperature was reduced from 28°C to 25°C; fermentation was continued at 25°C for 40-60 hours.

[0152] The purified non-naturally occurring truncated elastin was analyzed on an SDS-PAGE gel and a clear band of the expected size of 25 kilodaltons was observed at the position where no band was found in the control strain.

[0153] Table 2. 21 kDa truncated human elastin

[0154] According to the method of Example 1, non-naturally occurring truncated human elastin was synthesized.

[0155] The amino acid sequence of the 21 kDa non-naturally occurring truncated human elastin truncated at the N-terminus relative to full-length human elastin (SEQ ID NO: 20) is disclosed in SEQ ID NO: 8. The 21 kDa non-naturally occurring truncated human elastin has amino acids 1-522 deleted from full-length human elastin (SEQ ID NO: 20).

[0156] GPGGVAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAGADEGVRRSLSPELREGDPSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLG ALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK(SEQ ID NO:8)

[0157] The codon-optimized polynucleotide sequence encoding the 21 kDa non-naturally occurring truncated human elastin is disclosed in SEQ ID NO:9.

[0158] (SEQ ID NO:9)

[0159] As disclosed in Example 1, the cells were cultured and the polynucleotide of SEQ ID NO: 9 was expressed.

[0160] Example 3. 60 kDa truncated human elastin

[0161] The amino acid sequence of the 60.3 kDa non-naturally occurring truncated human elastin protein that is internally truncated relative to the full-length human elastin protein (SEQ ID NO: 20) is disclosed in SEQ ID NO: 10. The 60.3 kDa non-naturally occurring truncated human elastin protein has amino acids 461-527 deleted from the full-length human elastin protein (SEQ ID NO: 20).

[0162] GGVPGAIPGGVPGGVFYPGAGLGALGGGALGPGGKPLKPVPGGLAGAGLGAGLGAFPAVTFPGALVPGGVADAAAAYKAAKAGAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGAAGAGVLGGLVPGPQAAVPGVPGTGGVPGVGTPAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAGADEGVRRSLSPELREGDPSSSQHLPSTPSSPRVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK(SEQ ID NO:10)

[0163] A codon-optimized polynucleotide sequence encoding a 60.3 kDa non-naturally occurring truncated human elastin is disclosed in SEQ ID NO:11.

[0164]

[0165] As disclosed in Example 1, cells were cultured and the polynucleotide of SEQ ID NO: 11 was expressed.

[0166] Example 4. Effects of truncated elastin on fibroblast viability, tropocollagen synthesis and tropoelastin synthesis ring

[0167] Human fibroblast cultures were used to evaluate the ability of the non-naturally occurring truncated human elastin molecules of Example 1 to affect the synthesis of tropocollagen and tropoelastin. Human fibroblast cultures were also used to determine the viability of human fibroblasts following exposure to truncated elastin.

[0168] A 2% w / w stock solution of truncated elastin was prepared from the truncated elastin of Example 1. Aliquots from the 2% stock solution of truncated elastin were then used in the following experiments.

[0169] Preparation of fibroblasts

[0170] Fibroblasts were seeded into 0.5 mL of fibroblast growth medium (FGM) in each well of a 24-well plate and incubated overnight at 37 ± 2 ° C and 5 ± 1% CO2. The next day, the culture medium was removed by aspiration to eliminate any non-adherent cells and replaced with 0.5 mL of fresh FGM. The cells were grown until confluent, and the culture medium was replaced every 48 to 72 hours. After confluent, the cells were treated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 1.5% fetal bovine serum (FBS) for 24 hours to wash out any effects of growth factors contained in the normal culture medium. After a 24-hour washing period, the cells were treated with a specific concentration of truncated elastin dissolved in FGM containing 1.5% FBS. Transforming growth factor β (TGF-β) (20 ng / mL) was used as a positive control for collagen and elastin synthesis. Untreated cells (negative control) only received DMEM containing 1.5% FBS. Cells were incubated for 48 hours and at the end of the incubation period, cell culture medium was collected and either stored frozen (-75°C) or analyzed immediately. Materials were tested in triplicate.

[0171] MTT assay

[0172] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay is a colorimetric assay used to determine the metabolic activity of cells. Changes in cell number are assessed by the MTT assay. When cells are exposed to MTT, the reduction of MTT by mitochondria in viable cells results in the formation of insoluble purple formazin crystals, which are extracted from the cells with isopropanol and quantified by spectrophotometry. Non-viable cells cannot reduce MTT and therefore cannot produce purple formazin crystals. The intensity of the purple color is proportional to the number of viable cells (metabolically active cells). The intensity of the purple color is directly proportional to the metabolic activity of the cells and inversely proportional to the toxicity of the test material.

[0173] After the 2-day cultivation described above, the cell culture medium (see above) was removed and the fibroblasts were washed twice with phosphate buffered saline (PBS) to remove any remaining elastin molecules. After the last wash, 500 μL of DMEM supplemented with 0.5 mg / ml MTT was added to each well and the cells were incubated for 1 hour at 37 ± 2 ° C and 5 ± 1% CO2. After cultivation, the DMEM / MTT solution was removed, the cells were washed once again with PBS, and then 0.5 mL of isopropanol was added to the wells to extract the purple formazine crystals. 200 microliters of isopropyl extracts were transferred to a 96-well plate and the plate was read at 540 nm, using isopropanol as a blank control.

[0174] The mean MTT absorbance value of the negative control cells was calculated and used to represent 100% cell viability. The individual MTT absorbance values ​​from cells undergoing various treatments were then divided by the mean of the negative control cells and expressed as a percentage to determine the change in cell viability caused by each treatment.

[0175] Tropocollagen synthesis

[0176] Fibroblasts are the main source of extracellular matrix peptides, including the structural proteins collagen and elastin. Procollagen is a large peptide synthesized by fibroblasts in the dermis of the skin and is the precursor of collagen. When the peptide is processed to form mature collagen, the propeptide portion is cleaved off (type I C-peptide). The mature collagen and type I C-peptide fragments are then released into the extracellular environment. As collagen is synthesized, type I C-peptide fragments accumulate in the tissue culture medium. Since the stoichiometric ratio between the two parts of the procollagen peptide is 1:1, the determination of type I C-peptide reflects the amount of collagen synthesized. Type I C-peptide can be measured by an enzyme-linked immunosorbent assay (ELISA)-based method.

[0177] A series of type I C-peptide standards ranging from 0 ng / mL to 640 ng / mL were prepared. Next, an ELISA microplate was prepared by removing any unwanted bands from the plate frame and then adding 100 μL of peroxidase-labeled antigen collagen type IC peptide antibody to each well used in the assay. Twenty (20) μL of sample (collected tissue culture medium) or standard was then added to the appropriate wells, the microplate was covered and allowed to incubate at 37°C for 3 ± 0.25 hours. After incubation, the wells were aspirated and washed 3 times with 400 μL of wash buffer. After removing the last wash, 100 μL of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as chromogen) 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 was read at 450 nm using an ELISA reader.

[0178] To quantify the amount of each substance present, a standard curve was generated using known concentrations of each substance. Regression analysis was performed to establish a line that best fits these data points. The absorbance values ​​of the test material and the untreated sample were used to estimate the amount of each substance present in each sample.

[0179] Elastin synthesis

[0180] Elastin is the main component of the elastic fiber network that gives tissues the ability to rebound after brief stretching. This protein is released by fibroblasts (soluble elastin) into the extracellular space, where it then cross-links with other elastin proteins to form an extensive network of fibers and sheets (insoluble elastin). Soluble elastin can be easily measured from cell culture media by ELISA-based methods.

[0181] Soluble α-elastin was dissolved in 0.1 M sodium carbonate (pH 9.0) at a concentration of 1.25 μg / mL. 150 μL of this solution was then added to the wells of a 96-well Maxisorp Nunc plate, and the plate was incubated overnight at 4°C. The next day, the wells were filled with PBS containing 0.25% bovine serum albumin (BSA) and 0.05% Tween 20. The plate was then incubated with the blocking solution at 37°C for 1 hour and then washed twice with PBS containing 0.05% Tween 20.

[0182] Prepare a set of α-elastin standards ranging from 0 to 100 ng / mL. Then transfer 180 μL of the standards or truncated elastin to a 650 μL microcentrifuge tube. Prepare an anti-elastin antibody solution (antibody diluted 1:100 in PBS containing 0.25% BSA and 0.05% Tween 20) and add 20 μL of the solution to the tube. Then incubate the tube overnight at 4±2°C. The next day, transfer 150 μL from each tube to a 96-well elastin ELISA plate and incubate the plate at room temperature for 1 hour. Then wash the plate 3 times with PBS containing 0.05% Tween 20. After washing, add 200 μL of a solution containing a peroxidase-conjugated secondary antibody diluted in PBS containing 0.25% BSA and 0.05% Tween 20, and incubate the plate at room temperature for 1 hour. After washing the plate 3 times, 200 μL of substrate solution was added and the plate was incubated at room temperature in the dark for 10 to 30 minutes. After the final incubation, the plate was read at 460 nm using a plate reader.

[0183] Example 5. Effect of Truncated Elastin on Keratinocyte Proliferation and UVB Protection

[0184] The human keratinocyte cell culture model was used to evaluate the ability of the test materials to affect cell proliferation. In addition, the effect of the test materials on cell viability after exposure to UVB was also evaluated.

[0185] A 2% w / w stock solution of truncated elastin was prepared from the truncated elastin of Example 1. Aliquots from the 2% stock solution of truncated elastin were then used in the following experiments.

[0186] The study was conducted in two parts. In the first part, cultured keratinocytes were incubated with the test materials for 48 hours, after which changes in the number of viable cells were assessed using the MTT assay. In the second part of the study, cultured keratinocytes were irradiated with UVB and then treated with the test materials for 48 hours. At the end of 48 hours, the number of viable cells was again assessed by the MTT assay.

[0187] Changes in the number of viable cells can be determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. The MTT assay is a colorimetric analysis of cellular metabolic activity and is a reflection of the number of viable cells. The reduction of MTT by mitochondria in viable cells results in the formation of insoluble purple formazine crystals that are extracted from the cells with isopropanol and quantified by spectrophotometry. The intensity of the purple color is proportional to the number of metabolically viable cells.

[0188] Proliferation assay

[0189] For proliferation assays, keratinocytes were seeded into 96-well plates without growth factors and incubated for 24 hours at 37 ± 2 ° C and 5 ± 1% CO2. After the initial incubation, the culture medium was replaced with a culture medium supplemented with the test material. Normal culture medium (containing growth factors) was used as a positive control. After adding the test material, the cells were cultured for 48 hours as described above. At the end of the incubation period, the change in the number of viable cells was determined using the MTT assay.

[0190] UVB protection test

[0191] For UVB protection assays, keratinocytes were seeded into 96-well plates using normal culture medium and incubated for 24 h at 37 ± 2°C and 5 ± 1% CO2. After this initial incubation, the culture medium was replaced with 100 μL of phosphate buffered saline (PBS) and the cells were exposed to UVB (40 mJ / cm 2 After UVB exposure, PBS was replaced with fresh medium supplemented with test materials (100 μg / mL ascorbic acid as a positive control), and cells were incubated for 48 hours at 37±2°C and 5±1% CO2. At the end of the 48-hour incubation, cell viability was determined using the MTT assay.

[0192] Example 6. Effect of truncated elastin on thymine dimer formation

[0193] After exposure to ultraviolet radiation, the amount of thymine dimers (TT dimers) present in the DNA of cells increases. Increased TT dimer formation is associated with skin damage and certain types of cell proliferative diseases, including skin cancer.

[0194] The truncated elastin of Example 1 was tested to determine whether it could reduce the formation of TT dimers in human epidermal keratinocytes. Human keratinocytes were seeded into 12-well plates using normal culture medium and incubated at 37±2°C and 5±1% CO2 for 24 hours. After the initial incubation, the culture medium was replaced with 100 μL of phosphate buffered saline (PBS) and the cells were exposed to UVB (25 mJ / cm 2 ). After UVB exposure, PBS was replaced with fresh medium supplemented with test materials or Trolox (100 μL g / ml, positive control), and cells were cultured overnight at 37±2°C and 5±1% CO2. At the end of the incubation, cellular DNA was extracted and thymine dimer content was determined using an ELISA-based method.

[0195] 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 the PBS and proteinase K, 200 μL buffer AL was added to each well. After rotating the plate again to mix the reagents, the plate was incubated at 55 ± 2 ° C for 10 minutes. After the plate was cooled to room temperature, the DNA was precipitated by adding 200 μL 100% ethanol. The precipitated DNA mixture was then transferred to a DNEasy spin column in a 2mL collection tube and centrifuged at 8,000 RPM for 1 minute. The flow-through tube and collection tube were discarded, and 500 μL wash buffer one was added to the spin column, the column was placed in a new collection tube, and centrifuged at 8,000 RPM for 1 minute. The flow-through tube and collection tube were discarded again, 500 μL wash buffer two was added to the spin column, the column was placed in a new collection tube, and centrifuged at 14,000 RPM for 3 minutes. The spin column was then placed in a new 1.5 mL centrifuge tube and 110 μL of ultrapure water was added to the column. The column was incubated at room temperature for 1 minute and then centrifuged at 8,000 RPM for 1 minute.

[0196] The extracted DNA was quantified by fluorometry. In a 96-well plate, 2 μL aliquots of DNA sample were mixed with 100 μL Tris-EDTA (TE) buffer. A series of DNA standards were also transferred to the wells of the 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 measured using an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0197] Thymine dimer detection can be performed using OxiSelect TM UV-induced DNA damage ELISA kit (OxiSelect TM UV-Induced DNA Damage ELISA Kit) was used for determination.

[0198] Aliquots of genomic DNA samples or standards were converted to single-stranded DNA by incubating the samples at 95°C for 10 minutes and then cooling on ice. 100 μL of each sample or standard was transferred to a DNA binding ELISA plate and incubated overnight at 4°C. The next day, the wells were rinsed once with 100 μL PBS and then blocked with 150 μL of assay diluent at room temperature for 1 hour. After removing the assay diluent, 100 μL of anti-CPD antibody was added to each well and the plate was incubated for 1 hour at room temperature. After incubation, the plate was washed 3 times with 250 μL of wash buffer per well, and 150 μL of blocking agent was added to the plate. The plate was blocked again at room temperature for 1 hour and then washed 3 times as described above. 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 produce color in the plate. The color reaction was stopped by adding 100 μL of stop solution and the plate was read at 460 nm using a plate reader.

[0199] To quantify the amount of DNA present, a standard curve is generated using known concentrations of DNA and their respective fluorescence intensities (measured in RFU or relative fluorescence units). A regression analysis is performed to establish a line that best fits these data points. The relative fluorescence units (RFU) of each unknown sample are then used to estimate the amount of DNA.

[0200] A series of DNA standards with known amounts of thymine dimer content were used to generate a standard curve. This standard curve was used to determine the amount of DNA damage in the sample DNA. The mean values ​​of each treatment group were calculated and compared using analysis of variance (ANOVA).

[0201] Example 7. Protective effect of truncated human elastin on fibroblasts

[0202] The effects of non-naturally occurring truncated human elastin on fibroblast viability, procollagen synthesis and elastin synthesis were determined according to the method of Example 4.

[0203] The effects of non-naturally occurring truncated human elastin on keratinocyte proliferation and UVB protection were determined according to the method of Example 5.

[0204] The method of Example 6 was used to determine the effect of non-naturally occurring truncated human collagen on thymine dimer formation after exposure to UV radiation.

[0205] Example 8. Effect of truncated elastin on inflammatory cytokines

[0206] Keratinocytes and dermal fibroblasts play an important role in the immune response of the skin. In response to irritating chemicals or UV radiation (pro-inflammatory / pro-irritant stimuli), keratinocytes can release a large number of cytokines. These cytokines are thought to help bring immune cells to the site of inflammation. Cytokines released by keratinocytes include TNFα, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18, and IL-1RA.

[0207] The test model used for this study was MatTek EpiDerm. This skin model consists of normal human-derived epidermal keratinocytes that have been cultured to form a multilayered, highly differentiated model of human epidermis. Ultrastructural analysis revealed the presence of keratohyalin granules, tonofilaments, desmosomes, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in a pattern characteristic of the in vivo epidermis. Markers of mature epidermis-specific differentiation such as profilaggrin, K1 / K10 cytokeratin pairs, involucrin, and epidermal transglutaminase type I have been localized in this model. MatTek EpiDerm is also mitotically and metabolically active.

[0208] MatTek EpiDerm tissue was used to evaluate the ability of various test materials to inhibit the release of the inflammatory mediator IL-1α. The test materials were compared to an over-the-counter topical hydrocortisone preparation (positive control) as well as untreated tissue (negative control 1) and untreated non-inflamed tissue (negative control 2). The test was also used to evaluate the viability of tissue after exposure to the test materials.

[0209] 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 by colorimetric enzyme-linked immunosorbent assays (ELISAs). Briefly, antibodies covalently linked to a solid support can bind to IL-1α, IL-6, or IL-8 present in a sample of spent culture medium. A secondary antibody covalently linked to acetylcholinesterase can then detect the specifically bound cytokine. Upon addition of an appropriate colored substrate, acetylcholinesterase can produce a colored end product that can be measured spectrophotometrically.

[0210] MatTek EpiDerm tissues were purchased from MatTek and stored at 4°C until use. Prior to use, the tissues to be used were removed from the agarose transport tray and placed in a 6-well plate containing 0.9 mL of assay medium (37 ± 2°C) without hydrocortisone. The tissues were incubated overnight at 37 ± 2°C and 5 ± 1% CO2. After the initial incubation, the assay medium was replaced with 0.9 mL of fresh medium (37 ± 2°C) without hydrocortisone. Three tissues were prepared for each test material.

[0211] The inflammatory response of the tissue is induced by ultraviolet radiation (UVB). The UVB lamp is used to deliver 300mJ / cm 2 dose of UVB radiation. Immediately after application of the inflammatory stimulus, 50 μL or mg of the test material was applied directly to the tissue surface. Over-the-counter hydrocortisone cream was used as a positive control. For the negative control, the tissue was exposed to the inflammatory stimulus but was not treated with any type of anti-inflammatory material. An additional set of tissues was not exposed to the inflammatory stimulus to provide a baseline measurement of cytokines. The tissue was incubated at 37±2°C and 5±1% CO2 for 24 hours after exposure to the inflammatory stimulus. After 24 hours of incubation, the cell culture medium was collected and stored at -75°C until cytokine analysis.

[0212] Prepare ELISA plates by diluting the appropriate capture antibody in PBS. Next, add 100 μL of the diluted capture antibody to the wells of a 96-well ELISA plate and incubate the plate overnight at room temperature. The next day, wash the plate 3 times with 300 μL of wash buffer (0.05% Tween 20 in PBS) and then block by adding 300 μL of blocking buffer (1% BSA in PBS) to each well. Incubate the plate with blocking buffer for at least 1 hour. After incubation, remove the blocking buffer and wash the plate 3 times as described above.

[0213] Prepare a series of standards and distribute 100 μL of each of these standards to two wells in an appropriate 96-well plate (replicate). Subsequently, 100 μL of each sample is added to another well and the plate is incubated at room temperature for 2 hours. After incubation, the plate is washed 3 times as described above. After removing the last wash solution, 100 μL of biotin-coupled detection antibody is added. After the plate is incubated at room temperature for two hours, the plate is washed again as described above. Then 100 μL of HRP-streptavidin is added to each well and the plate is incubated at room temperature for 20 minutes. Next, 100 μL of substrate solution (hydrogen peroxide + tetramethylbenzidine as chromogen) is added to each well. Once sufficient color development occurs, 50 μL of stop solution (2N sulfuric acid) is added to each well and the plate is read at 460nm.

[0214] After 24 hours of incubation, the tissue was rinsed twice with at least 100 μL of phosphate-buffered saline to remove the test material, then transferred to a 6-well plate containing 1.0 mL of assay medium supplemented with MTT (1 mg / mL) and incubated at 37±2°C and 5±1% CO2 for 3±0.25 hours. After incubation, the tissue was rinsed at least twice with 100 μL of phosphate-buffered saline, wiped dry, and then placed in a 24-well plate containing 2 mL of isopropanol per well. The 24-well plate was covered and incubated on a rocking platform at room temperature for at least 2 hours to extract the reduced MTT from the tissue. After extraction, a 200 μL sample of the isopropanol / MTT mixture was transferred to a 96-well plate and the absorbance of the sample was read at 540 nm using a microplate reader, with 200 μL of isopropanol used as a blank control.

[0215] Example 9. Urban dust protection of truncated elastin

[0216] A keratinocyte culture model was used to evaluate the ability of truncated elastin to exert a protective effect by promoting cell survival after exposure to urban dust.

[0217] Human epidermal keratinocytes were pretreated with the test materials and then exposed to urban dust. At the end of the treatment period, changes in cell viability were determined by MTT assay.

[0218] Keratinocytes were seeded into 100 μL of culture medium in each well of a 96-well plate and incubated overnight at 37 ± 2°C and 5 ± 1% CO2. The next day, the medium was removed by aspiration to eliminate any non-adherent cells and replaced with 100 μL of fresh medium. Cells were grown until confluent, with medium changes every 48 to 72 hours.

[0219] Pre-treatment with test material followed by urban dust treatment

[0220] Test materials were prepared in cell culture medium at 2 times of their final desired concentration. City dust (NIST 1649B, purchased from Sigma Chemicals) was also prepared in 2 times solution. For pretreatment, 50 μL of 2 times test materials were mixed with 50 μL of culture medium, and cells were cultivated for 24 hours. At the end of the pretreatment period, the test materials containing culture medium were removed and replaced with 50 μL of 2 times city dust and 50 μL of culture medium. Another group of cells was treated with culture medium only (no dust exposure) and used as a reference control to represent 100% cell viability. The cells were then cultivated for 24 hours and then the MTT assay was performed to determine the changes in cell viability.

[0221] At the end of the treatment period, the cell culture medium was removed and the cells were washed with PBS. After washing, 100 μL of cell culture medium supplemented with 0.5 mg / mL MTT was added to each well, and the cells were incubated for 30 minutes at 37 ± 2 ° C and 5 ± 1% CO2. After incubation, the culture medium / MTT solution was removed, the cells were washed once again with PBS, and then 100 μL of isopropanol was added to the wells to extract the purple formazine crystals. The 96-well plate was then read at 540 nm, using isopropanol as a blank control.

[0222] The mean MTT absorbance value of cells not exposed to dust was calculated and used to represent the 100% value of cell number. Then, each MTT value from cells subjected to various treatments was divided by the mean value of cells not exposed to dust and expressed as a percentage to determine the change in cell number caused by each treatment.

[0223] Example 10. 23 kDa truncated human elastin

[0224] A non-naturally occurring truncated human elastin protein without a His tag, a linker, and a thrombin cleavage site is disclosed below. The codon-optimized nucleotide sequence and amino acid sequence encoding the truncated elastin protein are disclosed below. In SEQ ID NO: 12, the DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO: 13 and encodes amino acids 1-19 of SEQ ID NO: 13. In SEQ ID NO: 12, the truncated elastin protein sequence is encoded by nucleotides 58-843 of SEQ ID NO: 13 and encodes amino acids 20-281 of SEQ ID NO: 13.

[0225] The codon-optimized nucleotide sequence encoding this non-naturally occurring truncated elastin is provided in SEQ ID NO:12.

[0226] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGGCAGCAGCCGCAGCAGCGGCAAAAGCAGCAGCGAAATTTGGTGCGGGTGCAGCCGGTGTGCTGCCAGGCGTAGGTGGCGCTGGTGTACCTGGTGTCCCTGGCGCAATTCCAGGTATTGGCGGTATTGCAGGCGTTGGTACTCCGGCAGCTGCAGCCGCTGCCGCAGCCGCAGCTAAAGCAGCCAAATATGGTGCAGCGGCAGGCCTGGTTCCTGGCGGTCCAGGTTTTGGTCCGGGTGTTGTTGGCGTCCCAGGTGCCGGTGTGCCTGGTGTGGGTGTTCCAGGTGCGGGTATTCCGGTTGTTCCTGGCGCAGGCATTCCGGGTGCCGCAGTTCCGGGTGTAGTTAGCCCGGAATAA(SEQ ID NO:12)

[0227] The amino acid sequence of a 23 kDa non-naturally occurring truncated human elastin sequence including the DsbA secretion signal is disclosed in SEQ ID NO:13.

[0228] MKKIWLALAGLVLAFSASAAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGP GGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPE(SEQ ID NO:13)

[0229] A codon-optimized nucleotide sequence encoding a non-naturally occurring truncated human elastin protein without the DsbA secretion tag elastin protein is provided in SEQ ID NO:14.

[0230] GCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGGCAGCAGCCGCAGCAGCGGCAAAAGCAGCAGCGAAATTTGGTGCGGGTGCAGCCGGTGTGCTGCCAGGCGTAGGTGGCGCTGGTGTACCTGGTGTCCCTGGCGCAATTCCAGGTATTGGCGGTATTGCAGGCGTTGGTACTCCGGCAGCTGCAGCCGCTGCCGCAGCCGCAGCTAAAGCAGCCAAATATGGTGCAGCGGCAGGCCTGGTTCCTGGCGGTCCAGGTTTTGGTCCGGGTGTTGTTGGCGTCCCAGGTGCCGGTGTGCCTGGTGTGGGTGTTCCAGGTGCGGGTATTCCGGTTGTTCCTGGCGCAGGCATTCCGGGTGCCGCAGTTCCGGGTGTAGTTAGCCCGGAATAA(SEQ ID NO:14)

[0231] The amino acid sequence of a non-naturally occurring truncated human elastin without the DsbA secretion tag is disclosed in SEQ ID NO:15.

[0232] AGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKA GYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPE(SEQ ID NO:15)

[0233] The polynucleotide of SEQ ID NO: 12 was synthesized by Twist DNA. The overlapping sequence between the pET28 vector and SEQ ID NO: 12 was designed to be 20 to 30 bp long and the polynucleotide of SEQ ID NO: 12 was synthesized by PCR. GXL polymerase (www.takarabio.com / products / pcr / gc-rich-pcr / primestar-gxl-dna-polymerase) was added. Then, the opened pET28a vector and the insert DNA (SEQ ID NO: 12) were assembled into the final plasmid using In-Fusion cloning (www.takarabio.com / products / cloning / in-fusion-cloning). The plasmid sequence was then verified by Sanger sequencing using Genewiz (www.genewiz.com / en).

[0234] Transformed cells were grown in minimal medium from Example 1 and frozen in 1.5 ml aliquots with vegetable glycerol at a 50:50 ratio of cells:glycerol. A vial of this frozen culture was revived overnight in 50 ml minimal medium at 37°C, 200 rpm. Cells were transferred to 300 ml minimal medium and grown for 6-9 hours to reach an OD600 of 5-10.

[0235] A 23 kDa non-naturally occurring truncated human elastin protein was detected on a 12% BisTris protein gel. A photograph of the gel is depicted in Figure 1 The mass of the 23 kDa non-naturally occurring truncated human elastin was confirmed to be correct by intact mass spectrometry using liquid chromatography-mass spectrometry.

[0236] Example 11. 13 kDa truncated human elastin

[0237] The following discloses a non-naturally occurring truncated human elastin protein without a His tag, a linker, and a thrombin cleavage site. The following discloses a codon-optimized nucleotide sequence and an amino acid sequence encoding the elastin protein. In SEQ ID NO: 16, the DsbA secretion tag is encoded by nucleotides 1-57 and encodes amino acids 1-19 of SEQ ID NO: 17. In SEQ ID NO: 16, the truncated elastin protein sequence is encoded by nucleotides 58-489 and encodes amino acids 20-163 of SEQ ID NO: 17.

[0238] The codon-optimized nucleotide sequence encoding the elastin protein is provided in SEQ ID NO:16.

[0239] ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCGTTAGCGCCGGTGCAGTTGTTCCGCAGC CTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGC AGGCTGAAACCGAAAGCACCAGGTGTTGGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCT GGCGGTTATGGTCTGCCGTATAACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGTAA(SEQ ID NO:16)

[0240] The amino acid sequence of the 13 kDa non-naturally occurring truncated human elastin sequence including the DsbA secretion signal is disclosed in SEQ ID NO:17.

[0241] MKKIWLALAGLVLAFSASAAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQ(SEQ ID NO:17)

[0242] The codon-optimized nucleotide sequence encoding a non-naturally occurring truncated human elastin protein without the DsbA secretion tag is provided in SEQ ID NO:18.

[0243] GCCGGTCTGGGTGGCGTGCCTGGCGTTGGTGGCCTGGGCCGGTGCAGTTGTTCCGCAGCCTGGTGCGGGTGTTAAACCGGGTAAAGTTCCTGGTGTTGGTCTGCCTGGTGTTTATCCAGGCGGTGTTCTGCCAGGTGCGCGTTTTCCTGGCGTGGGTGTTCTGCCGGGTGTTCCGACCGGTGCAGGCGTGAAACCGAAAGCACCAGGTGTT GGTGGTGCATTTGCAGGTATTCCAGGTGTGGGTCCGTTTGGTGGTCCGCAGCCAGGCGTTCCGCTGGGTTATCCGATTAAAGCACCGAAACTGCCTGGCGGTTATGGTCTGCCGTATACCACCGGTAAACTGCCGTATGGTTATGGCCCTGGCGGTGTTGCCGGTGCGGCAGGTAAAGCAGGCTATCCGACCGGTACCGGTGTAGGTCCGCAGTAA(SEQ ID NO:18)

[0244] The amino acid sequence of a non-naturally occurring truncated human elastin without the DsbA secretion tag is disclosed in SEQ ID NO:19.

[0245] AGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQ(SEQ ID NO:19)

[0246] The polynucleotide of SEQ ID NO: 16 was synthesized by Twist DNA. The overlapping sequence between the pET28 vector and SEQ ID NO: 16 was designed to be 20 to 30 bp long and was synthesized by PCR. GXL polymerase (www.takarabio.com / products / pcr / gc-rich-pcr / primestar-gxl-dna-polymerase) was added. The opened pET28a vector and the insert DNA (SEQ ID NO: 16) were then assembled into the final plasmid using In-Fusion cloning (www.takarabio.com / products / cloning / in-fusion-cloning). The plasmid sequence was then verified by Sanger sequencing using Genewiz (www.genewiz.com / en).

[0247] Transformed cells were grown in minimal medium and frozen in 1.5 ml aliquots with vegetable glycerol at a 50:50 ratio of cells:glycerol. A vial of this frozen culture was revived overnight in 50 ml minimal medium at 37°C, 200 rpm. Cells were transferred to 300 ml minimal medium and grown for 6-9 hours to reach an OD600 of 5-10.

[0248] Detection of a 13 kDa non-naturally occurring truncated human elastin on a 12% BisTris protein gel. A photograph of the gel is depicted in Figure 1 The mass of the 13 kDa non-naturally occurring truncated human elastin was confirmed to be correct by intact mass spectrometry using liquid chromatography-mass spectrometry.

[0249] Example 12. Purity and homogeneity of 13 kDa non-naturally occurring truncated elastin

[0250] The polypeptide having the amino acid sequence of SEQ ID NO: 19 was analyzed by SDS-PAGE to assess purity and homogeneity. Figure 2 It was shown that the polypeptide having the amino acid sequence of SEQ ID NO: 19 appeared as a single band on the SDS-PAGE protein gel, indicating high purity and homogeneity. In contrast, commercially available elastin appeared as a smear on the protein gel, indicating high heterogeneity.

[0251] Example 13. In vitro studies of non-naturally occurring truncated human collagen

[0252] Non-naturally occurring truncated human elastin stimulates fibroblast production of type I collagen

[0253] Fibroblasts are a major source of extracellular matrix peptides, including the structural proteins collagen and elastin. Type I collagen secretion by human primary fibroblasts was assessed. Fibroblasts were cultured with the polypeptide of SEQ ID NO: 19 for 48 hours. The culture supernatant was analyzed by ELISA for procollagen type I C-peptide, a readout of total secreted type I collagen. Figure 3 The results showed that a 0.1% w / w solution of the polypeptide of SEQ ID NO: 19 ( Figure 3 , "B"), 0.05% w / w solution ( Figure 3 , "C") and 0.025% w / w solution ( Figure 3 Fibroblasts treated with "D" were significantly higher than those of untreated control fibroblasts ( Figure 3 , “A”) secrete higher levels of type I collagen.

[0254] Non-naturally occurring truncated human elastin demonstrates antioxidant capacity

[0255] The oxygen radical absorbance capacity (ORAC) assay was performed to analyze the antioxidant capacity of the polypeptide according to SEQ ID NO: 19. The ORAC assay is a cell-free assay that uses a fluorescence readout to measure antioxidant capacity. Data are reported in Trolox (vitamin E) equivalents (TE). Figure 4 As shown, a 0.0125% w / w solution of the polypeptide according to SEQ ID NO: 19 ( Figure 4 , "E"), 0.025% w / w solution ( Figure 4 , "D"), 0.05% w / w solution ( Figure 4 , "C")")), 0.1% w / w solution ( Figure 4 , "B") and 0.2% w / w solution ( Figure 4 , "A") each demonstrated antioxidant properties. For example, a 0.2% w / w solution of the polypeptide according to SEQ ID NO: 19 exhibited antioxidant properties comparable to 228 μM Trolox.

[0256] Non-naturally occurring truncated human elastin stimulates antioxidant genes in keratinocytes

[0257] Keratinocytes were incubated for 24 hours with medium alone or with a 0.03% w / w solution of the polypeptide according to SEQ ID NO: 19. RNA was extracted from the cells and global gene expression was analyzed by Clariom microarray (ThermoFisher). Figure 5 Compared with untreated cells ( Figure 5Compared with , "A", multiple antioxidant genes (including SOD2, GPX2, GPX4, GSTK1, GSTZ1, GSTA4, GSTM2, CCS, GPX1, GLRX, PRDX5, PRDX6 and PRDX2) were upregulated in keratinocytes treated with the polypeptide according to SEQ ID NO: 19 ( Figure 5 , “B”).

[0258] Non-naturally occurring truncated human elastin reduces expression of apoptotic genes in keratinocytes

[0259] Keratinocytes were incubated for 24 hours with medium alone (untreated) or with a 0.03% w / w solution of the polypeptide according to SEQ ID NO: 19. RNA was extracted from the cells and global gene expression was analyzed by Clariom microarray (ThermoFisher). Figure 6 Compared with untreated cells ( Figure 6 Compared with the above, multiple 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: 19 ( Figure 6 , “B”).

[0260] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and will suggest various modifications or changes made by those skilled in the art, and will be included in the spirit and scope of the present application and the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A non-naturally occurring polypeptide, wherein the non-naturally occurring polypeptide consists of the amino acid sequence of SEQ ID NO:

19.

2. A composition comprising 0.001% to 5% w / w of the non-naturally occurring polypeptide according to claim 1.

3. The composition according to claim 2, wherein the composition exhibits one or more properties selected from the group consisting of: stimulating the growth of fibroblasts, increasing the viability of fibroblasts or keratinocytes, stimulating the synthesis of tropoelastin, stimulating the synthesis of tropoelastin, stimulating the expression of one or more antioxidant genes, reducing the expression of one or more pro-apoptotic genes, reducing the production of one or more inflammatory cytokines, reducing the formation of thymine-thymine (TT) dimers, and any combination thereof.

4. The composition of claim 2, wherein the composition is formulated for topical application.

5. The composition of claim 4, wherein the composition comprises a topical carrier, a preservative, or both.

6. The composition of claim 5, wherein the topical carrier is selected from the group consisting of water, glyceryl ether-8 oil, glycerin, coconut alkanes, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, pentylene glycol, disodium ethylenediaminetetraacetic acid (EDTA), caprylyl glycol, chlorphenesin, phenoxyethanol, liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusted powders, biodegradable polymers, mineral oil, triglyceride oils, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and any combination thereof.

7. The composition according to claim 5, wherein the preservative is 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-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methyl parahydroxybenzoate, sorbic acid, rosemary extract, EDTA, and any combination thereof.

8. The composition according to claim 2, wherein the composition is a cosmetic.

9. The composition of claim 2, wherein the composition is formulated as a personal care product for application to the skin.

10. The composition of claim 9, wherein the personal care product is selected from the group consisting of: facial masks, skin cleansers, soaps, cleansing creams, cleansing lotions, facial cleansers, cleansing milks, cleansing pads, facial cleansers, facial creams, body creams, moisturizers, facial serums, facial masks, body masks, toners, facial sprays, eye creams, eye treatments, and exfoliating formulas.

11. The composition of claim 2, wherein the composition is formulated as a personal care product for application to the hair.

12. The composition of claim 11, wherein the personal care product is selected from the group consisting of: shampoo, conditioner, hair serum, scalp serum, hair mist and hair spray.

13. A method of providing a cosmetic benefit to the skin of a subject, the method comprising applying to the skin of a subject a composition according to any one of claims 2-10, thereby providing a cosmetic benefit to the skin of the subject.

14. The method of claim 13, wherein the cosmetic benefit is improving the appearance of the subject's skin, protecting the subject's skin, or both.

15. The method of claim 14, wherein the improving the subject's skin appearance is selected from the group consisting of increasing skin firmness, increasing skin elasticity, increasing skin hydration, increasing skin tactile texture, increasing skin or visual texture, and any combination thereof.

16. The method of claim 14, wherein the protecting the subject's skin is selected from the group consisting of reducing skin damage, promoting repair of damaged skin, protecting skin from ultraviolet (UV) damage, increasing the vitality of skin cells, protecting skin cells from the effects of urban dust exposure, and any combination thereof.

17. A polynucleotide comprising a nucleic acid sequence encoding the non-naturally occurring polypeptide of claim 1.

18. The polynucleotide of claim 17, wherein the polynucleotide is contained in a vector.

19. The polynucleotide of claim 17 or claim 18, wherein the polynucleotide further comprises a nucleic acid sequence encoding one or more of a secretion tag, a histidine tag, and a protease cleavage site.

20. The polynucleotide of claim 17 or claim 18, wherein the nucleic acid sequence encoding the non-naturally occurring polypeptide is codon optimized for expression in a host cell.

21. A recombinant cell comprising at least one copy of a heterologous nucleic acid sequence encoding the non-naturally occurring polypeptide of claim 1.

22. The recombinant cell of claim 21, wherein the recombinant cell is a microbial cell.

23. The recombinant cell of claim 22, wherein the microbial cell is a bacterial cell.

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