Recombinant collagen and elastin molecules and uses thereof

Abstract

The present disclosure provides non-naturally occurring collagen and elastin molecules. The non-naturally occurring collagen and elastin include truncated collagens, truncated elastins, and fusion proteins thereof. The non-naturally occurring collagen and elastin are useful in food, cosmetics, and many other products and uses.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880076990.3, filed on September 28, 2018, entitled “Recombinant Collagen and Elastin Molecules and Their Uses Thereof” (the corresponding PCT application was filed on September 28, 2018, and has the application number PCT / US2018 / 053601).

[0002] Cross-referencing and incorporation of related applications

[0003] This application claims priority to U.S. Patent Application No. 16 / 144,914, filed September 27, 2018, entitled “RECOMBINANT COLLAGEN AND ELASTIN MOLECULES AND USES THEREOF,” which claims the benefit of U.S. Provisional Patent Application No. 62 / 564,964, filed September 28, 2017, and U.S. Provisional Patent Application No. 62 / 657,591, filed April 13, 2018, both entitled “RECOMBINANT COLLAGEN AND ELASTIN MOLECULES AND USES THEREOF,” the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] This disclosure relates to non-naturally occurring full-length and truncated collagen molecules and full-length and truncated elastin molecules and their uses. Background Technology

[0005] Collagen and similar proteins are among the most abundant proteins in the biosphere. Collagen and elastin are structural proteins found in the skin, connective tissue, bones, and other tissues of animals. In humans, collagen accounts for about one-third of total protein and about three-quarters of the dry weight of the skin. Elastin is a highly elastic protein found in connective tissue and other types of tissue.

[0006] Collagen has a triple helix structure, in which three polypeptide chains coil together in a helical shape. Each polypeptide chain consists of a repeating triplet amino acid sequence named GLY-XY. X and Y can be any amino acid, while the third amino acid is glycine. High concentrations of the amino acids proline and hydroxyproline are found in collagen. The most common triplet is proline-hydroxyproline-glycine (Gly-Pro-Hyp), accounting for approximately 10.5% of collagen triplets.

[0007] Gelatin is a product obtained through the partial hydrolysis of collagen. Typically, gelatin is produced by acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis, or by heating collagen in an aqueous solution (e.g., boiling animal bones and skin, boiling fish scales, etc.).

[0008] Gelatin is used in many products, including cosmetics, food, pharmaceuticals, medical devices, photographic film, adhesives, and many others. The physical and chemical properties of gelatin are tailored to specific applications. These physical / chemical properties include gel strength, melting point temperature, viscosity, color, turbidity, pH, isoelectric point, etc.

[0009] Elastin is an elastic protein 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 the building block of elastin. Unlike collagen, which contains a family of genes, the human body has only one tropoelastin gene. When expressed, this single elastin gene is spliced ​​to produce different forms of tropoelastin. Many tropoelastin molecules associate together to form elastin.

[0010] L-forms, or L-type bacteria, are bacterial strains derived from their parent species (N-forms) that can grow as cell walls-less (protoplastoid type) or cell walls-free (protoplastic type). See Madoff S (Ed): The Bacterial L-Forms. New York: Marcel Dekker Inc., 1986; Mattmann LH (Ed): Cell Wall Deficient Forms. Boca Raton: CRC Press; 1993; and Gumpert J, Taubeneck U: Characteristic properties and biological significance of stable protoplasttype L-forms. In Protoplasts, Lecture Proceedings of the 6th International Protoplast Symposium: Basel. Experientia 1983, 46(suppl): 227-241.

[0011] The L-form of the protoplast type has been cultured in a cell wall-free state and represents a genetically stable mutant exhibiting extreme pleiotropic changes, including the inability to form a cell wall, capsule, flagella, pili, spores, and mesenchyme; altered colony and cell morphology; qualitative and quantitative changes in the lipid and protein components of the cytoplasmic membrane; lack of extracellular proteolytic activity; resistance to bacteriophages; and the inability to proliferate outside of laboratory conditions. See Gumpert and Taubeneck (ibid.); and Hoischen et al., Lipid and fatty acid composition of cytoplasmic membranes from Streptomyces hygroscopic and its stable protoplast type L-form. J Bacteriol 1997, 179:3430-3436. Summary of the Invention

[0012] On one hand, non-naturally occurring collagen produced by host cells is provided. Non-naturally occurring collagen includes jellyfish (hydra) collagen, human collagen, Chondrosia reniformis (kidney sponge) collagen, or Rhincodon typus (whale shark) collagen. In one embodiment, the non-naturally occurring collagen is full-length or truncated collagen. In one embodiment, the collagen is truncated internally between 50 and 500 amino acids. In another embodiment, the truncation occurs at the C-terminus or N-terminus of the collagen polypeptide. Non-naturally occurring collagens are SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110 or SEQ ID NO:112.

[0013] On the other hand, non-naturally occurring collagen also contains an amino acid sequence including a secretion tag, a histidine tag, a green fluorescent protein tag, a protease cleavage site, a β-lactamase, and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat. When non-naturally occurring collagen contains one or more amino acid trimer repeats of the glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK) sequence, the number of GEK and / or GDK trimer repeats can range from 2 to 50 trimer repeats. On one hand, the secretion tag is DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, or HylA, or a hybrid secretion tag that includes a portion of a secretion tag fused to a portion of a second secretion tag. An exemplary secretion tag is DsbA.

[0014] In one aspect, a composition is provided comprising non-naturally occurring collagen at a concentration between 0.005% and 30% w / w. The composition may also contain at least one additional ingredient, including a topical carrier or preservative.

[0015] On the one hand, compositions containing non-naturally occurring collagen are topical compositions applied to the skin. These topical compositions are used to reduce skin damage or promote the repair of damaged skin.

[0016] One approach provides a method for reducing skin damage or promoting the repair of damaged skin. This method involves applying a composition containing elastin to the skin of a subject. This method increases the activity of fibroblasts or keratinocytes in the subject's skin. In another aspect, the application of the composition increases the synthesis of procollagen by fibroblasts in the subject's skin. In yet another aspect, the topical application of the composition protects the skin or keratinocytes from UV damage. In yet another embodiment, the collagen or elastin disclosed herein reduces the formation of thymine-thymine (TT) dimers.

[0017] Another approach presented in this article is a method for increasing skin cell vitality. This method involves applying collagen or elastin molecules to the skin or skin cells. The collagen or elastin provided increases the vitality of keratinocytes and / or fibroblasts when exposed to UV radiation, urban dust, or other harmful stimuli.

[0018] Another aspect provided herein is a method for reducing the production of inflammatory cytokines in skin cells. In one embodiment, the skin cells are keratinocytes. The method includes applying collagen or elastin molecules to the skin cells. The production of inflammatory cytokines includes TNFα, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18, and IL-1RA.

[0019] On the other hand, a method for protecting skin cells from exposure to urban dust is provided. This method includes the step of applying the collagen or elastin disclosed herein to skin cells. Exposure of skin cells to collagen or elastin increases the vitality of the skin cells. In one embodiment, the skin cells are keratinocytes or fibroblasts.

[0020] On one hand, a non-naturally occurring elastin produced by the host cell is provided. The non-naturally occurring elastin is jellyfish elastin, human elastin, Chondrosia reniformis (kidney sponge) elastin, or Rhincodon typus elastin. In one embodiment, the non-naturally occurring elastin is a full-length or truncated elastin. In one embodiment, the elastin is truncated internally between 50 and 500 amino acids. In another embodiment, the truncation occurs at the C-terminus or N-terminus of the elastin polypeptide. The non-naturally occurring elastin is SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:98, or SEQ ID NO:110.

[0021] On the other hand, non-naturally occurring elastin also contains an amino acid sequence including a secretion tag, a histidine tag, a green fluorescent protein tag, a protease cleavage site, a β-lactamase, and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat. When non-naturally occurring collagen contains one or more amino acid trimer repeats of the glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK) sequence, the number of GEK and / or GDK trimer repeats can range from 2 to 50 trimer repeats. On one hand, the secretion tag is DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, or HylA, or a hybrid secretion tag that includes a portion of a secretion tag fused to a portion of a second secretion tag. An exemplary secretion tag is DsbA.

[0022] In another embodiment, a composition comprising between 0.005% and 30% w / w of non-naturally occurring elastin is provided. The composition may also contain at least one additional ingredient, including a topical carrier or preservative.

[0023] On one hand, compositions containing non-naturally occurring elastin are topical compositions applied to the skin. These topical compositions are used to reduce skin damage or promote the repair of damaged skin.

[0024] One embodiment provides a method for reducing skin damage or promoting the repair of damaged skin. The method includes applying a composition containing elastin to the skin of a subject. This method increases the activity of fibroblasts in the subject's skin. In another embodiment, the application of the composition increases the synthesis of procollagen by fibroblasts in the subject's skin. In yet another embodiment, the topical application of the composition protects the skin or keratinocytes from UV damage. In yet another embodiment, the collagen or elastin disclosed herein reduces the formation of thymine-thymine (TT) dimers.

[0025] Another embodiment provides a polynucleotide encoding non-naturally occurring collagen or elastin. This polynucleotide encodes collagen or elastin derived from jellyfish, humans, *Chondrosia reniformis* (kidney sponge), or *Rhincodon typus*. The encoded collagen or elastin can be full-length or truncated. In one embodiment, the collagen or elastin is truncated by an internal truncation between 50 and 500 amino acids.

[0026] In one embodiment, a polynucleotide encoding a fusion protein is provided, the fusion protein comprising a secretion tag, a histidine tag, a green fluorescent protein tag, a protease cleavage site, a β-lactamase, and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat, together with collagen or elastin. Non-naturally occurring collagen or elastin may comprise one or more amino acid trimer repeats of the glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK) sequence, the number of GEK and / or GDK trimer repeats ranging from 2 to 50. In one aspect, the secretion tag is DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, or HylA, or a hybrid secretion tag comprising a portion of a secretion tag fused to a portion of a second secretion tag. An exemplary embodiment uses DsbA as the secretion tag.

[0027] Polynucleotides and vectors can be used to transform host cells and express polynucleotides. Polynucleotides encoding non-naturally occurring collagen are provided, wherein the polynucleotide is SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, or SEQ ID NO:105. Provided are polynucleotides encoding non-naturally occurring elastin, wherein the polynucleotides are SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70 and SEQ ID NO:72, SEQ ID NO:99 or SEQ ID NO:101.

[0028] Host cells for expressing the polynucleotides of the present invention are disclosed. The host cell can be any host cell, including bacterial cells, yeast cells, fungal cells, insect cells, mammalian cells, plant cells, and any other cell used for expressing exogenous polynucleotides.

[0029] A bacterial host cell is provided, wherein the cell has been modified to inhibit cell division and increase periplasmic space. An exemplary host cell is *Escherichia coli* (E. coli).

[0030] One embodiment provides a method for producing non-naturally occurring collagen or non-naturally occurring elastin. The method includes the steps of: inoculating a culture medium with recombinant host cells containing polynucleotides encoding collagen or elastin; culturing the host cells; and isolating the non-naturally occurring collagen or non-naturally occurring elastin from the host cells. Attached Figure Description

[0031] Figure 1 The physiological differences between converted and unconverted cells are depicted. A) Unconverted E. coli cells. B) E. coli population identical to Figure A but physiologically converted. C) Physiological differences in converted E. coli cells containing cytoplasmic RFP and periplasmic GFP. D) Fluorescence imaging of the cells in Figure C illustrates the localization of targeted proteins.

[0032] Figure 2 Enhanced protein production in converted cells is depicted. AB) The target protein for T7-induced protein production is GFP expressed in the periplasm of *E. coli* BL21. The same cell population was used and induced at OD 1.1. A) Protein ladder (lane 1), IPTG-induced protein production (lane 2), IPTG-induced protein production with physiological conversion (lane 3). B) Two flasks of GFP-induced cell cultures, with IPTG-only on the left and IPTG+ conversion on the right. C) Expression of 22 kDa collagen from converted cells, showing the protein ladder (lane 1), supernatant after protein production (lane 2), and cell pellet (lane 3).

[0033] Figure 3 A time-shifted image depicting the transformation of E. coli cells over time was presented. Figure 4 Other organisms undergoing physiological transformation are shown. A) Normal physiology of *Agrobacterium tumefaciens*. B) Physiology of *Agrobacterium tumefaciens* after transformation. C) Normal physiology of *Pseudomonas aeruginosa* PAO1. D) Physiology of *Pseudomonas aeruginosa* PAO1 after transformation. E) Normal physiology of *Brevundimonas diminuta*. F) Physiology of *Brevundimonas diminuta* after transformation. G) Normal physiology of *Agrobacterium tumefaciens*. H) Physiology of *Agrobacterium tumefaciens* after transformation.

[0034] Figure 4Other organisms undergoing physiological transformation are shown. A) Normal physiology of *Agrobacterium tumefaciens*. B) Physiology of *Agrobacterium tumefaciens* after transformation. C) Normal physiology of *Pseudomonas aeruginosa* PAO1. D) Physiology of *Pseudomonas aeruginosa* PAO1 after transformation. E) Normal physiology of *Agrobacterium tumefaciens*. F) Physiology of *Agrobacterium tumefaciens* after transformation. G) Normal physiology of *Agrobacterium tumefaciens*. H) Physiology of *Agrobacterium tumefaciens* after transformation.

[0035] Figure 5 The study demonstrated a reduction in TT dimer formation by treating human keratinocytes with truncated collagen. Detailed Implementation

[0036] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of this disclosure. However, those skilled in the art will understand that this disclosure can be practiced without these details.

[0037] As used in this article, the term “about” means ±10%.

[0038] The term "composed of" means "including and limited to".

[0039] The term "consistently made up of" means that a composition, method, or structure may include other ingredients, steps, and / or portions, provided that such other ingredients, steps, and / or portions do not materially alter the essential characteristics and novelty of the claimed composition, method, or structure.

[0040] As used herein, the singular forms “an,” “a,” and “the” include the plural forms unless the context clearly indicates otherwise. For example, the terms “compound” or “at least one compound” can include a variety of compounds, including mixtures thereof.

[0041] Throughout this application, various embodiments of this disclosure may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of this disclosure. Therefore, the scope description should be considered to specifically disclose all possible sub-scopes and the individual values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered to specifically disclose sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those scopes, such as 1, 2, 3, 4, 5, and 6. This applies to any scope width.

[0042] Whenever a range of values ​​is indicated herein, it is intended to include any referenced numerical value (fraction or integer) within the indicated range. The phrases “between” and “from” the first indicated number to the second indicated number are used interchangeably herein and are intended to include the first and second indicated numbers and all fractions and integers between them.

[0043] As used herein, the term “method” means the manner, means, techniques and processes used to accomplish a given task, including but not limited to those manner, means, techniques and processes known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or those manner, means, techniques and processes that practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine can readily develop from known manner, means, techniques and processes.

[0044] As used herein, the term "collagen" or "collagen-like protein" refers to a monomeric polypeptide that can associate with one or more collagen or collagen-like polypeptides to form a quaternary structure. Collagen can be treated with acids, alkalis, or heat to prepare gelatin. The quaternary structure of natural collagen is a triple helix, which is typically composed of three polypeptides. Of the three polypeptides that form natural collagen, two are usually identical and are referred to as the α chain. The third polypeptide is referred to as the β chain. Thus, typical natural collagen can be referred to as AAB, where the collagen consists of two α ("A") chains and one β ("B") chain. As used herein, the term "procollagen" refers to a cellularly produced polypeptide that can be processed into naturally occurring collagen.

[0045] As used herein, the term "elastin" refers to a polypeptide that is elastic and has the ability to stretch or contract and return to its original shape. Elastin is naturally found in connective tissue.

[0046] As used herein, the term "expression vector" or "vector" refers to a nucleic acid assembly capable of directing the expression of a foreign gene. An expression vector may contain a promoter operatively linked to a foreign gene, a restriction endonuclease site, a nucleic acid encoding one or more selectable markers, and other nucleic acids that can be used in recombinant technology practices.

[0047] As used in this article, the term "fibroblast" refers to cells that synthesize procollagen and other structural proteins. Fibroblasts are widely distributed throughout the body and are found in the skin, connective tissue, and other tissues.

[0048] The term "fluorescent protein" refers to a protein commonly used in genetic engineering as a reporter molecule for the expression of exogenous polynucleotides. This protein fluoresces when exposed to ultraviolet or blue light, emitting bright visible light. Proteins that emit green light are called green fluorescent protein (GFP), while those that emit red light are called red fluorescent protein (RFP).

[0049] As used herein, the term "gelatin" refers to collagen that has been further processed by exposure to acids, alkalis, or heat. While not wishing to be bound by theories or mechanisms, it is understood that treating collagen with acids, alkalis, or heat denatures the collagen peptides. Aqueous solutions of denatured collagen form reversible gels used in food, cosmetics, pharmaceuticals, industrial products, medical products, laboratory culture media, and many other applications.

[0050] As used herein, the term “gene” refers to a polynucleotide that encodes a specific protein, which may refer alone to the coding region or may include regulatory sequences preceding (5' non-coding sequence) and following (3' non-coding sequence).

[0051] The term "histidine tag" refers to a string of 2-30 consecutive histidine residues on a recombinant polypeptide.

[0052] The term "host cell" refers to a cell that has been engineered to express introduced exogenous polynucleotides.

[0053] The term "keratinocyte" refers to cells that produce keratin, a protein found in the epidermis of the skin.

[0054] As used herein, the term "lactamase" refers to an enzyme that hydrolyzes antibiotics containing a lactam (cyclic amide) moiety. "Beta-lactamase" or "β-lactamase" refers to an enzyme that hydrolyzes antibiotics containing a β-lactam moiety.

[0055] As used herein, the term "non-naturally occurring" refers to collagen or elastin that is not normally found in nature. Non-naturally occurring collagen or elastin is prepared by recombinant synthesis. The non-naturally occurring collagen or elastin is recombinant collagen or recombinant elastin. In one embodiment, the non-naturally occurring collagen is a truncated collagen. Other non-naturally occurring collagen peptides include chimeric collagen. A chimeric collagen is a peptide in which a portion of a collagen peptide is adjacent to a portion of a second collagen peptide. For example, a collagen molecule comprising a portion of jellyfish collagen adjacent to a portion of human collagen is a chimeric collagen. In another embodiment, the non-naturally occurring collagen comprises a fusion peptide containing additional amino acids, such as a secretion tag, a histidine tag, green fluorescent protein, a protease cleavage site, a GEK repeat, a GDK repeat, and / or a β-lactamase. In one embodiment, the non-naturally occurring elastin is a truncated elastin. Other non-naturally occurring elastin peptides include chimeric elastin. Chimeric elastin is a polypeptide in which a portion of an elastin polypeptide is adjacent to a portion of a second elastin polypeptide. For example, a collagen molecule comprising a portion of jellyfish elastin adjacent to a portion of human elastin is a chimeric elastin. In another embodiment, a non-naturally occurring elastin comprises a fusion polypeptide containing additional amino acids, such as a secretion tag, a histidine tag, green fluorescent protein, a protease cleavage site, and / or a β-lactamase. Chimeric gelatin or chimeric elastin may contain additional amino acids, such as a secretion tag, a histidine tag, green fluorescent protein, a protease cleavage site, a GEK repeat, a GDK repeat, and / or a β-lactamase.

[0056] The term "protease cleavage site" refers to the amino acid sequence that is cleaved by a specific protease.

[0057] The term "secretory tag" or "signal peptide" refers to an amino acid sequence that recruits cellular machinery from the host cell to transport expressed proteins to specific locations or organelles within the host cell.

[0058] The term "truncated collagen" refers to a monomeric polypeptide smaller than full-length collagen, in which one or more portions of the full-length collagen are absent. Collagen polypeptides are truncated at the C-terminus or N-terminus, or by removing internal portions of the full-length collagen polypeptide.

[0059] The term "truncated elastin" refers to a monomeric polypeptide smaller than full-length elastin, in which one or more portions of the full-length elastin are absent. Elastin polypeptides are truncated at the C-terminus or N-terminus, or by removing internal portions of the full-length elastin polypeptide.

[0060] An expression system using modified bacterial cells (converted cells) is disclosed in commonly owned application PCT / US17 / 24857, which, by reference, inhibits cell division and significantly enhances the growth of the periplasmic space. In this expression system, the expressed protein targets the periplasmic space. The production of recombinant proteins is significantly increased in these converted cells compared to non-converted cells. Structurally, the cell includes an inner and outer membrane but lacks a functional peptidoglycan cell wall, and the cell shape is spherical with an increasing volume over time. Notably, while the periplasmic space typically comprises only 10-20% of the total cell volume, the periplasmic compartments in the converted state described herein can comprise more than 20%, 30%, 40%, or 50% of the total cell volume, and up to 60%, 70%, 80%, or 90%.

[0061] The modified bacterial cells of PCT / US17 / 24857 are derived from Gram-negative bacteria, such as those selected from γ-Proteobacteria and α-Proteobacteria. In some embodiments, the bacteria are selected from Escherichia coli, Vibrio natriegens, Pseudomonas fluorescens, Caurobacter crescentus, Agrobacterium tumefaciens, and Defective shortwave monoclonal bacteria. In a particular embodiment, the bacteria are Escherichia coli, such as strain BL21(DE3).

[0062] On the other hand, the host bacterial cells have increased periplasmic space in a culture medium containing magnesium salts, wherein the concentration of magnesium ions in the medium is at least about 3, 4, 5, or 6 mM. In a further embodiment, the concentration of magnesium ions in the culture medium is at least about 7, 8, 9, or 10 mM. In some embodiments, the concentration of magnesium ions in the culture medium is between about 5 mM and 25 mM, about 6 mM, and / or about 20, 15, or 10 mM. In some embodiments, the magnesium salt is selected from magnesium sulfate and magnesium chloride.

[0063] In other embodiments, the culture medium further comprises an osmotic stabilizer, including, for example, sugars (e.g., arabinose, glucose, sucrose, glycerol, sorbitol, mannitol, fructose, galactose, sucralose, maltotriose, erythritol, ribitol, pentaerythritol, arabinitol, galactitol, xylitol, idoteol, maltotriose, etc.), betaine (e.g., trimethylglycine), proline, and sodium chloride, wherein the concentration of the osmotic stabilizer in the culture medium is at least about 4%, 5%, 6%, or 7% (w / v). In further embodiments, the concentration of the osmotic stabilizer is at least about 8%, 9%, or 10% (w / v). In some embodiments, the concentration of the osmotic stabilizer in the culture medium is between about 5% and about 20% (w / v).

[0064] In some embodiments, the cell culture may further comprise ammonium chloride, ammonium sulfate, calcium chloride, amino acids, ferrous(II) sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate, and yeast extract.

[0065] The culture of host bacterial cells can be continuous or discontinuous; it can be a batch process, a fed-batch process, or a repeated fed-batch process.

[0066] In some embodiments, the antibiotic is selected from: β-lactam antibiotics (e.g., penicillin, cephalosporins, carbapenems, and monocyclic lactams), phosphonate antibiotics, peptide antibiotics, and glycopeptide antibiotics. In specific embodiments, the antibiotic is selected from arafonide, amoxicillin, ampicillin, aztreonam, bacitracin, carbenicillin, cefamandole, cefotaxime, cefsulfuron-methyl, cefotaxime, phosphatamicin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, fosfomycin, primaxin, and vancomycin.

[0067] Without being bound by theory, the cell morphology that promotes recombinant protein production and inhibits cell division appears to be driven by the removal of the cell wall under the aforementioned culture conditions. In some embodiments, the method of removing / inhibiting cell wall synthesis can be carried out by using antibiotics that inhibit peptidoglycan synthesis (such as ampicillin, carbenicillin, penicillin, or fosfomycin), or other methods known in the art.

[0068] When a suitable periplasmic targeting signal sequence is present, the recombinant peptide can be secreted into the periplasmic space of bacterial cells. (Joly, JC and Laird, MW, in The Periplasmed. Ehrmann, M., ASM Press, Washington DC, (2007) 345-360.) The chemically oxidative environment of the periplasm favors the formation of disulfide bonds, thereby facilitating the proper folding of the peptide for its function.

[0069] Typically, the signal sequence can be a component of the expression vector or a portion of a foreign gene inserted into the vector. The selected signal sequence should be one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For bacterial host cells that do not recognize and process the natural signal sequence of the foreign gene, this signal sequence is replaced by any well-known bacterial signal sequence. In some embodiments, the DsbA signal sequence can be used to target the recombinant polypeptide to the periplasmic space. (Dinh and Bernhardt, J Bacteriol, Sept. 2011, 4984-4987).

[0070] On one hand, it provides non-naturally occurring collagen or elastin produced by host cells. These non-naturally occurring collagen or elastin are jellyfish collagen or elastin, human collagen or elastin, Chondrosia reniformis (kidney sponge) collagen or elastin, or Rhincodon typus collagen or elastin. The non-naturally occurring collagen or elastin is truncated collagen. This truncation is internal truncation, truncation at the N-terminal portion of the collagen or elastin, or truncation at the C-terminal portion of the collagen or elastin. Collagen or elastin is truncated to a length between 50 and 1000 amino acids, between 50 and 950 amino acids, between 50 and 900 amino acids, between 50 and 850 amino acids, between 50 and 800 amino acids, between 50 and 750 amino acids, between 50 and 700 amino acids, between 50 and 650 amino acids, between 50 and 600 amino acids, between 50 and 550 amino acids, between 50 and 500 amino acids, between 50 and 450 amino acids, between 50 and 400 amino acids, between 50 and 350 amino acids, between 50 and 300 amino acids, between 50 and 250 amino acids, between 50 and 200 amino acids, between 50 and 150 amino acids, or between 50 and 100 amino acids. In another embodiment, collagen or elastin is truncated to 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, or 35 mm. 0, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acids. Non-naturally occurring collagen or elastin is encoded by a portion or the entire polynucleotide sequence disclosed herein.

[0071] The non-naturally occurring collagen or elastin also contains an amino acid sequence with a secretion tag. The secretion tag directs the collagen or elastin into the periplasmic space of the host cell. In a particular embodiment, the signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, or HylA, or a hybrid secretion tag comprising a portion of a secretion tag fused to a portion of a second secretion tag. On one hand, the secretion tag is attached to the non-naturally occurring collagen or elastin. On the other hand, the secretion tag is cleaved from the non-naturally occurring collagen or elastin.

[0072] Non-naturally occurring collagen or elastin also contains a histidine tag. A histidine tag or multiple histidine tags are sequences of 2 to 20 histidine residues attached to collagen or elastin. Histidine tags may contain 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. Histidine tags can be used to purify proteins using chromatographic methods with nickel-based chromatographic media. Exemplary fluorescent proteins include green fluorescent protein (GFP) or red fluorescent protein (RFP). Fluorescent proteins are well known in the art. In one embodiment, the non-naturally occurring collagen or elastin contains GFP and / or RFP. In one embodiment, superfolded GFP is fused to a non-naturally occurring collagen or elastin. Superfolded GFP is a GFP that can fold correctly even when fused to a weakly folded peptide. On one hand, a histidine tag is attached to the non-naturally occurring collagen or elastin. On the other hand, the histidine tag is cleaved from the non-naturally occurring collagen or elastin.

[0073] Non-naturally occurring collagen or elastin also contains protease cleavage sites. These sites can be used to cleave recombinant collagen or elastin to remove portions of the polypeptide. Removable portions of the polypeptide include secretory tags, histidine tags, fluorescent protein tags, and / or β-lactamases. Proteases include endopeptides, exopeptides, serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamate proteases, and metalloproteinases. Exemplary protease cleavage sites include amino acids cleaved by thrombin, TEV protease, factor Xa, intestinal peptidase, and rhinovirus 3C protease. On one hand, the cleavage tag is attached to the non-naturally occurring collagen or elastin. On the other hand, the cleavage tag is removed from the non-naturally occurring collagen or elastin by a suitable protease.

[0074] Non-naturally occurring collagen or elastin also contains enzymes that act as β-lactamases. β-lactamases can be used as selection markers. On one hand, β-lactamases attach to non-naturally occurring collagen or elastin. On the other hand, β-lactamases are cleaved from non-naturally occurring collagen or elastin.

[0075] Non-naturally occurring collagen or elastin also contains GEK amino acid trimer repeats and / or GDK amino acid trimer repeats. GEK and GDK trimer repeats contribute to the gelation of collagen and / or gelatin. In one embodiment, the non-naturally occurring collagen or elastin contains 2-50 GEK and / or 2-50 GDK trimer repeats, 2-40 GEK and / or 2-40 GDK trimer repeats, 2-30 GEK and / or 2-30 GDK trimer repeats, 2-20 GEK and / or 2-20 GDK trimer repeats, 2-15 GEK and / or 2-1 The repeats can be 0 GEK and / or 2-10 GDK trimers, 2-9 GEK and / or 2-9 GDK trimers, 2-8 GEK and / or 2-8 GDK trimers, 2-7 GEK and / or 2-7 GDK trimers, 2-6 GEK and / or 2-6 GDK trimers, 2-5 GEK and / or 2-5 GDK trimers, or 2-4 GEK and / or 2-4 GDK trimers. On one hand, the GEK trimer or GDK trimer repeats attach to non-naturally occurring collagen or elastin. On the other hand, the GEK trimer or GDK trimer repeats cleave from non-naturally occurring collagen or elastin.

[0076] This article provides compositions comprising non-naturally occurring collagen and / or non-naturally occurring elastin in the range of 0.005% to 30% w / w. The composition comprises 0.005% to 20% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin, 0.005% to 10% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin, 0.005% to 5% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin, 0.005% to 2% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin, 0.005% to 1% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin, 0.005% to 0.5% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin, and 0.005% to 0.2% w / w of non-naturally occurring collagen and / or non-naturally occurring elastin.

[0077] Compositions containing non-naturally occurring collagen and / or non-naturally occurring elastin are personal care products. In some embodiments, the composition is formulated for topical application. The composition may contain other cosmetic ingredients suitable for human use. The personal care product can be used to prevent or treat damage to human skin or hair caused by ultraviolet radiation. The personal care product is suitable for the skin or hair. The composition includes, for example, face masks, skin cleansers such as soaps, cleansing creams, cleansing lotions, cleansing creams, facial puffs, facial cleansers, shampoos, conditioners, and shower gels.

[0078] Compositions containing non-naturally occurring collagen and / or non-naturally occurring elastin may also contain at least one additional ingredient, including a topical carrier or preservative. Topical carriers include those selected from liposomes, biodegradable microcapsules, emulsions, sprays, aerosols, powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbate, cetyl wax, cetearyl alcohol, 2-octyldodecanool, benzyl alcohol, cyclomethyl silicone oil, cyclopentasiloxane, and water. Preservatives include those selected from tocopherol, diiodomethyl-p-tolyl sulfone, 2-bromo-2-nitropropane-1,3-diol, cis isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azamonoadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA.

[0079] A method is provided to reduce skin damage, promote the repair of damaged skin, protect skin from UV damage, and protect skin cells from exposure to urban dust. The method includes applying a composition containing non-naturally occurring collagen and / or non-naturally occurring elastin to the skin of a subject. Unbound by any particular theory or mechanism, the collagen and / or elastin in the composition reduce skin damage by protecting against UV damage and / or promote the repair of damaged skin by increasing cell vitality and / or increasing procollagen synthesis when applied to the skin, and / or promote skin cell vitality. In one aspect, collagen and elastin reduce the formation of thymine-thymine (TT) dimers.

[0080] One aspect provides a polynucleotide encoding non-naturally occurring collagen or elastin. This polynucleotide encodes collagen or elastin derived from jellyfish, humans, *Chondrosia reniformis* (kidney sponge), or *Rhincodon typus*. This polynucleotide encodes full-length or truncated collagen or elastin.

[0081] Another aspect provides polynucleotides encoding collagen or elastin fusion proteins. Elastin or collagen fusion proteins contain secretion tags, histidine tags, fluorescent protein tags, protease cleavage sites, β-lactamases, and / or GEK amino acid trimer repeats and / or GDK amino acid trimer repeats, along with collagen or elastin.

[0082] On one hand, polynucleotides are carriers used to transform host cells and express the polynucleotide. Polynucleotides also contain nucleic acids encoding enzymes that allow the host organism to grow in the presence of selectants. Selectants include certain sugars, including galactose-containing sugars, or antibiotics, including ampicillin, hygromycin, G418, etc. Enzymes used to confer resistance to selectants include β-galactosidases or β-lactamases.

[0083] In one aspect, a host cell for expressing the polynucleotides of the present invention is provided. The host cell can be any host cell, including Gram-negative bacterial cells, Gram-positive bacterial cells, yeast cells, insect cells, mammalian cells, plant cells, or any other cell used for expressing exogenous polynucleotides. An exemplary Gram-negative host cell is *Escherichia coli*.

[0084] The bacterial host cell was taught to inhibit cell division and increase periplasmic space. As discussed herein and taught in Example 1, β-lactam antibiotics can be used as conversion agents to transform wild-type bacterial cells into modified bacterial cells in which cell replication is inhibited and periplasmic space is increased. Exemplary β-lactam antibiotics include penicillins, cephalosporins, carbapenems, and monocyclic lactams.

[0085] The transition form (L-form) of bacteria is cultured in a medium containing certain salts and other nutrients. Salt and medium compositions tested to support physiological transition physiology include M63 salt medium, M9 salt medium, PYE medium, and Luria-Bertani (LB) medium. In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may be included, alone or in mixtures with other supplements or mediums (such as complex nitrogen sources), at appropriate concentrations. In some embodiments, the medium also contains one or more components selected from: ammonium chloride, ammonium sulfate, calcium chloride, casein amino acids, ferrous(II) sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate, and yeast extract.

[0086] β-lactamases are enzymes that confer resistance to lactam antibiotics to prokaryotic cells. Typically, when β-lactamases are expressed in bacterial host cells, the expressed β-lactamase protein also contains a targeting sequence (secretion tag) that directs the β-lactamase protein into the periplasmic space. Unless the β-lactamase is transported into the periplasmic space, it is not functional. β-lactamases that can target the periplasmic space without using a separate secretion tag are provided. By creating fusion proteins in which a periplasmic secretion tag is added to the N-terminus of a protein (such as GFP, collagen, or a GFP / collagen chimera), the function of β-lactamases lacking the native secretion tag can be used to select for the complete translation and secretion of the N-terminal fusion protein. Using this method, we have used the DsbA-GFP-collagen-β-lactamase fusion to select truncated products that are conducive to translation and secretion in target collagen.

[0087] Another embodiment provides a method for generating non-naturally occurring collagen or non-naturally occurring elastin. The method includes the steps of: inoculating a culture medium with recombinant host cells containing polynucleotides encoding collagen or elastin, culturing the host cells, and isolating the non-naturally occurring collagen or non-naturally occurring elastin from the host cells.

[0088] A process for preparing proteins by fermentation is provided. The process includes the following steps:

[0089] a) Culture recombinant Gram-negative bacterial cells in a medium containing magnesium salts, wherein the concentration of magnesium ions in the medium is at least about 6 mM, and wherein the bacterial cells contain a foreign gene encoding the protein.

[0090] b) Add an antibiotic to the culture medium, wherein the antibiotic inhibits the biogeneration of peptidoglycan in bacterial cells; and

[0091] c) Harvesting proteins from the culture medium.

[0092] Bacteria can be cultured continuously, for example as described in WO 05 / 021772, or discontinuously in a batch process (batch culture), a fed-batch process, or a repeated fed-batch process, for the purpose of producing target proteins. In some embodiments, protein production is carried out on a large scale. Various large-scale fermentation programs can be used for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1,000 liters, preferably from about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (preferred carbon / energy source). Small-scale fermentation typically refers to fermentation in fermenters with a volume capacity not exceeding about 20 liters.

[0093] To accumulate target proteins, host cells are cultured under conditions sufficient for their accumulation. Such conditions include, for example, temperature, nutrients, and cell density conditions that allow the cells to express and accumulate proteins. Furthermore, as those skilled in the art will recognize, these conditions are those under which the cells can perform essential cellular functions such as transcription, translation, and, for the purpose of secreting proteins, transport proteins from one cellular compartment to another.

[0094] Bacterial cells are cultured at a suitable temperature. For example, for the growth of *E. coli*, 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 in a non-converted or converted state are cultured at one temperature and then converted to another temperature to induce protein production. Host cells are first cultured at one temperature to proliferate, and then cultured at a lower temperature to induce protein production. The first temperature is 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 is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C. The incubation at the second temperature was carried out for 1 to 100 hours, 5 to 90 hours, 5 to 80 hours, 5 to 70 hours, 10 to 70 hours, 15 to 70 hours, 15 to 65 hours, 15 to 60 hours, 20 to 60 hours, 20 to 55 hours, 20 to 50 hours, 24 to 50 hours, 24 to 48 hours, 30 to 50 hours, 30 to 45 hours, or 30 to 40 hours.

[0095] The pH of the culture medium can be any pH, approximately 5–9, depending primarily on the host organism. For *E. coli*, the pH is approximately 6.8 to approximately 7.4, or approximately 7.0.

[0096] To induce gene expression, cells are typically cultured until a certain optical density is reached, for example, an OD 600 of approximately 1.1, at which point induction begins (e.g., by adding an inducer, by depleting repressors, inhibitors, or culture medium components) to induce the expression of a foreign gene encoding a target protein. In some embodiments, the expression of the foreign gene can be induced by an inducer selected from, for example, isopropyl-β-d-1-thiogalactopyranoside, lactose, arabinose, maltose, tetracycline, dehydrotetracycline, 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 during fermentation during cell proliferation is between 10% and 30%. To induce gene expression, the dissolved oxygen level is reduced to below 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%. In host cells, whether in a physiological or transitional state, protein production can be induced by lowering the fermentation temperature, as disclosed herein.

[0097] After product accumulation, the cells are vortexed and centrifuged to induce lysis and release of recombinant proteins. Most proteins are found in the supernatant, but any residual membrane-bound proteins can be released using a detergent such as Triton X-100.

[0098] In subsequent steps, the target protein, as a soluble or insoluble product released from the cellular matrix, is recovered in a manner that minimizes the co-recovery of cell debris and product. Recovery can be performed in any manner, but in one embodiment, recovery may include purification via histidine tagging of a nickel column. See, for example, Purification of Proteins Using Polyhistidine Affinity Tags, Methods Enzymology. 2000; 326:245-254.

[0099] This invention provides, but is not limited to, the following embodiments:

[0100] 1. A non-naturally occurring collagen, selected from jellyfish collagen, human collagen, Chondrosia reniformis (kidney sponge) collagen, and Rhincodon typus (whale shark) collagen.

[0101] 2. The non-naturally occurring collagen according to Embodiment 1, wherein the non-naturally occurring collagen is truncated.

[0102] 3. The non-naturally occurring truncated collagen according to embodiment 2, wherein the collagen is truncated internally by 50 to 300 amino acids.

[0103] 4. The non-naturally occurring collagen according to any one of embodiments 1 to 3, wherein the amino acid sequence of the collagen is selected from SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110 and SEQ ID NO:112.

[0104] 5. A non-naturally occurring collagen according to any one of embodiments 1 to 4, wherein the collagen further comprises an amino acid sequence selected from secretion tags, histidine tags, green fluorescent protein, protease cleavage sites, and β-lactamase proteins.

[0105] 6. The non-naturally occurring collagen according to embodiment 5, wherein the secretion tag is DsbA.

[0106] 7. A non-naturally occurring collagen according to any one of embodiments 1 to 6, wherein the collagen further comprises one or more amino acid trimer repeats of the glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK) sequence.

[0107] 8. The non-naturally occurring collagen according to embodiment 7, wherein the non-naturally occurring collagen comprises a trimer repeat of 2 to 50 amino acids.

[0108] 9. The non-naturally occurring collagen according to any one of embodiments 1 to 8, wherein the non-naturally occurring collagen is jellyfish collagen.

[0109] 10. A composition comprising, as described in any one of embodiments 1 to 9, a non-naturally occurring collagen at a concentration of 0.005% to 30% w / w, wherein the composition stimulates fibroblast growth and / or stimulates procollagen synthesis and / or reduces thymine-thymine (TT) dimer formation.

[0110] 11. The composition according to embodiment 10, wherein the composition contains between 0.005% and 1% of non-naturally occurring collagen.

[0111] 12. The composition according to embodiment 10, wherein the composition contains between 0.01% and 1% of non-naturally occurring collagen.

[0112] 13. The composition according to embodiment 10, wherein the composition contains between 0.02% and 0.5% of non-naturally occurring collagen.

[0113] 14. The composition according to any one of embodiments 10 to 13, wherein the composition is a topical composition, and the topical composition further comprises at least one additional component, the additional component including a topical carrier and / or a preservative.

[0114] 15. The composition according to embodiment 14, wherein the topical composition is a face mask.

[0115] 16. The topical composition according to embodiment 14 or 15, wherein the topical carrier is selected from liposomes, biodegradable microcapsules, emulsions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbate, cetyl wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, cyclomethyl silicone oil, cyclopentadiene, and water.

[0116] 17. The topical composition according to any one of embodiments 14 to 16, wherein 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-azamonoadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA.

[0117] 18. A method for reducing skin damage, promoting the repair of damaged skin, protecting skin from UV damage, or increasing skin cell vitality, the method comprising the step of applying the composition of any one of embodiments 1 to 17 to the skin or skin cells of a subject.

[0118] 19. The method according to embodiment 18, wherein the activity of fibroblasts present in the skin of the subject is increased.

[0119] 20. The method according to embodiment 18, wherein fibroblasts present in the skin of the subject increase the synthesis of procollagen.

[0120] 21. An elastin that is not naturally occurring, selected from jellyfish elastin, human elastin, Chondrosia reniformis (kidney sponge) elastin or Rhincodon typus elastin.

[0121] 22. The non-naturally occurring elastin according to embodiment 21, wherein the non-naturally occurring elastin is truncated.

[0122] 23. The non-naturally occurring truncated elastin according to embodiment 22, wherein the elastin is truncated by at least 50 amino acids.

[0123] 24. A non-naturally occurring elastin according to any one of embodiments 21 to 23, wherein the amino acid sequence of the elastin is selected from SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:98 and SEQ ID NO:100.

[0124] 25. A non-naturally occurring elastin according to any one of embodiments 21 to 24, wherein the elastin further comprises an amino acid sequence selected from secretion tags, histidine tags, green fluorescent protein, protease cleavage sites, and β-lactamase proteins.

[0125] 26. The non-naturally occurring elastin according to embodiment 25, wherein the secretion tag is DsbA.

[0126] 27. A non-naturally occurring elastin according to any one of embodiments 21 to 26, wherein the elastin further comprises one or more amino acid trimer repeats of the glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK) sequence.

[0127] 28. The non-naturally occurring elastin according to embodiment 27, wherein the non-naturally occurring elastin comprises a trimer repeat of 2 to 50 amino acids.

[0128] 29. A non-naturally occurring elastin according to any one of embodiments 21 to 28, wherein the non-naturally occurring elastin is human elastin.

[0129] 30. A composition comprising, as described in any one of embodiments 21 to 29, a non-naturally occurring elastin at a concentration of 0.005% to 30% w / w, wherein the composition stimulates fibroblast growth and / or stimulates procollagen synthesis.

[0130] 31. The composition according to embodiment 30, wherein the composition contains between 0.005% and 1% of non-naturally occurring elastin.

[0131] 32. The composition according to embodiment 30, wherein the composition contains between 0.01% and 1% of non-naturally occurring elastin.

[0132] 33. The composition according to embodiment 30, wherein the composition contains between 0.02% and 0.5% of non-naturally occurring elastin.

[0133] 34. The composition according to any one of embodiments 30 to 33, wherein the composition is a topical composition, and the topical composition further comprises at least one additional component, the additional component including a topical carrier and / or a preservative.

[0134] 35. The composition according to embodiment 34, wherein the topical composition is a face mask.

[0135] 36. The topical composition according to embodiment 34 or 35, wherein the topical carrier is selected from liposomes, biodegradable microcapsules, emulsions, sprays, aerosols, powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbate, cetyl wax, cetearyl alcohol, 2-octyldodecanool, benzyl alcohol, cyclomethyl silicone oil, cyclopentadiene, and water.

[0136] 37. The topical composition according to any one of embodiments 34 to 36, wherein the preservative is selected from tocopherol, diiodomethyl-p-tolyl sulfone, 2-bromo-2-nitropropane-1,3-diol, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract and EDTA.

[0137] 38. A method for reducing skin damage, promoting the repair of damaged skin, or protecting skin from UV damage, the method comprising applying the composition of any one of embodiments 21 to 37 to the skin of a subject.

[0138] 39. The method according to embodiment 38, wherein the activity of fibroblasts or keratinocytes present in the skin of the subject is increased.

[0139] 40. The method according to embodiment 38, wherein fibroblasts present in the skin of the subject increase the synthesis of procollagen.

[0140] 41. A polynucleotide encoding a non-naturally occurring collagen, wherein the polynucleotide is selected from SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:105.

[0141] 42. The polynucleotide according to embodiment 41, wherein the polynucleotide is a carrier.

[0142] 43. The polynucleotide according to embodiment 41 or 42, wherein the polynucleotide further comprises a nucleic acid selected from nucleic acids encoding secretion tags, histidine tags, green fluorescent proteins, protease cleavage sites, and β-lactamase proteins.

[0143] 44. The polynucleotide according to any one of embodiments 41 to 43, wherein the polynucleotide further comprises a polynucleotide encoding one or more amino acid trimer repeats of the sequence glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK).

[0144] 45. A host cell comprising a polynucleotide according to any one of embodiments 41 or 45.

[0145] 46. ​​The host cell according to embodiment 48, wherein the host cell transports the non-naturally occurring collagen to the periplasmic space of the host cell.

[0146] 47. The host cell according to embodiment 45 or 46, wherein the host cell produces jellyfish collagen.

[0147] 48. The host cell according to any one of embodiments 45 to 47, wherein the host cell is a Gram-negative bacterium.

[0148] 49. The method according to embodiment 48, wherein the host cell is Escherichia coli.

[0149] 50. The method according to embodiment 49, wherein the Escherichia coli is a transformed Escherichia coli.

[0150] 51. A method for producing non-naturally occurring collagen, the method comprising the steps of:

[0151] a. Culturing the host cells described in any one of embodiments 45 to 50 in a culture medium; and

[0152] b. Isolate the non-naturally occurring collagen from the host cells.

[0153] 52. A polynucleotide encoding a non-naturally occurring elastin, wherein the polynucleotide is selected from SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:99 and SEQ ID NO:101.

[0154] 53. The polynucleotide according to embodiment 52, wherein the polynucleotide is a carrier.

[0155] 54. The polynucleotide according to embodiment 52 or 53, wherein the polynucleotide further comprises a nucleic acid selected from nucleic acids encoding secretion tags, histidine tags, green fluorescent protein, protease cleavage sites, and β-lactamase proteins.

[0156] 55. The polynucleotide according to any one of embodiments 52 to 54, wherein the polynucleotide further comprises a polynucleotide encoding one or more amino acid trimer repeats of the sequence glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK).

[0157] 56. A host cell comprising a polynucleotide according to any one of embodiments 52 to 55.

[0158] 57. The host cell according to embodiment 56, wherein the host cell transports the non-naturally occurring collagen to the periplasmic space of the host cell.

[0159] 58. The host cell according to embodiment 56 or 57, wherein the host cell produces jellyfish collagen.

[0160] 59. The host cell according to any one of embodiments 56 to 58, wherein the host cell is a Gram-negative bacterium.

[0161] 60. The method according to embodiment 59, wherein the host cell is Escherichia coli.

[0162] 61. The method according to embodiment 60, wherein the Escherichia coli is a transformed Escherichia coli.

[0163] 62. A method for producing non-naturally occurring elastin, the method comprising the steps of:

[0164] a. Culturing the host cells described in any one of embodiments 56 to 61 in a culture medium; and

[0165] b. Isolate the non-naturally occurring collagen from the host cells.

[0166] 63. A method for reducing inflammatory cytokines produced by skin cells, the method comprising the step of applying the composition of any one of embodiments 1 to 17 to the skin cells.

[0167] 64. The method according to embodiment 63, wherein the inflammatory cytokine is IL-1α.

[0168] 65. The method according to embodiment 63, wherein the skin cells are keratinocytes.

[0169] 66. A method for increasing skin cell vitality, the method comprising the step of applying the composition of any one of embodiments 1 to 17 to the skin or skin cells of a subject.

[0170] 67. The method according to embodiment 66, wherein the activity of keratinocytes present in the skin of the subject is increased.

[0171] 68. The method according to embodiment 66, wherein the activity of fibroblasts present in the skin of the subject is increased.

[0172] 69. A method for protecting skin cells from exposure to urban dust, the method comprising the step of applying a composition according to any one of embodiments 1 to 17 to skin cells, wherein the vitality of the skin cells is increased.

[0173] 70. The method according to embodiment 69, wherein the skin cells are keratinocytes or fibroblasts.

[0174] Example

[0175] Example 1: Expression System

[0176] Materials and methods:

[0177] Strains:

[0178] The physiological transitions and protein production tested:

[0179] Escherichia coli BL21(DE3) - from NEB, product #c2527

[0180] Escherichia coli K12 NCM3722 - from The Coli Genetic Stock Center, CGSC#12355

[0181] Physiological shifts in testing:

[0182] γ-Proteobacteria:

[0183] Sodium-dependent Vibrio natriegens - from ATCC, product #14048

[0184] Fluorescent Pseudomonas - from ATCC, Product #31948

[0185] Pseudomonas aeruginosa PAO1 - from ATCC, product #BAA-47

[0186] α-Proteobacteria:

[0187] Crested serogroup - from ATCC, product #19089

[0188] Agrobacterium tumefaciens / Radiobacterium radiobacter - from ATCC, product #33970

[0189] Defective shortwave monoclonal bacteria - from ATCC, product #13184

[0190] Culture medium composition:

[0191] 1 liter of 5x M63 salt:

[0192] 10g (NH4)2SO4 - from P212121, product #7783-20-2

[0193] 68g KH2PO4 - from P212121, product #7778-77-0

[0194] 2.5mg FeSO4·7H2O - from Sigma Aldrich, product #F7002

[0195] Increase the volume to 1 liter using milliQ water.

[0196] Adjust the pH to 7 using KOH (from P212121, product #1310-58-3).

[0197] Autoclave the mixture

[0198] 1 liter 1M mgSO4: 246.5g MgSO4 7H2O - from P212121, (Sigma Aldrich, Product #10034-99-8)

[0199] Increase the volume to 1 liter using milliQ water.

[0200] The mixture is autoclaved.

[0201] 1 liter of transition medium 1:

[0202] 133.4 mL 5X m63 salt

[0203] 10 mL lM MgSO4

[0204] 38.6g glucose - from P212121, product #50-99-7

[0205] 66.6g sucrose - from P212121, product #57-50-1

[0206] 8.33g LB Mixture - from P212121, Product #lb-miller

[0207] Increase the volume to 1 liter using milliQ water.

[0208] The mixture was sterilized by filtration through a 0.22 μM pore vacuum filter (Sigma Aldrich, product #CLS430517).

[0209] 1 liter of transition medium 2:

[0210] 133.4 mL 5X m63 salt

[0211] 10 mL lM MgSO4

[0212] 38.6g glucose - from P212121, product #50-99-7

[0213] 66.6g sucrose - from P212121, product #57-50-1

[0214] 10g Yeast Extract - from FisherSci.com, product #J60287A1

[0215] Increase the volume to 1 liter using milliQ water.

[0216] The mixture was sterilized by filtration through a 0.22 μM pore vacuum filter (Sigma Aldrich, product #CLS430517).

[0217] For use in bioreactor growth:

[0218] 5 liters of MGZ12 culture medium for bioreactor:

[0219] 1) Autoclave 1L of glucose at a concentration of 500g / L in DI water. (VWR, Product #97061-170)

[0220] 2) Autoclave 1L of sucrose at a concentration of 500g / L in DI water. (Geneseesci.com, Product #62-112).

[0221] 3) Autoclave in 3946 mL DI water:

[0222] 20g (NH4)2HPO4. (VWR, Product #97061-932).

[0223] 66.5g KH2PO4. (VWR, Product #97062-348).

[0224] 22.5g H3C6H5O7. (VWR, product #BDH9228-2.5KG).

[0225] 2.95g MgSO4·7H2O. (VWR, Product #97062-134).

[0226] 10 mL of trace metal (Teknova), 1000x. (Teknova, product #T1001). After autoclaving, add 400 mL of (1) to (3), add 65 mL of 10M NaOH (VWR, product #97064-480) to (3), and add 666 mL of (2) to (3). If necessary, a glucose feed of 500 g / L can be used during fermentation.

[0227] Add the following during induction:

[0228] 50 mL of 1 M MgSO4·7H2O was added to a 5 L bioreactor containing 1 to 10 mM IPTG. (carbosynth.com, product #EI05931).

[0229] Add fosfomycin (50 μg / mL or higher) and carbenicillin (100 μg / mL or higher). Physiological transition:

[0230] Optimal physiological conversion was initiated at an OD 600 of 1 to 1.1, used to grow *E. coli* in shake flasks at volumes up to 1 L. For other tested species, cultures were grown in conversion medium and subcultured once the maximum OD 600 was reached. In all cases, physiological conversion was initiated by adding 100–200 μg / mL carbenicillin (from P212121, product #4800-94-6) and 50–100 μg / mL fosfomycin (from P212121, product #26016-99-9). Most populations were in the conversion state within a few hours. To confirm that the cells had undergone physiological transformation, the cells were imaged on a Nikon Ti-E camera equipped with a perfect focusing system, a Nikon CFI60 Plan Apo 100X NA 1.45 objective lens, Prior automatic filter wheel and stage, LED-CFP / YFP / mCherry and LED-DA / FI / TX filter kit (Semrock), Lumencor Sola II SE LED illumination system and Hamamatsu Flash 4.0V2 CMOS camera.

[0231] Image analysis of physiological transitions:

[0232] ImageJ was used to analyze the image to measure dimensions. In the transformed state, the spherical outline of the outer membrane was treated as a sphere to calculate the total volume (V = (4 / 3)πr³). The cytoplasmic volume was calculated as an ellipsoid existing within the sphere (V = (4 / 3)π * (longest radius) * (shortest radius)²). To calculate the periplasmic volume, the cytoplasmic volume was subtracted from the total cell volume.

[0233] Protein expression and quantification:

[0234] Escherichia coli BL21(DE3) (NEB product #c2527) containing pET28a (emd Millipore product #69864) and its derivatives carrying GFP or collagen derivatives were grown overnight at 37°C in a shaking incubator on conversion medium containing 50 mg / mL kanamycin (p212121, product #2251180). The next day, the overnight culture was diluted 1:10 to fresh conversion medium containing 50 mg / mL kanamycin and subculture was initiated. The cultures were then physiologically converted, and protein production was induced simultaneously at an OD 600 of 1 to 1.1 (read from a Molecular Devices Spectramax M2 microplate reader). Physiological conversion and protein production were induced by the addition of 100 μg / mL carbenicillin, 50 μg / mL fosfomycin, and 100 μg / mL IPTG (p212121, product #367-93-1). In the transition state, protein expression continued for 8 hours to overnight on an orbital shaker at room temperature (approximately 22°C). To quantify total protein levels, a mixture of cultures was prepared using Quick Start. TM The Bradford protein assay was used, and the standard curve was quantified on a Molecular Devices Spectramax M2 microplate reader. To quantify the relative intensity of the target protein production relative to the rest of the protein population, a mixed portion of the culture was... TGX TM Samples were run on the gel and treated with Bio-Safe. TM Coomassie staining.

[0235] Induction of protein production:

[0236] Protein production was induced under physiological conditions following standard procedures. We consistently used the BL21(DE3) strain containing plasmid pET28a, which drives the production of IPTG / lactose-induced recombinant proteins, and targeted these recombinant proteins to the periplasmic space using the DsbA signaling sequence. Using GFP proteins that target the periplasmic space as described above, we have demonstrated the ability to obtain and (compared to) a 5-fold increase in protein yield within the same timeframe (Figure). Induction was optimal at an OD 600 of 1.1, and induction was continued for 10 hours, at which point approximately 200 mg / mL of protein was measured.

[0237] Example 2: Production of full-length collagen

[0238] Full-length jellyfish collagen was produced using the expression system discussed in Example 1 of this document. The wild-type full-length amino acid sequence of Podocoryna carnea (jellyfish or hydra) collagen is provided in SEQ ID NO:1.

[0239]

[0240] / / www.ncbi.nlm.nih.gov / protein / 4379341?report=genbank&log$=protalign&blast_rank=1&RID=T1N9ZEUW014

[0241] The noncodon-optimized polynucleotide sequence encoding full-length jellyfish collagen is disclosed in SEQ ID NO:2.

[0242]

[0243] https: / / www.ncbi.nlm.nih.gov / nucleotide / 3355656?report=genbank&log$=nuclalign&blast_rank=l&RID=TSYP7CMV014

[0244] Two distinct codon-optimized polynucleotide sequences encoding wild-type full-length jellyfish collagen were synthesized. The two polynucleotide sequences differ slightly due to slight differences in codon optimization methods. In addition to the untruncated full-length jellyfish collagen, the polynucleotides also encode a secretion tag, a 9-amino acid histidine tag, a short linker, and a thrombin cleavage site. The DsbA secretion tag is encoded by nucleotides 1-71. The histidine tag containing 9 histidine residues is encoded by nucleotides 73-99, encoding amino acids 25-33. The linker is encoded by nucleotides 100-111. The thrombin cleavage tag is encoded by nucleotides 112-135, encoding amino acids 38-45. The truncated collagen is encoded by nucleotides 136-1422. These two polynucleotides are disclosed in SEQ ID NO:3 and 4 below.

[0245]

[0246]

[0247] The amino acid sequences encoded by the polynucleotides of SEQ ID NO:3 and SEQ ID NO:4 are disclosed in SEQ ID NO:5 below. In SEQ ID NO:5, the DsbA secretion tag is encoded by nucleotides 1-71 and encodes amino acids 1-24; the histidine tag containing 9 histidine residues is encoded by nucleotides 73-99 and encodes amino acids 25-33; the linker is encoded by nucleotides 100-111 and encodes amino acids 34-37; the thrombin cleavage tag is encoded by nucleotides 112-135 and encodes amino acids 38-45; and the full-length collagen is encoded by nucleotides 136-1422 and encodes amino acids 46-474.

[0248]

[0249] Full-length jellyfish collagen without DsbA secretion tag, histidine tag, linker and thrombin cleavage site is disclosed in SEQ ID NO:89.

[0250]

[0251] The polynucleotides in SEQ ID NO:3 and SEQ ID NO:4 were synthesized from Gen9 DNA (now synthesized in-house at Ginkgo Bioworks). The pET28 vector was designed to overlap SEQ ID NO:3 and SEQ ID NO:4 to be 30 to 40 bp in length and added using PCR with the enzyme PrimeStar GXL polymerase (http: / / www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase?sitex=10020:22372:US). The opened pET28a vector and the insert DNA (SEQ ID NO:3 or SEQ ID NO:4) were then assembled together using SGI Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc) to form the final plasmid. The plasmid sequence was then verified using Sanger sequencing from Eurofins Genomics (www.eurofinsgenomics.com).

[0252] Transformed cells were cultured in basal medium and frozen in 1.5 aliquots containing glycerol at a cell-to-glycerol ratio of 50:50. One vial of this frozen culture was incubated overnight in 50 ml of basal medium at 37°C and 200 rpm. Cells were then transferred to 300 ml of basal medium and grown for 6–9 hours to achieve an OD600 of 5–10.

[0253] The basic culture medium used in this embodiment and throughout this application was prepared as follows. The basic culture medium (Table 1) was divided into several portions for autoclaving. These portions consisted of a salt mixture (diammonium hydrogen phosphate, potassium dihydrogen phosphate, anhydrous citric acid, magnesium sulfate heptahydrate), 500 g / L sucrose, 55% glucose, trace metal TM5 (Table 2), and 10 M sodium hydroxide. After autoclaving in a hood, the basic culture medium was mixed together at the above concentrations.

[0254] Table 1. Basic culture medium formulations for shake flask cultures

[0255] chemical substances Chemical formula MW Concentration (g / L) Diammonium hydrogen phosphate <![CDATA[(NH4)2HPO4]]> 133 4 Potassium dihydrogen phosphate <![CDATA[KH2PO4]]> 137 13.3 Anhydrous citric acid <![CDATA[H3C6H5O7]]> 192.14 4.5 Magnesium sulfate heptahydrate <![CDATA[MgSO4.7H2O]]> 246 0.59 Trace Metal TM5 2 glucose <![CDATA[C6H 12 O6]]> 500 40 Sodium hydroxide 10M NaOH 400 5.2 500g / L of sucrose <![CDATA[C 12 H 22 O 11 ]]> 500 66.6

[0256] Table 2. Composition of Trace Metal TM5

[0257] chemical substances Chemical formula MW Conc(g / L) Ferrous sulfate heptahydrate <![CDATA[FeSO4·7H2O]]> 278.02 27.8 Calcium chloride <![CDATA[CaCl2·2H2O]]> 147 2.94 manganese chloride <![CDATA[MnCl2]]> 125.84 1.26 Zinc sulfate <![CDATA[ZnSO4·H2O]]> 179.5 1.8 Nickel chloride <![CDATA[NiCl2·6H2O]]> 237.69 0.48 Sodium molybdate <![CDATA[Na2MoO4·2H2O]]> 241.95 0.48 Sodium selenite <![CDATA[Na2SeO3]]> 172.94 0.35 Boric acid <![CDATA[H3BO3]]> 61.83 0.12

[0258] The harvested cells were homogenized twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen and other proteins.

[0259] Collagen was purified by acid treatment of homogenized cell broth. The pH of the homogenized broth was lowered to 3 using 6M hydrochloric acid. The acidified cell broth was incubated overnight at 4°C with mixing, followed by centrifugation. The supernatant of the acidified broth was tested on a polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitate. The collagen broth thus obtained had a high salt content. To achieve a reduction in volume and salt, a gel with 0.1m... 2 The ultrafiltration cartridges utilize an EMDMillipore tangential flow filtration system for concentration and percolation steps. When two cartridges are used in parallel, the total filtration area is 0.2 m². 2 A 5x volume reduction and a 19x salt reduction were achieved during the TFF stage. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was then dried for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0260] The purified collagen was analyzed on an SDS-PAGE gel, and a thick, clear band was observed at the expected size of 42 kDa. The purified collagen was also analyzed by mass spectrometry, confirming that the 42 kDa protein was jellyfish collagen.

[0261] Fermentation was carried out at various temperatures ranging from 25°C to 28°C. For some fermentations, the fermentation temperature was maintained at a constant temperature, and collagen was purified immediately after fermentation was complete (OD600 of 5-10). For other fermentations, the fermentation temperature was maintained for the desired time, and when the cell density reached an OD600 of 5-10, the temperature was lowered to induce protein production. Typically, the temperature was lowered from 28°C to 25°C. Fermentation was continued at 25°C for 40-60 hours, after which collagen was isolated.

[0262] Other full-length jellyfish collagen

[0263] Full-length jellyfish collagen lacking a His tag, linker, and thrombin cleavage site is disclosed below. The two codon-optimized nucleotide sequences encoding this collagen are provided in SEQ ID NO:6 and SEQ ID NO:7. The difference in nucleotide sequences is due to different codon optimization strategies, but they encode the same protein. The amino acid sequence is disclosed in SEQ ID NO:8. The DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24. The collagen sequence is encoded by nucleotides 73-1359 and encodes amino acids 25-453.

[0264]

[0265]

[0266]

[0267] Example 3: Production of truncated collagen

[0268] A codon-optimized DNA sequence optimized for expression in *E. coli* was synthesized and expressed, encoding a truncated jellyfish collagen with 240 internal amino acids. The DNA sequence is shown in SEQ ID NO:9 below. In SEQ ID NO:9, the DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24 of SEQ ID NO:10. The histidine tag containing 9 histidine residues is encoded by nucleotides 73-99 and encodes amino acids 25-33 of SEQ ID NO:10. The linker is encoded by nucleotides 100-111 and encodes amino acids 34-37 of SEQ ID NO:10. The thrombin cleavage site is encoded by nucleotides 112-135 and encodes amino acids 38-45 of SEQ ID NO:10. The truncated collagen is encoded by nucleotides 136-822 and encodes amino acids 46-274 of SEQ ID NO:10.

[0269]

[0270] The truncated collagen is approximately 54% of the full-length collagen and is disclosed in SEQ ID NO:10 below.

[0271]

[0272] A polynucleotide encoding truncated jellyfish collagen that does not have a DsbA secretion tag, histidine tag, linker, and thrombin cleavage site is disclosed in SEQ ID NO:85.

[0273]

[0274] Truncated jellyfish collagen without DsbA secretion tag, histidine tag, linker and thrombin cleavage site is disclosed in SEQ ID NO:86.

[0275]

[0276] The polynucleotide of SEQ ID NO:9 was codon-optimized and synthesized from Gen9 DNA (now synthesized in-house at Ginkgo Bioworks). The overlap between the pET28 vector and SEQ ID NO:9 was designed to be 30 to 40 bp long and added using PCR with the enzyme PrimeStar GXL polymerase (http: / / www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase?sitex=10020:22372:US). The opened pET28a vector and the insert DNA (SEQ ID NO:9) were then assembled together using SGIGibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc) to form the final plasmid. The plasmid sequence was then verified by Sanger sequencing from Eurofins Genomics (www.eurofinsgenomics.com).

[0277] Transformed cells were cultured in basal medium and frozen in 1.5 aliquots containing glycerol at a cell-to-glycerol ratio of 50:50. One vial of this frozen culture was incubated overnight in 50 ml of basal medium at 37°C and 200 rpm. Cells were then transferred to 300 ml of basal medium and grown for 6–9 hours to achieve an OD600 of 5–10.

[0278] A bioreactor was prepared using 2.7 L of basal medium plus glucose, and 300 mL of culture with an OD600 of 5-10 was added to bring the initial volume to 3 L. Cells were grown at 28 °C, pH 7, and dissolved oxygen maintained at 20% saturation using a cascade containing agitation, air, and oxygen. The pH was controlled using a 28% w / w ammonium hydroxide solution. Once the initial 40 g / L bolus was depleted after approximately 13 hours, fermentation was initiated in fed-batch mode using a dissolved oxygen constant (DO-stat) feeding algorithm. After 24-26 hours of initial growth, the OD600 reached above 100. At this point, 300 mL of 500 g / L sucrose was added, and the temperature was lowered to 25 °C. High-density culture was induced to produce protein using 1 mM IPTG. Fermentation continued for another 20-24 hours, and cells were harvested using a benchtop centrifuge at 9000 rcf and 15 °C for 60 minutes. The cell pellet recovered from centrifugation was resuspended at a 2x buffer weight ratio of 1x cells in a buffer solution containing 0.5M NaCl and 0.1M KH2PO4 at pH 8.

[0279] The harvested cells were homogenized twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen and other proteins.

[0280] Fermentation was carried out at various temperatures ranging from 25°C to 28°C. For some fermentations, the fermentation temperature was maintained at a constant temperature, and collagen was purified immediately after fermentation was complete (OD600 of 5-10). For other fermentations, the fermentation temperature was maintained for the desired time, and when the cell density reached an OD600 of 5-10, the temperature was lowered to induce protein production. Typically, the temperature was lowered from 28°C to 25°C. Fermentation was continued at 25°C for 40-60 hours, after which collagen was isolated.

[0281] Collagen was purified by acid treatment of homogenized cell broth. Additionally, the non-homogenized whole cells recovered from the bioreactor after centrifugation and resuspending in the aforementioned buffer were also acid-treated. The pH of the homogenized slurry of the resuspended whole cells was lowered to 3 using 6M hydrochloric acid. The acidified cell slurry was incubated overnight at 4°C with mixing, followed by centrifugation. The supernatant of the acidified slurry was tested on a polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitate. The resulting collagen slurry had a high salt content. To achieve volume and salt reduction, a gel with 0.1m... 2 The EMD Millipore tangential flow filtration system uses ultrafiltration cartridges for concentration and percolation steps. When two cartridges are used in parallel, the total filtration area is 0.2 m². 2 A 5x volume reduction and a 19x salt reduction were achieved during the TFF stage. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was then dried for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0282] Purified truncated collagen from homogenized cell broth or heterogenized cells was analyzed on SDS-PAGE gel, and a thick, clear band was observed at the expected size of 27 kDa. The purified collagen was also analyzed by mass spectrometry, confirming that the 27 kDa protein was jellyfish collagen.

[0283] The following section provides another method for purifying full-length and truncated collagen.

[0284] The fermented broth was mixed with 0.3–0.5% w / v polyethyleneimine (PEI). After incubation with PEI for 15 minutes, the fermented broth was centrifuged at 9000 rcf for 15 minutes to recover the supernatant, which contained collagen. The cell-containing pellet was discarded, and the PEI-treated collagen-containing supernatant was mixed with sodium bentonite (final w / v 0.2%). Mix with bentonite and centrifuge. Discard the precipitate containing bentonite and recover the supernatant.

[0285] The bentonite-treated supernatant was concentrated 3-6 times using a tangential flow filtration (TFF) system (EMD Millipore) with a 5 kDa cartridge. Collagen was retained with minimal loss in the permeate stream. To remove salt, the residue from the concentration step was permeated using the same TFF apparatus. The final conductivity of the protein solution was <10 mSiemens. Typical conductivity ranges from 400 μSiemens to 1.5 mSiemens. Highly concentrated collagen solutions exhibit even higher conductivity, approaching 4 mSiemens. Those skilled in the art will understand that conductivity above 10 mSiemens can be observed depending on the collagen concentration. Next, the desalted and concentrated protein was treated with activated carbon using WL 9000 10x40 granular resin (Carbon Activated Corporation). 5% w / v of carbon resin was mixed with the collagen-containing protein feed and gently stirred at 45-50°C. With or without filter aids such as diatomaceous earth (Sigma Aldrich), the carbon-treated slurry was filtered using a Buchner funnel lined with Ertel M-953 filter press pads (Ertel Alsop). Following filtration, the collagen solution was passed through a 0.2-micron filter and then filtered with sodium bentonite (final w / v 0.2%). Wyoming Bentonite) treatment for one to several hours followed by centrifugation at 9000 rcf for 15–30 minutes yields a high-purity, clear, and particle-free collagen solution. When endotoxin protein removal is required, the protein is passed through a chromatographic filter such as Sartobind-Q (Sartorius-Stedim) for specific removal of endotoxin proteins.

[0286] The purified collagen was analyzed on an SDS-PAGE gel, and a thick, clear band was observed at 30 kDa. This upward shift in size is attributed to the structure of the collagen molecule and its high glycine / proline amino acid content. The purified collagen was also analyzed by mass spectrometry, confirming that the 30 kDa protein was a truncated collagen.

[0287] The truncated collagen was further analyzed by HPLC using an Agilent 1100 series HPLC system. The column was a 50 mm Agilent PLRP-S reversed-phase column with an inner diameter of 4.6 mm, a particle size of μM, and a pore size of 1000 Å.

[0288] Samples were prepared by diluting 1:1 in 0.04% sodium azide solution in HPLC-grade water. After dilution, the resulting mixture was filtered through a 0.45 μm filter to remove any large particles that might clog the HPLC column. For analysis, the sample was appropriately diluted with 20 mM ammonium acetate buffer in HPLC-grade water at approximately pH 4.5. After mixing, the sample was transferred to a 300 μl microvolume vial and then placed in an autosampler. Analytical parameters such as sample flow rate, column temperature, mobile phase flow rate, and mobile phase composition could be changed using the HPLC software ChemStation. In an exemplary but non-limiting analysis, the parameters were: sample flow rate of 1 mL / min, column temperature of 80 °C, column pressure of 60–70 bar, mobile phase composition of 97.9% water / 1.9% acetonitrile and 0.2% trifluoroacetic acid; UV wavelength for analysis of 214.4 nm; injection volume of 10 μL; and sample run time of 10 min.

[0289] Under these conditions, the truncated jellyfish collagen of SEQ ID NO:91 had an elution time of approximately 5.4 minutes. ChemStation quantified the peak area of ​​the elution peak and calculated the protein concentration using a calibration curve that directly correlates the peak area with the protein concentration. The calibration curve was generated using a known collagen solution that was serially diluted to contain collagen concentrations ranging from 0.06 mg / mL to 1.00 mg / mL.

[0290] Truncated collagen lacking His-tag-connector-thrombin cleavage site

[0291] A truncated jellyfish collagen lacking a His tag, linker, and thrombin cleavage site is disclosed below. The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:11. The amino acid sequence is disclosed in SEQ ID NO:12. The DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24. The truncated collagen sequence is encoded by nucleotides 73-639 and encodes amino acids 25-213.

[0292]

[0293] A polynucleotide encoding truncated jellyfish collagen without a His tag, linker, and thrombin cleavage site is disclosed in SEQ ID NO:90.

[0294]

[0295] Truncated jellyfish collagen without His tag, connector and thrombin cleavage site is disclosed in SEQ ID NO:91.

[0296]

[0297] truncated collagen with GEK repeats

[0298] Jellyfish collagen with GEK repeats is disclosed below. The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:13. The amino acid sequence is disclosed in SEQ ID NO:14. The DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24. The GEK repeat is encoded by nucleotides 73-126 and encodes the GEK repeat of amino acids 25-42. The truncated collagen sequence is encoded by nucleotides 127-693 and encodes amino acids 43-231.

[0299]

[0300] The polynucleotide of SEQ ID NO:13 was codon-optimized and synthesized from Gen9 DNA (now synthesized in-house by GinkgoBioworks). The overlap between the pET28 vector and SEQ ID NO:13 was designed to be 30 to 40 bp long and added using PCR with the enzyme PrimeStar GXL polymerase (http: / / www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase?sitex=10020:22372:US). The opened pET28a vector and the insert DNA (SEQ ID NO:13) were then assembled together using SGIGibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc) to form the final plasmid. The plasmid sequence was then verified using Sanger sequencing from Eurofins Genomics (www.eurofinsgenomics.com).

[0301] Transformed cells were cultured in basal medium and frozen in 1.5 aliquots containing glycerol at a cell-to-glycerol ratio of 50:50. One vial of this frozen culture was incubated overnight in 50 ml of basal medium at 37°C and 200 rpm. Cells were then transferred to 300 ml of basal medium and grown for 6–9 hours to achieve an OD600 of 5–10.

[0302] A bioreactor was prepared using 2.7 L of basal medium plus glucose, and 300 mL of culture with an OD600 of 5-10 was added to bring the initial volume to 3 L. Cells were grown at 28°C, pH 7, and dissolved oxygen maintained at 20% saturation using a cascade containing agitation, air, and oxygen. The pH was controlled using a 28% w / w ammonium hydroxide solution. Once the initial clump of 40 g / L was depleted after approximately 13 hours, fermentation was initiated in fed-batch mode using a constant dissolved oxygen feeding algorithm. After 24-26 hours of initial growth, the OD600 reached above 100. At this point, 300 mL of 500 g / L sucrose was added, and the temperature was lowered to 25°C. High-density culture was induced to produce protein using 1 mM IPTG. Fermentation continued for another 20-24 hours, and cells were harvested using a benchtop centrifuge at 9000 rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended at a 2x buffer weight ratio of 1x cells in a buffer solution containing 0.5M NaCl and 0.1M KH2PO4 at pH 8.

[0303] The harvested cells were homogenized twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen and other proteins.

[0304] As described above, collagen was purified by acidifying whole cells recovered from the bioreactor after centrifugation and resuspending in buffer. The pH of the homogenized slurry or resuspended suspension was lowered to 3 using 6M hydrochloric acid. The acidified cell slurry was incubated overnight at 4°C with mixing, followed by centrifugation. The supernatant of the acidified slurry was tested on a polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitate. The collagen slurry thus obtained had a high salt content. To achieve a reduction in volume and salt, a gel with 0.1m³ of each was used. 2 The EMD Millipore tangential flow filtration system uses ultrafiltration cartridges for concentration and percolation steps. When two cartridges are used in parallel, the total filtration area is 0.2 m². 2 A 5x volume reduction and a 19x salt reduction were achieved during the TFF stage. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was then dried for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0305] Purified collagen was analyzed on an SDS-PAGE gel and observed to have an apparent molecular weight of 35 kDa. The 35 kDa band did not conform to the expected size of 22 kDa. This upward shift in the apparent size was attributed to the interaction of the GEK repeat with the gel matrix. Mass spectrometry confirmed that the 35 kDa band was indeed collagen with GEK repeats.

[0306] Truncated collagen with GDK repeats

[0307] A jellyfish collagen with GDK repeats is disclosed below. The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:15. The amino acid sequence is disclosed in SEQ ID NO:16. The DsbA secretion tag is encoded by nucleotides 1-72, encoding amino acids 1-24. The GDK repeat is encoded by nucleotides 73-126, encoding the GDK repeat of amino acids 25-42. The truncated collagen sequence is encoded by nucleotides 127-693, encoding amino acids 43-231.

[0308]

[0309]

[0310] The polynucleotide of SEQ ID NO:15 was codon-optimized and synthesized from Gen9 DNA (now synthesized in-house at GinkgoBioworks). The overlap between the pET28 vector and SEQ ID NO:15 was designed to be 30 to 40 bp long and added using PCR with the enzyme PrimeStar GXL polymerase (http: / / www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase?sitex=10020:22372:US). The opened pET28a vector and the insert DNA (SEQ ID NO:15) were then assembled together using SGIGibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc) to form the final plasmid. The plasmid sequence was then verified using Sanger sequencing from Eurofins Genomics (www.eurofinsgenomics.com).

[0311] Transformed cells were cultured in basal medium and frozen in 1.5 aliquots containing glycerol at a cell-to-glycerol ratio of 50:50. One vial of this frozen culture was incubated overnight in 50 ml of basal medium at 37°C and 200 rpm. Cells were then transferred to 300 ml of basal medium and grown for 6–9 hours to achieve an OD600 of 5–10.

[0312] A bioreactor was prepared using 2.7 L of basal medium plus glucose, and 300 mL of culture with an OD600 of 5-10 was added to bring the initial volume to 3 L. Cells were grown at 28°C, pH 7, and dissolved oxygen maintained at 20% saturation using a cascade containing agitation, air, and oxygen. The pH was controlled using a 28% w / w ammonium hydroxide solution. Once the initial clump of 40 g / L was depleted after approximately 13 hours, fermentation was initiated in fed-batch mode using a constant dissolved oxygen feeding algorithm. After 24-26 hours of initial growth, the OD600 reached above 100. At this point, 300 mL of 500 g / L sucrose was added, and the temperature was lowered to 25°C. High-density culture was induced to produce protein using 1 mM IPTG. Fermentation continued for another 20-24 hours, and cells were harvested using a benchtop centrifuge at 9000 rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended at a 2x buffer weight ratio of 1x cells in a buffer solution containing 0.5M NaCl and 0.1M KH2PO4 at pH 8.

[0313] The harvested cells were homogenized twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen and other proteins.

[0314] As described above, collagen was purified by acidifying whole cells recovered from the bioreactor after centrifugation and resuspending in buffer. The pH of the homogenized slurry was lowered to 3 using 6M hydrochloric acid. The acidified cell slurry was incubated overnight at 4°C with mixing, followed by centrifugation. The supernatant of the acidified slurry was tested on a polyacrylamide gel and found to contain a relatively high abundance of collagen compared to the initial precipitate. The collagen slurry thus obtained had a high salt content. To achieve a reduction in volume and salt, a gel with 0.1m³ of each was used. 2 The EMD Millipore tangential flow filtration system uses ultrafiltration cartridges for concentration and percolation steps. When two cartridges are used in parallel, the total filtration area is 0.2 m². 2 A 5x volume reduction and a 19x salt reduction were achieved during the TFF stage. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was then dried for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0315] Purified collagen was analyzed on an SDS-PAGE gel and observed to have an apparent molecular weight of 35 kDa. The 35 kDa band did not conform to the expected size of 22 kDa. This upward shift in the apparent size was attributed to the interaction of GDK repeats with the gel matrix. Mass spectrometry confirmed that the 35 kDa band was indeed collagen with GDK repeats.

[0316] Truncated collagen with DsbA secretion tag-His tag-connector-thrombin cleavage site and GFP β-lactamase fusion (version 1):

[0317] A jellyfish collagen fusion of a DsbA secretion tag-His tag-connector-thrombin cleavage site and a GFP β-lactamase is disclosed below. The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:17. The amino acid sequence is disclosed in SEQ ID NO:18. The DsbA secretion tag is encoded by nucleotides 1-72, encoding amino acids 1-24. The His tag is encoded by nucleotides 73-99, encoding a 9-histidine tag of amino acids 25-33. The connector is encoded by nucleotides 100-111, encoding amino acids 34-37. The thrombin cleavage site is encoded by nucleotides 112-135, encoding amino acids 38-45. The green fluorescent protein (GFP) with the connector is encoded by nucleotides 136-873, encoding amino acids 46-291. The truncated collagen sequence is encoded by nucleotides 874-1440, encoding amino acids 292-480. The β-lactamase with the linker is encoded by nucleotides 1441-2232 and amino acids 481-744. Even without an independent secretion tag, the β-lactamase correctly targets the periplasmic space. The DsbA secretion tag directs the entire transcript (a truncated collagen protein with a DsbA secretion tag-His tag-linker-thrombin cleavage site and a GFP β-lactamase fusion protein) into the periplasmic space, and the β-lactamase functions normally.

[0318]

[0319]

[0320] The polynucleotide SEQ ID NO:17 was constructed by assembling several DNA fragments. The sequence containing collagen was codon-optimized and synthesized from Gen9 DNA (now synthesized in-house by Ginkgo Bioworks). The GFP was also synthesized from Gen9. The β-lactamase was cloned from plasmid pKD46 (http: / / cgsc2.biology.yale.edu / Strain.php7ID=68099) using PCR with the enzyme PrimeStar GXL polymerase (http: / / www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase?sitex=10020:22372:US). The overlap between the pET28 vector, GFP, collagen, and β-lactamase was designed to be 30 to 40 bp long and added using PCR with the enzyme PrimeStar GXL polymerase. The opened pET28a vector and the insert fragment were then assembled using SGI Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc) to form the final plasmid. The plasmid sequence was then verified using Sanger sequencing from Eurofins Genomics (www.eurofinsgenomics.com).

[0321] Transformed cells were cultured in basal medium and frozen in 1.5 aliquots containing glycerol at a cell-to-glycerol ratio of 50:50. One vial of this frozen culture was incubated overnight in 50 ml of basal medium at 37°C and 200 rpm. Cells were then transferred to 300 ml of basal medium and grown for 6–9 hours to achieve an OD600 of 5–10.

[0322] A bioreactor was prepared using 2.7 L of basal medium plus glucose, and 300 mL of culture with an OD600 of 5-10 was added to bring the initial volume to 3 L. Cells were grown at 28°C, pH 7, and dissolved oxygen maintained at 20% saturation using a cascade containing agitation, air, and oxygen. The pH was controlled using a 28% w / w ammonium hydroxide solution. Once the initial clump of 40 g / L was depleted after approximately 13 hours, fermentation was initiated in fed-batch mode using a constant dissolved oxygen feeding algorithm. After 24-26 hours of initial growth, the OD600 reached above 100. At this point, 300 mL of 500 g / L sucrose was added, and the temperature was lowered to 25°C. High-density culture was induced to produce protein using 1 mM IPTG. Fermentation continued for another 20-24 hours, and cells were harvested using a benchtop centrifuge at 9000 rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended at a 2x buffer weight ratio of 1x cells in a buffer solution containing 0.5M NaCl and 0.1M KH2PO4 at pH 8.

[0323] The harvested cells were homogenized twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen and other proteins.

[0324] Collagen was purified by acid treatment of homogenized whole cells recovered from the bioreactor after centrifugation and resuspending in the aforementioned buffer. The pH of the resuspended suspension was lowered to 3 using 6M hydrochloric acid. The acidified cell plasma was incubated overnight at 4°C with mixing, followed by centrifugation. The pH was then raised to 9 using 10N NaOH, and the supernatant of the plasma was tested on a polyacrylamide gel, revealing a relatively high abundance of collagen compared to the initial precipitate. The resulting collagen plasma had a high salt content. To achieve a reduction in volume and salt, a 0.1m... 2 The ultrafiltration cartridges utilize an EMDMillipore tangential flow filtration system for concentration and percolation steps. When two cartridges are used in parallel, the total filtration area is 0.2 m². 2 A 5x volume reduction and a 19x salt reduction were achieved during the TFF stage. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. This slurry was dried for 3 days using a multi-tray freeze dryer to obtain a white, fluffy collagen powder.

[0325] The purified collagen-GFP-β-lactamase fusion protein was analyzed on an SDS-PAGE gel and observed to have an apparent molecular weight of 90 kDa. The expected size of the fusion protein was 85 kDa. Mass spectrometry confirmed that the 90 kDa band was indeed the collagen fusion protein.

[0326] Truncated collagen with DsbA secretion tag-His tag-connector-thrombin cleavage site and GFP β-lactamase fusion (version 2):

[0327] A jellyfish collagen fusion of a DsbA secretion tag-His tag-connector-thrombin cleavage site and a GFP β-lactamase is disclosed below. The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:19. The amino acid sequence is disclosed in SEQ ID NO:20. The DsbA secretion tag is encoded by nucleotides 1-72, encoding amino acids 1-24. The His tag is encoded by nucleotides 73-99, encoding a 9-histidine tag of amino acids 25-33. The connector is encoded by nucleotides 100-111, encoding amino acids 34-37. The thrombin cleavage site is encoded by nucleotides 112-135, encoding amino acids 38-45. The green fluorescent protein (GFP) with the connector is encoded by nucleotides 136-873, encoding amino acids 46-291. The truncated collagen sequence is encoded by nucleotides 874-1440, encoding amino acids 292-480. The β-lactamase with the linker is encoded by nucleotides 1441-2232 and amino acids 481-744.

[0328]

[0329]

[0330] Example 4: Production of full-length elastin

[0331] The full-length human elastin is expressed as described below. The wild-type full-length amino acid sequence of human elastin is provided below.

[0332]

[0333] http: / / www.uniprot.org / uniprot / P15502

[0334] The non-codon-optimized polynucleotide sequence encoding full-length elastin is disclosed below. In SEQ ID NO:22, nucleotides 1-78 encode the DsbA secretion tag, and nucleotides 79-2358 encode full-length human elastin.

[0335]

[0336] Elastin with codon-optimized DsbA secretion tag-His tag-connector-thrombin cleavage site

[0337] A codon-optimized polynucleotide sequence encoding a full-length human elastin protein having a DsbA secretion tag-His tag-connector-thrombin cleavage site is disclosed below. In SEQ ID NO:23: nucleotides 1-72 encode a DsbA secretion tag, which encodes amino acids 1-24 of SEQ ID NO:24; nucleotides 73-99 encode a 9His tag, which...

[0338] His tag encodes amino acids 25-33 of SEQ ID NO:24; nucleotides 100-111 encode a linker that encodes amino acids 34-37 of SEQ ID NO:24; nucleotides 112-135 encode a thrombin cleavage site that encodes amino acids 38-45 of SEQ ID NO:24; and nucleotides 136-2415 encode amino acids 46-805 of the full-length human elastin of SEQ ID NO:24.

[0339]

[0340]

[0341] A polynucleotide encoding a full-length human elastin without a natural sequence tag is disclosed in SEQ ID NO:87.

[0342]

[0343] The full-length human elastin sequence without a natural sequence tag is disclosed in SEQ ID NO:88.

[0344]

[0345] Codon-optimized elastin with DsbA secretion tag

[0346] A codon-optimized polynucleotide sequence encoding the full-length human elastin with a DsbA secretion tag is disclosed in SEQ ID NO:25. In SEQ ID NO:25: nucleotides 1-72 encode the DsbA secretion tag, which encodes amino acids 1-24 of SEQ ID NO:26; nucleotides 73-2355 encode amino acids 25-785 of the full-length human elastin of SEQ ID NO:26.

[0347]

[0348]

[0349] The polynucleotide of SEQ ID NO:22 was codon-optimized and synthesized from Gen9 DNA (now synthesized in-house by GinkgoBioworks). The overlap between the pET28 vector and SEQ ID NO:22 was designed to be 30 to 40 bp long and added using PCR with the enzyme PrimeStar GXL polymerase (http: / / www.clontech.com / US / Products / PCR / GC_Rich / PrimeSTAR_GXL_DNA_Polymerase?sitex=10020:22372:US). The opened pET28a vector and the insert DNA (SEQ ID NO:22) were then assembled together using SGIGibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc) to form the final plasmid. The plasmid sequence was then verified using Sanger sequencing from Eurofins Genomics (www.eurofinsgenomics.com).

[0350] Transformed cells were cultured in basal medium and frozen in 1.5 aliquots containing glycerol at a cell-to-glycerol ratio of 50:50. One vial of this frozen culture was incubated overnight in 50 ml of basal medium at 37°C and 200 rpm. Cells were then transferred to 300 ml of basal medium and grown for 6–9 hours to achieve an OD600 of 5–10.

[0351] A bioreactor was prepared using 2.7 L of basal medium plus glucose, and 300 mL of culture with an OD600 of 5-10 was added to bring the initial volume to 3 L. Cells were grown at 28°C, pH 7, and dissolved oxygen maintained at 20% saturation using a cascade containing agitation, air, and oxygen. The pH was controlled using a 28% w / w ammonium hydroxide solution. Once the initial clump of 40 g / L was depleted after approximately 13 hours, fermentation was initiated in fed-batch mode using a constant dissolved oxygen feeding algorithm. After 24-26 hours of initial growth, the OD600 reached above 100. At this point, 300 mL of 500 g / L sucrose was added, and the temperature was lowered to 25°C. High-density culture was induced to produce protein using 1 mM IPTG. Fermentation continued for another 20-24 hours, and cells were harvested using a benchtop centrifuge at 9000 rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended at a 2x buffer weight ratio of 1x cells in a buffer solution containing 0.5M NaCl and 0.1M KH2PO4 at pH 8.

[0352] Fermentation was carried out at various temperatures ranging from 25°C to 28°C. For some fermentations, the fermentation temperature was maintained at a constant temperature, and elastin was purified immediately after fermentation was complete (OD600 of 5–10). For other fermentations, the fermentation temperature was maintained for the desired time, and when the cell density reached an OD600 of 5–10, the temperature was lowered to induce protein production. Typically, the temperature was lowered from 28°C to 25°C. Fermentation was continued at 25°C for 40–60 hours, after which elastin was isolated.

[0353] The harvested cells were homogenized twice in a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen and other proteins.

[0354] The supernatant from homogenized cells was analyzed on an SDS-PAGE gel, and a clear band corresponding to the expected size of 68 kDa was observed at approximately 70 kDa. The purified elastin was analyzed by mass spectrometry.

[0355] A full-length elastin protein with a DsbA secretion tag-His tag-connector-thrombin cleavage site and a GFP β-lactamase fusion.

[0356] A human elastin with a DsbA secretion tag-His tag-connector-thrombin cleavage site and a GFP β-lactamase fusion is disclosed below. The codon-optimized nucleotide sequence encoding this elastin is provided in SEQ ID NO:27. The amino acid sequence is disclosed in SEQ ID NO:28. The DsbA secretion tag is encoded by nucleotides 1-72, encoding amino acids 1-24. The His tag is encoded by nucleotides 73-99, encoding a 9-histidine tag of amino acids 25-33. The connector is encoded by nucleotides 100-111, encoding amino acids 34-37. The thrombin cleavage site is encoded by nucleotides 112-135, encoding amino acids 38-45. The green fluorescent protein (GFP) with the connector is encoded by nucleotides 136-873, encoding amino acids 46-291. The full-length elastin sequence is encoded by nucleotides 874-3153, encoding amino acids 292-1051. The β-lactamase with the linker is encoded by nucleotides 3154-3945 and amino acids 1052-1315.

[0357]

[0358]

[0359] Example 5: Production of truncated elastin

[0360] The truncated human elastin was generated using the expression system described in Example 4. The full-length amino acid sequence lacking the natural secretion tag is disclosed in SEQ ID NO:29.

[0361]

[0362] A codon-optimized polynucleotide sequence encoding full-length human elastin lacking a natural secretion tag is disclosed in SEQ ID NO:30.

[0363]

[0364] The amino acid sequence of a 60.7 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:31. The 60.7 kDa truncated elastin has amino acids 706-761 deleted from the full-length elastin.

[0365]

[0366] The codon-optimized polynucleotide sequence encoding a truncated 60.7 kDa human elastin is disclosed in SEQ ID NO:32.

[0367]

[0368] The amino acid sequence of a 58.8 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:33. The 58.8 kDa truncated elastin has amino acids 2-85 deleted from the full-length elastin.

[0369]

[0370] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 58.8 kDa is disclosed in SEQ ID NO:34.

[0371]

[0372] The amino acid sequence of a 57 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:35. The 57 kDa truncated elastin has amino acids 661-761 deleted from the full-length elastin.

[0373]

[0374] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 57 kDa is disclosed in SEQ ID NO:36.

[0375]

[0376] The amino acid sequence of a 53.9 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:37. The 53.9 kDa truncated elastin has amino acids 624-761 deleted from the full-length elastin.

[0377]

[0378] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 53.9 kDa is disclosed in SEQ ID NO:38.

[0379]

[0380] The amino acid sequence of a 45.3 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:39. The 45.3 kDa truncated elastin has amino acids 529-761 deleted from the full-length elastin.

[0381]

[0382] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 45.3 kDa is disclosed in SEQ ID NO:40.

[0383]

[0384] The amino acid sequence of a 44.4 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:41. The 44.4 kDa truncated elastin has amino acids 2-246 deleted from the full-length elastin.

[0385]

[0386] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 44.4 kDa is disclosed in SEQ ID NO:42.

[0387]

[0388] The amino acid sequence of a 40.4 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:43. The 40.4 kDa truncated elastin has amino acids 2-295 deleted from the full-length elastin.

[0389]

[0390] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 40.4 kDa is disclosed in SEQ ID NO:44.

[0391]

[0392] The amino acid sequence of a 39.8 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:45. The 39.8 kDa truncated elastin has amino acids 462-761 deleted from the full-length elastin.

[0393]

[0394] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 39.8 kDa is disclosed in SEQ ID NO:46.

[0395]

[0396] The amino acid sequence of a 36.1 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:47. The 36.1 kDa truncated elastin has amino acids 418-761 deleted from the full-length elastin.

[0397]

[0398] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 36.1 kDa is disclosed in SEQ ID NO:48.

[0399]

[0400] The amino acid sequence of a 34.9 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:49. The 34.9 kDa truncated elastin has amino acids 2-360 deleted from the full-length elastin.

[0401]

[0402] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 34.9 kDa is disclosed in SEQ ID NO:50.

[0403]

[0404] The amino acid sequence of a 32kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:51. The 32kDa truncated elastin has amino acids 373-761 deleted from the full-length elastin.

[0405]

[0406] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 32 kDa is disclosed in SEQ ID NO:52.

[0407]

[0408] The amino acid sequence of a 29.9 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:53. The 60.7 kDa truncated elastin has amino acids 347-761 deleted from the full-length elastin.

[0409]

[0410] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 29.9 kDa is disclosed in SEQ ID NO:54.

[0411]

[0412] The amino acid sequence of a 29.4 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:55. The 29.4 kDa truncated elastin has amino acids 2-425 deleted from the full-length elastin.

[0413]

[0414] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 29.4 kDa is disclosed in SEQ ID NO:56.

[0415]

[0416] The amino acid sequence of a 25.3 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:57. The 25.3 kDa truncated elastin has amino acids 2-473 deleted from the full-length elastin.

[0417]

[0418] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 25.3 kDa is disclosed in SEQ ID NO:58.

[0419]

[0420] The amino acid sequence of a 24.1 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:59. The 24.1 kDa truncated elastin has amino acids 277-761 deleted from the full-length elastin.

[0421]

[0422] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 24.1 kDa is disclosed in SEQ ID NO:60.

[0423]

[0424] The amino acid sequence of a 20.3 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO:61. The 20.3 kDa truncated elastin has amino acids 229-761 deleted from the full-length elastin.

[0425]

[0426] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 20.3 kDa is disclosed in SEQ ID NO:62.

[0427]

[0428] The amino acid sequence of a 19.6 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:63. The 19.6 kDa truncated elastin has amino acids 2-542 deleted from the full-length elastin.

[0429]

[0430] A codon-optimized polynucleotide sequence encoding a 19.6 kDa truncated human elastin is disclosed in SEQ ID NO:64.

[0431]

[0432] The amino acid sequence of an 11 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:65. The 11 kDa truncated elastin has amino acids 2-635 deleted from the full-length elastin.

[0433]

[0434] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 11 kDa is disclosed in SEQ ID NO:66.

[0435]

[0436] The amino acid sequence of a 7.9 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:67. The 7.9 kDa truncated elastin has amino acids 2-674 deleted from the full-length elastin.

[0437]

[0438] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 7.9 kDa is disclosed in SEQ ID NO:68.

[0439]

[0440] The amino acid sequence of a 6.3 kDa human elastin truncated at the C-terminus is disclosed in SEQ ID NO: 69. The 6.3 kDa truncated elastin has amino acids 74-761 deleted from the full-length elastin.

[0441]

[0442] A codon-optimized polynucleotide sequence encoding a truncated human elastin protein of 6.3 kDa is disclosed in SEQ ID NO:70:

[0443]

[0444] The amino acid sequence of a 4.3 kDa human elastin truncated at the N-terminus is disclosed in SEQ ID NO:71. The 4.3 kDa truncated elastin has amino acids 2-717 deleted from the full-length elastin.

[0445]

[0446] A codon-optimized polynucleotide sequence encoding a truncated human elastin of 4.3 kDa is disclosed in SEQ ID NO:72.

[0447]

[0448] Truncated human elastin 1 with DsbA secretion and FLAG tagging

[0449] The amino acid sequence of a truncated human elastin 1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:98. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:99, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:98. The elastin nucleotide sequence is nucleotides 58-657 of SEQ ID NO:99, and the amino acid sequence is amino acids 20-219 of SEQ ID NO:98. The FLAG nucleotide sequence is nucleotides 658-684 of SEQ ID NO:99, and the amino acid sequence is amino acids 220-228 of SEQ ID NO:98.

[0450]

[0451] The nucleic acid sequence of truncated human elastin 1 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:99.

[0452]

[0453] As described herein, the polynucleotide of SEQ ID NO:99 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated elastin was purified. The purified elastin produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 25 kDa (Western). In the absence of this protein expression, no band appeared at this location on the gel.

[0454] Truncated human elastin 2 with DsbA secretion and FLAG tagging

[0455] The amino acid sequence of a truncated human elastin type 2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:100. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:101, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:100. The elastin nucleotide sequence is nucleotides 58-657 of SEQ ID NO:101, and the amino acid sequence is amino acids 20-219 of SEQ ID NO:100. The FLAG nucleotide sequence is nucleotides 658-684 of SEQ ID NO:101, and the amino acid sequence is amino acids 220-228 of SEQ ID NO:100.

[0456]

[0457] The nucleic acid sequence of truncated human elastin 2 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:101.

[0458]

[0459] As described herein, the polynucleotide of SEQ ID NO:101 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated elastin was purified. The purified elastin produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 25 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0460] Example 6: Effects of truncated collagen on fibroblast cell viability, procollagen synthesis, and elastin synthesis.

[0461] Human fibroblast cell cultures were used to evaluate the truncated jellyfish collagen molecules of Example 2 to determine their role in collagen and elastin synthesis. Human fibroblast cell cultures were also used to determine the viability of human fibroblasts after exposure to the truncated jellyfish collagen.

[0462] A stock solution of 2% w / w truncated collagen was prepared from the histidine-labeled truncated collagen of Example 3. Aliquots from the 2% truncated collagen stock solution were then used in the experiments described below.

[0463] Preparation of fibroblasts

[0464] Fibroblasts were seeded into each well of a 24-well plate with 0.5 ml of fibroblast growth medium (FGM) 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 0.5 ml of fresh FGM. Cells were allowed to grow until confluence, with the medium changed every 48 to 72 hours. After confluence, cells were treated with DMEM supplemented with 1.5% FBS for 24 hours to eliminate any effects from growth factors present in the normal medium. After the 24-hour cleansing period, cells were treated with truncated jellyfish collagen dissolved in FGM containing 1.5% FBS at a specified concentration. Transforming growth factor β (TGF-β) (20 ng / ml) was used as a positive control for collagen and elastin synthesis. Untreated cells (negative control) received only DMEM containing 1.5% FBS. Cells were incubated for 48 hours, and at the end of the incubation period, the cell culture medium was collected and frozen (-75 °C) or immediately analyzed. Three copies of the materials were used for testing.

[0465] MTT assay

[0466] MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazolium) 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, reduction of MTT by mitochondria in living cells results in the formation of insoluble purple formazin crystals. These crystals are then extracted from the cells with isopropanol and quantified spectrophotometrically. Lifeless cells cannot reduce MTT and therefore do not produce purple formazin crystals. The intensity of the purple color is directly proportional to the number of living cells (metabolic 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.

[0467] Following the 2-day incubation period discussed above, the cell culture medium was removed (see above), and the fibroblasts were washed twice with PBS to remove any residual jellyfish glycogen molecules. After the final wash, 500 μl of DMEM supplemented with 0.5 mg / ml MTT was added to each well, and the cells were incubated at 37 ± 2 °C and 5 ± 1% CO2 for 1 hour. After incubation, the DMEM / MTT solution was removed, and the cells were washed once more with PBS. Then, 0.5 ml of isopropanol was added to the wells to extract purple formazan crystals. 200 μL of the isopropanol extract was transferred to a 96-well plate, and the plate was read at 540 nm using isopropanol as a blank.

[0468] The mean MTT absorbance value of the negative control cells was calculated and used to represent 100% cell viability. Then, the individual MTT absorbance values ​​from cells that underwent various treatments were 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.

[0469] In Tables 1, 2, and 3 of this embodiment, experiments were conducted using designated aliquots of 2% truncated collagen stock solution in the assay. For example, in the sample testing “10% collagen solution,” an aliquot of 2% truncated collagen sufficient to provide 10% of the assay volume was used. For a total assay volume of 1.0 ml, 100 μl of 2% truncated collagen stock solution was used. In Tables 1, 2, and 3, "10% collagen solution" is 0.2% collagen, "5% collagen solution" is 0.1% collagen, "1% collagen solution" is 0.02% collagen, "0.5% collagen solution" is 0.01% collagen, "0.1% collagen solution" is 0.002% collagen, "0.05% collagen solution" is 0.001% collagen, "0.01% collagen solution" is 0.0002% collagen, and "0.005% collagen solution" is 0.0001% collagen.

[0470] The results of the MTT assay are shown in Table 3. These values ​​are expressed as mean viability percentage ± deviation from the mean.

[0471] Table 3. MTT Measurement

[0472]

[0473]

[0474] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0475] Histidine-tagged truncated jellyfish collagen exhibits a protective effect by increasing the cell viability of human fibroblasts. As shown in Table 3, the highest values ​​were observed in the MTT assay when fibroblasts were exposed to 0.02% to 0.2% truncated jellyfish collagen.

[0476] Pre-collagen synthesis

[0477] Fibroblasts are the primary 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 a precursor to collagen. When the peptide is processed to form mature collagen, the procollagen fraction is cleaved (type I C peptide). Both the mature collagen and the type I C peptide fragment are then released into the extracellular environment. During collagen synthesis, the type I C peptide fragment accumulates in the tissue culture medium. Because the stoichiometry between the two parts of the procollagen peptide is 1:1, measuring type I C peptide will reflect the amount of collagen synthesized. Type I C peptide can be measured using an ELISA-based method.

[0478] A range of type I C peptide standards ranging from 0 ng / ml to 640 ng / ml were prepared. Next, ELISA microplates were prepared by removing any unwanted strips from the plate frame and adding 100 μl of peroxidase-labeled anti-procollagen type I C peptide proantibody to each well used for assay. Twenty (20) μl of sample (collected tissue culture medium) or standard was then added to the appropriate wells, the microplates were capped, and incubated at 37°C for 3 ± 0.25 h. After incubation, the wells were aspirated and washed three times with 400 μl of wash buffer. After removing the final wash buffer, 100 μl of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as chromogen) was added to each well, and the plate was incubated at room temperature for 15 ± 5 min. After incubation, 100 μl of stop solution (1N sulfuric acid) was added to each well, and the plate was read at 450 nm using a microplate reader.

[0479] To quantify the amount of each substance present, a standard curve was generated using the known concentration 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.

[0480] The results of the ELISA assay are shown in Table 4.

[0481] Table 4. Type I collagen assay. Values ​​shown are average concentration (ng / ml) ± deviation from the average.

[0482] Handling method Type I C peptide (ng / ml) Unprocessed 1718±94 20ng / ml TGF-B 3028±332* 10% Collagen Solution 1940±100 5% Collagen Solution 2394±125* 1% Collagen Solution 1773±183 0.5% collagen solution 1127±19* 0.1% collagen solution 1158±10* 0.05% collagen solution 1416±64 0.01% collagen solution 1835±404 0.005% collagen solution 1551±149

[0483] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0484] A biphasic effect of truncated histidine-labeled jellyfish collagen on collagen synthesis was observed. Collagen synthesis increased at 1%, 5%, and 10% levels. At a concentration of 5%, truncated jellyfish collagen significantly increased collagen synthesis (p < 0.05).

[0485] elastin synthesis

[0486] Elastin is a major component of the elastic fiber network that gives tissues the ability to rebound after brief stretching. This protein is released into the extracellular space by fibroblasts (soluble elastin), which then cross-links with other elastins in the extracellular space to form an extensive network of fibers and sheets (insoluble elastin). Soluble elastin can be readily measured from cell culture medium using an ELISA-based method.

[0487] 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 applied 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 saturated with PBS containing 0.25% BSA and 0.05% Tween 20. The plate was then incubated with this blocking solution at 37°C for 1 hour, followed by washing twice with PBS containing 0.05% Tween 20.

[0488] A series of α-elastin standards ranging from 0 to 100 ng / ml were generated. 180 μl of the standards or truncated jellyfish collagen were then transferred to 650 μl microcentrifuge tubes. An anti-elastin antibody solution was prepared (the antibody was diluted 1:100 in PBS containing 0.25% BSA and 0.05% Tween 20), and 20 μl of this solution was added to the tube. The tube was then incubated overnight at 4 ± 2 °C. On the second day, 150 μl was transferred from each tube to a 96-well elastin ELISA plate, and the plate was incubated at room temperature for 1 hour. The plate was then washed three times with PBS containing 0.05% Tween 20. After washing, 200 μl of a solution containing a peroxidase-conjugated secondary antibody diluted in PBS containing 0.25% BSA and 0.05% Tween 20 was added, and the plate was incubated at room temperature for 1 hour. After washing the plate three times, add 200 μl of substrate solution and incubate the plate in the dark at room temperature for 10 to 30 minutes. After this final incubation, read the plate at 460 nm using a microplate reader.

[0489] Table 5. These values ​​are also expressed as average concentration (ng / ml) ± deviation from the average.

[0490] Handling method Elastin (ng / ml) Unprocessed 79±19 20ng / ml TGFB1 243±35* 10% Collagen Solution 68±18 5% Collagen Solution 99±13 1% Collagen Solution 126±21 0.5% collagen solution 145±21* 0.1% collagen solution 76±14 0.05% collagen solution 58±6 0.01% collagen solution 53±5 0.005% collagen solution 56±24

[0491] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0492] As shown in Table 5, truncated his-labeled jellyfish collagen significantly increased elastin production when used at a concentration of 0.5%.

[0493] Example 7: Effects of truncated collagen on keratinocyte proliferation and UVB protection

[0494] A human keratinocyte cell culture model was used to evaluate the ability of the test material to influence cell proliferation. Additionally, the effect of the test material on cell viability after UVB exposure was assessed.

[0495] A 2% w / w truncated collagen stock solution was prepared from the truncated collagen of Example 1. Aliquots from this 2% truncated collagen stock solution were then used in the following experiments.

[0496] This study was conducted in two parts. In the first part, cultured keratinocytes were incubated with test materials for 48 hours, and then the change in the number of viable cells was assessed using an MTT assay. In the second part of the study, cultured keratinocytes were irradiated with UVB and then treated with test materials for 48 hours. At the end of the 48-hour period, the number of viable cells was again assessed using an MTT assay.

[0497] Changes in the number of viable cells can be determined using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazolium). The MTT assay is a colorimetric analysis of cellular metabolic activity, reflecting the number of viable cells. Reduction of MTT by mitochondria in viable cells results in the formation of insoluble purple formazan crystals, which are then extracted from the cells with isopropanol and quantified spectrophotometrically. The intensity of the purple color is directly proportional to the number of metabolically active cells.

[0498] Proliferation assay

[0499] For proliferation assays, keratinocytes were seeded into 96-well plates without growth factors and incubated at 37±2°C and 5±1% CO2 for 24 hours. After this initial incubation, the medium was replaced with medium supplemented with the test material. Normal medium (containing growth factors) was used as a positive control. As described above, cells were cultured for 48 hours after the addition of the test material. At the end of the incubation period, changes in viable cell count were determined using an MTT assay.

[0500] UVB protection measurement

[0501] For the UVB protection assay, keratinocytes were seeded into 96-well plates using normal culture medium and incubated at 37±2℃ and 5±1% CO2 for 24 hours. After this initial incubation, the medium was replaced with 100 μl phosphate-buffered saline (PBS), and the cells were exposed to UVB (40 mJ / cm2). After UVB exposure, the PBS was replaced with fresh medium supplemented with test material (100 μg / ml ascorbic acid as a positive control), and the cells were cultured at 37±2℃ and 5±1% CO2 for 48 hours. At the end of the 48-hour incubation, cell viability was determined using an MTT assay.

[0502] MTT assay

[0503] After 48 hours of incubation, the cell culture medium was removed and replaced with 200 μl of medium supplemented with 0.5 mg / ml MTT. The wells were incubated at 37 ± 2 °C and 5 ± 1% CO2 for 1 hour. After incubation, the MTT solution was removed, and the cells were washed once with phosphate-buffered saline. Then, 200 μl of isopropanol was added to the wells to extract purple formazan crystals. Using isopropanol as a blank, the 96-well plates were read at 540 nm.

[0504] The mean absorbance value of cells that were not treated with the test material (proliferation assay: untreated group) or not exposed to UVB (UVB protection assay: group not exposed to UVB) was calculated and used to represent 100% cell viability. Then, the individual absorbance values ​​of cells treated with each method were divided by the mean absorbance value representing 100% cell viability and expressed as a percentage to determine the change in cell viability caused by each treatment.

[0505] The results of the proliferation assay using his-labeled truncated jellyfish collagen are shown in Table 6. The results of the UVB protection assay using his-labeled truncated jellyfish collagen are shown in Table 7. The values ​​for both assays are expressed as mean viability ± standard deviation.

[0506] Table 6. Proliferation Assay

[0507] Unprocessed 100±3.4 Positive control (growth factor) 139±3.8* 10% Collagen Solution 103±9.4 5% Collagen Solution 97±7.3 1% Collagen Solution 94±5.0 0.5% collagen solution 93±7.0 0.1% collagen solution 95±2.5 0.05% collagen solution 99±6.0 0.01% collagen solution 96±6.4 0.005% collagen solution 96±2.8

[0508] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0509] Table 7. UVB Protection Measurement

[0510] No exposure to UVB 100±1.7* Unprocessed 77±1.8 100ug / ml Trolox 92±2.0* 10% collagen solution 76±8.6 5% Collagen Solution 92±3.9* 1% Collagen Solution 91±2.9* 0.5% collagen solution 100±4.5* 0.1% collagen solution 86±4.8 0.05% collagen solution 91±1.6* 0.01% collagen solution 83±7.5 0.005% collagen solution 82±4.7

[0511] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0512] For proliferation assays, the untreated group was used to represent 100% cell viability. Values ​​above 100% reflect an increase in the number of live cells and thus indicate cell proliferation. In this study, no promotion of cell proliferation was observed in the test material.

[0513] In addition, keratinocyte proliferation was measured using truncated collagen from SEQ ID NO:91. 1% and 0.5% collagen solutions of 5% stock solution were prepared according to Example 8 and tested. The keratinocyte viability values ​​of the truncated collagen from SEQ ID NO:91 were 102 ± 2.9 and 102 ± 2.0, respectively. The observed values ​​were statistically significant (p < 0.05).

[0514] In addition to its effect on cell proliferation, the test material was screened to determine its impact on cell recovery after UVB exposure. In this study, UVB exposure was observed to lead to a significant reduction in the number of viable cells 48 hours post-exposure. However, treatment with the test material prevented this decrease in cell viability. The effect was significant at concentrations between 0.05% and 5%, with the optimal effect observed at 0.05%. Within this concentration range, cell viability was significantly higher than in the untreated group (the only exception being a 0.01% concentration), indicating that the material has a UVB protective effect. Since this material is added after UVB exposure, it can be used to reduce the damaging effects of UVB irradiation or to help damaged cells recover more quickly. For the latter, truncated collagen is beneficial when applied topically to the skin and has a regenerative effect on UVB-damaged skin cells.

[0515] In addition, UVB protection was measured using truncated collagen from SEQ ID NO:91. The keratinocyte viability values ​​for 1% and 0.5% collagen solutions of SEQ ID NO:91 were 80 ± 4.6 and 78 ± 2.5, respectively. The observed values ​​were statistically significant (p < 0.05).

[0516] Example 8: Effect of truncated collagen on thymine dimer formation

[0517] Following exposure to ultraviolet radiation, the levels of thymine dimer (TT dimer) in cellular DNA increase. Increased TT dimer formation is associated with skin damage and certain types of proliferative diseases, including skin cancer.

[0518] The polynucleotide of SEQ ID NO:11 was expressed in the expression system of Example 1 and purified as described in this example. The encoded polypeptide includes a DsbA secretion tag. When the polypeptide is processed via the secretory pathway, amino acids 1-24 of the DsbA tag of SEQ ID NO:12 are cleaved by the host cell. Truncated collagen without the DsbA secretion tag is provided in SEQ ID NO:91.

[0519] The truncated collagen of SEQ ID NO:91 was tested to determine whether it could reduce TT dimer formation in human epidermal keratinocytes. For this study, cells were exposed to UVB (25 mJ / cm²). Following exposure, cells were treated with test material or Trolox (100 μg / ml) and incubated overnight. On the second day, cellular DNA was extracted, and thymine dimer levels were determined using an ELISA-based method.

[0520] Human keratinocytes were seeded into 12-well plates using normal culture medium and incubated at 37±2℃ and 5±1% CO2 for 24 hours. After this initial incubation, the culture medium was replaced with 100 μl of phosphate-buffered saline (PBS), and the cells were exposed to UVB (25 mJ / cm2). After UVB exposure, the PBS was replaced with fresh culture medium supplemented with test material or Trolox (100 μg / ml, as a positive control), and the cells were cultured overnight at 37±2℃ and 5±1% CO2. At the end of the incubation, cellular DNA was extracted.

[0521] After overnight incubation, remove the cell culture medium from the wells and replace it with 200 μl of PBS and 20 μl of proteinase K. After swirl the plate to mix the PBS and proteinase K, add 200 μl of buffer AL to each well. After swirl the plate again to mix the reagents, incubate the plate at 55 ± 2 °C for 10 min. After cooling the plate to room temperature, precipitate the DNA by adding 200 μl of 100% ethanol. Then transfer the precipitated DNA mixture to a DNEasy SpinColumn in a 2 ml collection tube and centrifuge at 8,000 RPM for 1 min. Discard the flow-through and collection tube, add 500 μl of wash buffer 1 to the spin column, place the column in a new collection tube, and centrifuge at 8,000 RPM for 1 min. Discard the flow-through and collection tube again, add 500 μl of wash buffer 2 to the spin column, place the column in a new collection tube, and centrifuge at 14,000 RPM for 3 min. Then, place the column into a new 1.5 ml centrifuge tube and add 110 μl of ultrapure water to the column. Incubate the column at room temperature for 1 minute, then centrifuge at 8,000 RPM for 1 minute.

[0522] Extracted DNA was quantified using a fluorescence assay. Two μl aliquots of the DNA sample were mixed with 100 μl of TE buffer in a 96-well plate. 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 determined using an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0523] Using OxiSelect TM UV-induced DNA damage ELISA kit for thymine dimer detection.

[0524] Genome 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 washed once with 100 μl of PBS and then blocked for 1 hour at room temperature with 150 μl of assay diluent. 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 this incubation, the plate was washed three times with 250 μl of wash buffer per well, and then 150 μl of blocking agent was added to the plate. The plate was blocked again for 1 hour at room temperature, and then washed three times as previously described. Then 100 μl of secondary antibody was added to each well, and the plate was incubated for 1 hour at room temperature. After washing the plate again, 100 μl of substrate was added to each well, and the plate was incubated for 5–20 minutes to allow color development. The color reaction was stopped by adding 100 μl of stop solution, and the plate was read at 460 nm using a microplate reader.

[0525] To quantify the amount of DNA present, a standard curve was generated using known concentrations of DNA and their respective fluorescence intensities (measured in RFU or relative fluorescence units). Regression analysis was performed to establish a line that best fits these data points. The amount of DNA was then estimated using the relative fluorescence units (RFU) of each unknown sample.

[0526] A series of DNA standards with known levels of thymine dimer were used to generate a standard curve. This standard curve was used to determine the amount of DNA damage in the sample DNA. Mean values ​​for each treatment group were calculated and compared using ANOVA. See Table 8 and... Figure 5 In this process, the standard 5% collagen solution (5g of truncated collagen in 95ml of deionized water) was further diluted with phosphate-buffered saline (PBS) to the indicated solution percentage.

[0527] Table 8. Thymine Dimer Assay

[0528] deal with Thymine dimer, ng / ml No exposure to UVB 0.1±0.2* Unprocessed 10.7±1.4* 100μg / ml Trolox 1.6±0.3* 5% truncated collagen 3.5±0.3* 1% truncated collagen 5.5±1.0* 0.5% truncated collagen 8.9±0.8 0.1% truncated collagen 9.2±0.9 0.05% truncated collagen 9.2±0.3

[0529] * indicates a statistically significant difference from the untreated group, p < 0.05.

[0530] Table 8 shows that 5% and 1% truncated collagen solutions significantly reduced TT dimer formation (p < 0.05). The data are presented graphically. Figure 5 middle.

[0531] The experiment was repeated with another batch of truncated collagen (SEQ ID NO: 91). The amount of TT dimer (ng / ml) was 1.3 ± 1.2 in unexposed UVB cells, 18.1 ± 0.4 in untreated cells, 7.9 ± 0.3 in cells treated with 100 μg / ml Trolox, 13.1 ± 0.2 for 5% collagen, and 17.4 ± 0.7 for 1% collagen. The reduction in TT dimer formation was statistically significant (p < 0.05) for unexposed UVB cells, Trolox-treated cells, 5% collagen-treated cells, and 1% collagen-treated cells compared to untreated cells.

[0532] Example 9: Human Collagen

[0533] Shortened human type 21 collagen α1

[0534] A truncated human type 21 collagen α1 lacking a His tag, linker, and thrombin cleavage site is disclosed below. The codon-optimized nucleotide and amino acid sequences encoding this collagen are disclosed below. In SEQ ID NO:73 and 74, the DsbA secretion tag is encoded by nucleotides 1-72 and amino acids 1-24. In SEQ ID NO:73 and 74, the truncated collagen sequence is encoded by nucleotides 73-633 and amino acids 25-211.

[0535] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:73.

[0536]

[0537] The amino acid sequence is disclosed in SEQ ID NO:74.

[0538]

[0539] The codon-optimized nucleotide sequence encoding a truncated human type 21 collagen α1 collagen that does not have a DsbA secretion tag is provided in SEQ ID NO:75.

[0540]

[0541] The amino acid sequence encoding a truncated human type 21 collagen α1 without the DsbA secretion tag is provided in SEQ ID NO:76.

[0542]

[0543] Shortened human type 1 collagen α2(1)

[0544] A truncated human type 1 collagen α2 lacking a His tag, linker, and thrombin cleavage site is disclosed below. The codon-optimized nucleotide and amino acid sequences are disclosed below. In SEQ ID NO:78 and 79, the DsbA secretion tag is encoded by nucleotides 1-72 and amino acids 1-24. The truncated collagen sequence is encoded by nucleotides 73-636 and amino acids 25-212.

[0545] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:77.

[0546]

[0547] The amino acid sequence is disclosed in SEQ ID NO:78.

[0548]

[0549] The nucleic acid sequence encoding a truncated human type 1 collagen α2(1) without a DsbA secretion tag is disclosed in SEQ ID NO:79.

[0550]

[0551] The amino acid sequence encoding a truncated human type 1 collagen α2(1) without a DsbA secretion tag is disclosed in SEQ ID NO:80.

[0552]

[0553] Shortened human type 1 collagen α2(2)

[0554] A truncated human type 1 collagen α2 lacking a His tag, linker, and thrombin cleavage site is disclosed below. The codon-optimized nucleotide and amino acid sequences are disclosed below. In SEQ ID NO:82 and 83, the DsbA secretion tag is encoded by nucleotides 1-72 and amino acids 1-24. The truncated collagen sequence is encoded by nucleotides 73-609 and amino acids 25-203.

[0555] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:81.

[0556]

[0557] The amino acid sequence is disclosed in SEQ ID NO:82.

[0558]

[0559] The nucleic acid sequence of a truncated human type 1 collagen α2(2) without the DsbA secretion tag is disclosed in SEQ ID NO:83.

[0560]

[0561] The amino acid sequence of a truncated human type 1 collagen α2(2) without the DsbA secretion tag is disclosed in SEQ ID NO:84.

[0562]

[0563] The polynucleotide of SEQ ID NO:73, 77, or 81 was subcloned into vector pET28a to prepare a transformation vector. The vector was used to transform host cells to express the polynucleotide as described in Example 2.

[0564] After fermentation, truncated human collagen was purified from the fermented broth using the procedure disclosed in Example 3. The purified truncated human collagen was analyzed using SDS-PAGE and HPLC as disclosed in Example 3.

[0565] In SDS-PAGE analysis, all three truncated human collagen samples were run at their expected molecular weights. When analyzing the truncated human collagen samples using HPLC, the standard curve for jellyfish collagen from Example 3 was utilized. The retention times of the human collagen samples differed slightly from those of the jellyfish collagen. The retention time for SEQ ID NO:76 was 5.645 minutes, the retention time for SEQ ID NO:80 was 5.631 minutes, and SEQ ID NO:84 was run at two peaks with retention times of 5.531 and 5.7 minutes.

[0566] Truncated human type 1 collagen α2 with DsbA secretion and FLAG tagging 5

[0567] The truncated amino acid sequence of human type 1 collagen α2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:92. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:93, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:92. The collagen nucleotide sequence is nucleotides 58-657 of SEQ ID NO:93, and the amino acid sequence is amino acids 20-219 of SEQ ID NO:92. The FLAG nucleotide sequence is nucleotides 658-684 of SEQ ID NO:93, and the amino acid sequence is amino acids 220-228.

[0568]

[0569] The nucleic acid sequence of a truncated human type 1 collagen α2 truncated 5 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:93.

[0570]

[0571] As described herein, the polynucleotide of SEQ ID NO:93 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated collagen was purified. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 100 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0572] Truncated human type 1 collagen α2 with DsbA secretion and FLAG tagging 6

[0573] The truncated amino acid sequence of human type 1 collagen α2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:94. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:95, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:94. The collagen nucleotide sequence is nucleotides 58-657 of SEQ ID NO:95, and the amino acid sequence is amino acids 20-219 of SEQ ID NO:94. The FLAG nucleotide sequence is nucleotides 658-684 of SEQ ID NO:95, and the amino acid sequence is amino acids 220-228 of SEQ ID NO:94.

[0574]

[0575] The nucleic acid sequence of a truncated human type 1 collagen α2 truncated 6 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:95.

[0576]

[0577] As described herein, the polynucleotide of SEQ ID NO:94 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated collagen was purified. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 25 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0578] Truncated human type 1 collagen α2 with DsbA secretion and FLAG tagging 7

[0579] The truncated amino acid sequence of human type 1 collagen α2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:96. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:97, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:96. The collagen nucleotide sequence is nucleotides 58-759 of SEQ ID NO:96, and the amino acid sequence is amino acids 20-253 of SEQ ID NO:96. The FLAG nucleotide sequence is nucleotides 760-786 of SEQ ID NO:97, and the amino acid sequence is amino acids 254-262 of SEQ ID NO:96.

[0580]

[0581] The nucleic acid sequence of a truncated human type 1 collagen α2 truncated 7 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:97.

[0582]

[0583] As described herein, the polynucleotide of SEQ ID NO:97 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated collagen was purified. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 30 kDa. In the absence of this protein expression, no band was observed at this location on the gel.

[0584] Example 10: Protective effect of truncated human collagen on fibroblasts

[0585] The effects of truncated human collagen on fibroblast viability, procollagen synthesis, and elastin synthesis were determined according to the method in Example 6.

[0586] The effects of truncated human collagen on keratinocyte proliferation and UVB protection were determined according to the method in Example 7.

[0587] The effect of truncated collagen on thymine dimer formation after exposure to UV radiation was determined according to the method in Example 8.

[0588] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes that can be made based thereon will be conceived by those skilled in the art and are included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0589] Example 11: Effects of truncated collagen on inflammatory cytokines

[0590] Keratinocytes and dermal fibroblasts play important roles in the skin's immune response. In response to irritating chemicals or UV radiation (pro-inflammatory / pro-stimulatory stimuli), keratinocytes release large amounts of cytokines. These cytokines are believed to help bring immune cells into contact with sites 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.

[0591] The test model used in this study was MatTek EpiDerm. This skin model consisted of normal human epidermal keratinocytes that had been cultured to form a multilayered, highly differentiated human epidermal model. Ultrastructural analysis revealed the presence of keratinocyte granules, tonofilament bundles, desmosomes, and a multilayered stratum corneum containing an intercellular layer of lamellae arranged according to in vivo epidermal characteristics. Markers of mature epidermal-specific differentiation, such as filaggrinogen, K1 / K10 cytokeratin pairs, inner laminarin, and type I epidermal transglutaminase, were localized in this model. MatTek EpiDerm also exhibited mitotic and metabolic activity.

[0592] 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 with an over-the-counter topical hydrocortisone preparation (positive control), untreated tissue (negative control 1), and untreated non-inflammatory tissue (negative control 2). The test was also used to assess tissue viability after exposure to the test materials.

[0593] 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 directly measured in tissue culture media using a colorimetric enzyme-linked immunosorbent assay (ELISA). In short, an antibody covalently linked to a solid support binds to IL-1α, IL-6, or IL-8 present in the used culture medium sample. A secondary antibody covalently linked to acetylcholinesterase then detects the specifically bound cytokines. Upon addition of an appropriate colored substrate, acetylcholinesterase produces a colored end product measurable spectrophotometrically.

[0594] MatTek EpiDerm tissues were purchased from MatTek and stored at 4°C until use. Before 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 hydrocortisone-free assay medium (37±2°C). The tissues were allowed to incubate overnight at 37±2°C and 5±1% CO2. After this initial incubation, the assay medium was replaced with 0.9 ml of fresh hydrocortisone-free medium (37±2°C). Three aliquots of each test material were prepared.

[0595] Inflammatory responses are induced in tissues by UV irradiation (UVB). A UV lamp is used to deliver 300 mJ / cm² to the tissue. 2 Dose of UVB radiation. Immediately after application of the inflammatory stimulus, 50 μl or mg of test material was applied directly to the tissue surface. Over-the-counter hydrocortisone cream was used as a positive control. For the negative control, tissue was exposed to the inflammatory stimulus but not treated with any type of anti-inflammatory material. Another group of tissues was not exposed to the inflammatory stimulus to provide baseline measurements for cytokines. After exposure to the inflammatory stimulus, tissues were incubated at 37 ± 2 °C and 5 ± 1% CO2 for 24 hours. After 24 hours of incubation, cell culture medium was collected and stored at -75 °C until cytokine analysis.

[0596] ELISA plates were prepared by diluting the appropriate capture antibody in PBS. Next, 100 μl of the diluted capture antibody was added to each well of a 96-well ELISA plate, and the plate was incubated overnight at room temperature. The next day, the plate was washed three times with 300 μl of wash buffer (PBS containing 0.05% Tween 20), and then blocked by adding 300 μl of blocking buffer (PBS containing 1% BSA) to each well. The plate was incubated with the blocking buffer for at least one hour. After incubation, the blocking buffer was removed, and the plate was washed three times as described above.

[0597] Prepare a series of standards and dispense 100 μl of each standard into two wells of an appropriate 96-well plate (in duplicate). Then, add 100 μl of each sample to the other wells and incubate the plate at room temperature for two hours. After incubation, wash the plate three times as described above. Once the final wash buffer is removed, add 100 μl of biotin-conjugated detection antibody. Incubate the plate at room temperature for two hours, then wash the plate again as described above. Then, add 100 μl of HRP-streptavidin to each well and incubate the plate at room temperature for 20 minutes. Once the final wash buffer is removed, add 100 μl of substrate solution (hydrogen peroxide + tetramethylbenzidine as chromogen) to each well. Once sufficient color development has occurred, add 50 μl of stop solution (2N sulfuric acid) to each well and read at 460 nm.

[0598] After 24 hours of incubation, the tissue was rinsed twice with at least 100 μl of phosphate-buffered saline to remove test material, and then transferred to a 6-well plate containing 1.0 ml of assay medium supplemented with MTT (1 mg / ml), and allowed to incubate 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, blotted dry, and then placed in a 24-well plate containing 2 ml of isopropanol per well. The 24-well plate was covered and allowed to incubate at room temperature on a shaking platform for at least 2 hours to extract reduced MTT from the tissue. After extraction, 200 μl of the isopropanol / MTT mixture sample 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 as a blank. The MTT assay is described in Example 6 of this document. The cell viability results of the MTT assay were similar to those obtained in Example 6.

[0599] The results of the IL-1α assay are shown in Table 9 below. Sample 4 is a 2% stock solution of jellyfish collagen (SEQ ID NO: 91), while Sample 3 is a 2% stock solution of truncated jellyfish collagen (SEQ ID NO: 10). In Table 9 below, the percentages shown are the percentage dilutions of the stock solutions used for testing. For example, a 1% treatment of Sample 4 is a 1% solution of a 2% truncated collagen stock solution. Untreated cells produced 18.2 pg / ml of IL-1α. After treatment with truncated collagen, all samples showed a decrease in IL-1α production. The 1% treatment of Sample 4 reduced IL-1α production to 13.4 pg / ml, which was significant (p < 0.05). The decrease in IL-1α production indicates that the truncated collagen has an anti-inflammatory effect.

[0600] Table 9 IL-1a Detection

[0601]

[0602]

[0603] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0604] Example 12: Urban Dust Protection Using Truncated Collagen

[0605] use keratinocytes Culture models were used to assess the ability of truncated collagen to exert a protective effect by promoting cell survival after exposure to urban dust.

[0606] Human epidermal keratinocytes were pretreated with test material and then exposed to urban dust. Changes in cell viability were then determined by MTT assay at the end of the treatment period.

[0607] 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. On the second day, the culture medium was removed by aspiration to eliminate any non-adherent cells and replaced with 100 μl of fresh culture medium. Cells were allowed to grow until confluence, with the culture medium changed every 48 to 72 hours.

[0608] The test materials were pretreated before urban dust treatment.

[0609] Test material was prepared at twice the final desired concentration (2x) in cell culture medium. Urban dust (NIST 1649B from Sigma Chemicals) was also prepared as a 2x solution. For pretreatment, 50 μl of 2x test material was combined with 50 μl of culture medium, and cells were incubated for 24 h. At the end of pretreatment, the test material containing culture medium was removed and replaced with 50 μl of 2x urban dust and 50 μl of culture medium. Another group of cells was treated with culture medium only (without dust exposure) and used as a reference control to represent 100% cell viability. Cells were then incubated for 24 h, followed by MTT assay to determine changes in cell viability.

[0610] 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 at 37 ± 2 °C and 5 ± 1% CO2 for 30 min. After incubation, the culture medium / MTT solution was removed, and the cells were washed again with PBS. Then, 100 μl of isopropanol was added to the wells to extract purple formazan crystals. Isopropanol was then used as a blank, and the 96-well plate was read at 540 nm.

[0611] The mean MTT absorbance value of the dust-free exposed cells was calculated and used as a 100% value to represent the cell number. Then, the individual MTT values ​​from cells subjected to various treatments were divided by the mean of the dust-free exposed cells and expressed as a percentage to determine the change in cell number caused by each treatment.

[0612] The MTT results for pretreatment of test materials followed by dust treatment are shown in Table 10. Table 10 shows that cell viability increased after pretreatment with truncated collagen and subsequent exposure to urban dust, with increasing amounts of collagen treatment. These results demonstrate that truncated collagen can prevent the decline in cell viability associated with urban dust exposure.

[0613] Table 10 MTT assay, pretreatment of truncated collagen

[0614]

[0615] * indicates a value that is significantly different from the untreated group (p < 0.05).

[0616] Example 12: Truncated Chondrosia reniformis collagen with DsbA secretion and FLAG tagging. Truncated Chondrosia reniformis fibrous collagen 1

[0617] The amino acid sequence of truncated Chondrosia reniformis fibrous collagen 1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:102. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:103, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:102. The nucleotide sequence of the fibrous collagen is nucleotides 58-792 of SEQ ID NO:103, and the amino acid sequence is amino acids 20-264 of SEQ ID NO:102. The FLAG nucleotide sequence is nucleotides 793-819 of SEQ ID NO:103, and the amino acid sequence is amino acids 265-273 of SEQ ID NO:102.

[0618]

[0619] The nucleic acid sequence of truncated Chondrosia reniformis fibrous collagen 1 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:103.

[0620]

[0621] As described herein, the polynucleotide of SEQ ID NO:103 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated *Chondrosia reniformis* fibrous collagen 1 was purified. The purified fibrous collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 40 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0622] truncated Chondrosia reniformis (renal sponge) fibrous collagen 2 with DsbA secretion and FLAG tagging

[0623] The amino acid sequence of truncated Chondrosia reniformis fibrous collagen 2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:104. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:105, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:104. The nucleotide sequence of the fibrous collagen is nucleotides 58-1323 of SEQ ID NO:105, and the amino acid sequence is amino acids 20-441 of SEQ ID NO:104. The FLAG nucleotide sequence is nucleotides 1324-1350 of SEQ ID NO:105, and the amino acid sequence is amino acids 442-450 of SEQ ID NO:104.

[0624]

[0625] The amino acid sequence of truncated Chondrosia reniformis fibrous collagen 2 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:105.

[0626]

[0627] As described herein, the polynucleotide of SEQ ID NO:105 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated *Chondrosia reniformis* fibrous collagen 2 was purified. The purified fibrous collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 55 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0628] Truncated Chondrosia reniformis (renal sponge) non-fibrous collagen 1 with DsbA secretion and FLAG tagging

[0629] The amino acid sequence of a truncated Chondrosia reniformis non-fibrous collagen 1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:106. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:107, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:106. The nucleotide sequence of the non-fibrous collagen is nucleotides 58-831 of SEQ ID NO:107, and the amino acid sequence is amino acids 20-277 of SEQ ID NO:106. The FLAG nucleotide sequence is nucleotides 832-858 of SEQ ID NO:107, and the amino acid sequence is amino acids 278-286 of SEQ ID NO:106.

[0630]

[0631] The nucleic acid sequence of a truncated Chondrosia reniformis non-fibrous collagen 1 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:107.

[0632]

[0633] As described herein, the polynucleotide of SEQ ID NO:107 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated *Chondrosia reniformis* non-fibrous collagen 1 was purified. The purified non-fibrous collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 30 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0634] Truncated Chondrosia reniformis (renal sponge) non-fibrous collagen 2 with DsbA secretion and FLAG tagging

[0635] The amino acid sequence of truncated Chondrosia reniformis non-fibrous collagen 2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:108. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:109, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:108. The nucleotide sequence of the non-fibrous collagen is nucleotides 58-1509 of SEQ ID NO:109, and the amino acid sequence is amino acids 20-503 of SEQ ID NO:108. The FLAG nucleotide sequence is nucleotides 1510-1536 of SEQ ID NO:109, and the amino acid sequence is amino acids 504-512 of SEQ ID NO:108.

[0636]

[0637] The nucleic acid sequence of truncated Chondrosia reniformis non-fibrous collagen 2 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:109.

[0638]

[0639] As described herein, the polynucleotide of SEQ ID NO:109 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated *Chondrosia reniformis* non-fibrous collagen 2 was purified. The purified fibrous collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 60 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0640] Example 13: Truncated Rhincodon typus (whale shark) collagen with DsbA secretion and FLAG tagging; Truncated Rhincodon typus (whale shark) type 1 collagen α1 truncated 1

[0641] The amino acid sequence of a truncated Rhincodon typus type 1 collagen with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:110. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:111, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:110. The collagen nucleotide sequence is nucleotides 58-630 of SEQ ID NO:111, and the amino acid sequence is amino acids 20-210 of SEQ ID NO:110. The FLAG nucleotide sequence is nucleotides 631-657 of SEQ ID NO:111, and the amino acid sequence is amino acids 211-219 of SEQ ID NO:110.

[0642]

[0643] The nucleic acid sequence of a truncated Rhincodon typus type 1 collagen with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:111.

[0644]

[0645] As described herein, the polynucleotide of SEQ ID NO:111 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated Rhincodon typus type 1 collagen was purified. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 25 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0646] Truncated Rhincodon typus (whale shark) type 6 collagen α1 truncated 2 with DsbA secretion and FLAG tag

[0647] The amino acid sequence of a truncated Rhincodon typus type 6 collagen with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:112. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:113, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:112. The collagen nucleotide sequence is nucleotides 58-684 of SEQ ID NO:113, and the amino acid sequence is amino acids 20-228 of SEQ ID NO:112. The FLAG nucleotide sequence is nucleotides 685-711 of SEQ ID NO:113, and the amino acid sequence is amino acids 229-237 of SEQ ID NO:112.

[0648]

[0649] The nucleic acid sequence of a truncated Rhincodon typus type 6 collagen truncated 2 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:113.

[0650]

[0651] As described herein, the polynucleotide of SEQ ID NO:113 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated Rhincodon typus type 6 collagen was purified. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 35 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

[0652] Truncated Rhincodon typus (whale shark) type 6 collagen α1 with DsbA secretion and FLAG tagging

[0653] The truncated amino acid sequence of Rhincodon typus type 6 collagen α1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:114. The DsbA secretion tag is encoded by nucleotides 1-57 of SEQ ID NO:115, and the amino acid sequence is amino acids 1-19 of SEQ ID NO:114. The collagen nucleotide sequence is nucleotides 58-735 of SEQ ID NO:115, and the amino acid sequence is amino acids 20-245 of SEQ ID NO:114. The FLAG nucleotide sequence is nucleotides 736-762 of SEQ ID NO:115, and the amino acid sequence is amino acids 246-254 of SEQ ID NO:114.

[0654]

[0655] The nucleic acid sequence of truncated Rhincodon typus type 6 collagen α1 truncated 3 with DsbA secretion and FLAG tagging is disclosed in SEQ ID NO:115.

[0656]

[0657] As described herein, the polynucleotide of SEQ ID NO:115 was subcloned into the vector pET28a, expressed in host *E. coli* cells, and the truncated Rhincodon typus type 1 collagen was purified. The purified collagen produced a clear band on SDS-PAGE, and an anti-FLAG protein blot was observed at approximately 25 kDa. In the absence of this protein expression, no band appeared at this location on the gel.

Claims

1. A recombinant polypeptide comprising the amino acid sequence shown in SEQ ID NO: 91 and optionally a secretion tag, histidine tag, protease cleavage site or β-lactamase protein.

2. The recombinant polypeptide according to claim 1, wherein the secretion tag is DsbA.

3. A recombinant polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 91 and green fluorescent protein.

4. A composition comprising the recombinant polypeptide of any one of claims 1 to 3, ranging from 0.005% to 30% w / w.

5. The composition according to claim 4, wherein the composition is a topical composition.

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

7. The composition according to claim 6, wherein the local carrier is selected from biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, sorbitan monostearate, polysorbate, cetyl wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, water, and any combination thereof.

8. The composition according to claim 7, wherein the silicone oil is selected from cyclomethyl silicone oil and cyclopentadioxane.

9. The composition according to claim 6, wherein the local carrier is selected from liposomes, biodegradable microcapsules, aerosols, powders, and any combination thereof.

10. The composition of claim 4, wherein the composition further comprises an emulsion and / or a spray.

11. The composition according to claim 4, wherein the composition further comprises wax, alcohol and / or emulsifier.

12. The composition according to claim 6, wherein 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-azamonoadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, EDTA, and any combination thereof.

13. The composition according to any one of claims 6 to 12, wherein the composition is a personal care product.

14. Use of the composition of any one of claims 6 to 13 in the preparation of a personal care product for treating the skin of a subject, wherein the personal care product results in an increase in the activity of the subject's keratinocytes after exposure to UV radiation.

15. The use according to claim 14, wherein the personal care product results in a reduction in the production of inflammatory cytokines.

16. A host cell comprising a heterologous nucleic acid sequence encoding a recombinant polypeptide according to any one of claims 1 to 3.

17. The host cell according to claim 16, wherein the host cell is a bacterial cell.

18. The host cell of claim 17, wherein the bacterial cell is a species of Escherichia coli.

19. The host cell according to any one of claims 16 to 18, wherein the heterologous nucleic acid sequence comprises SEQ ID NO:

90.

20. The host cell according to any one of claims 16 to 18, wherein the heterologous nucleic acid sequence is codon-optimized for expression in the cell.

21. A composition comprising a host cell according to any one of claims 16 to 20 and a culture medium comprising a recombinant polypeptide according to any one of claims 1 to 3.

22. A method for generating a recombinant polypeptide, the method comprising the following steps: a. Culture the host cells according to any one of claims 16 to 20 in a culture medium; as well as b. Isolate the recombinant polypeptide from the host cell.

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