Recombinant collagen and elastin molecules and uses thereof

Abstract

To provide recombinant collagen and elastin molecules, and uses thereof.SOLUTION: This disclosure provides non-naturally occurring collagen and elastin molecules. The non-naturally occurring collagens and elastins include truncated collagens, truncated elastins, as well as fusion proteins thereof. The non-naturally occurring collagen and elastin are useful in foods, cosmetics and many other products and uses. In one aspect, a non-naturally occurring collagen produced by a host cell is provided. The non-naturally occurring collagen is jellyfish (Hydrozoan) collagen, human collagen, Chondrosia renformis (kidney sponge) collagen, or Rhincodon typus (whale shark) collagen.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND APPLICATIONS INCORPORATED BY REFERENCE This application claims priority to U.S. Provisional Patent Application No. 16 / 144,914, entitled "RECOMBINANT COLLAGEN AND ELASTIN MOLECULES AND USES THEREOF," filed September 27, 2018, which claims the benefit under 35 U.S.C. 119(e) 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 of which are entitled "RECOMBINANT COLLAGEN AND ELASTIN MOLECULES AND USES THEREOF," the contents of which are incorporated herein by reference in their entireties.

[0002] The present disclosure relates to non-naturally occurring full-length and truncated collagen and elastin molecules and uses thereof. [Background technology]

[0003] Collagen and similar proteins are the most abundant proteins in the biosphere. Collagen and elastin are structural proteins found in the skin, connective tissue and bones of animals and other tissues. In humans, the amount of collagen present in the body is about one-third of the total protein and accounts for about three-quarters of the dry weight of skin. Elastin is a highly elastic protein found in connective tissue and other types of tissues.

[0004] The structure of collagen is a triple helix, with three polypeptide chains forming a helical coil together. Each polypeptide chain is composed of a repeating triplet amino acid sequence, designated GLY-XY. X and Y can be any amino acid, and the third amino acid is glycine. The amino acids proline and hydroxyproline are found in high concentrations in collagen. The most common triplet is proline-hydroxyproline-glycine (Gly-Pro-Hyp), which accounts for approximately 10.5% of the triplets in collagen.

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

[0006] Gelatin is used in many products including cosmetics, food, pharmaceuticals, medical devices, photographic film, adhesives, binders, etc. The physical and chemical properties of gelatin are tailored to the specific application. These physical / chemical properties include gel strength, melting temperature, viscosity, color, turbidity, pH, isoelectric point, etc.

[0007] Elastin is an elastic protein that is important for the proper functioning of arteries, lungs, tendons, ligaments, skin and other tissues. Elastin provides tissues with the ability to stretch and return to their original shape. The protein tropoelastin is a component of elastin. In contrast to collagen, which contains a family of genes, in humans there is one tropoelastin gene. When expressed, the single elastin gene is spliced ​​to produce different forms of tropoelastin protein. Many tropoelastin molecules associate with each other to form elastin.

[0008] L-form bacteria, or L-forms, are bacterial strains derived from a parent species (N-form) that can grow as cell wall-deficient (spheroplast type) cells or as cell wall-free (protoplast type) cells. 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 protoplast type L-forms. In Protoplasts, Lecture See Proceedings of the 6th International Protoplast Symposium: Basel. Experientia 1983, 46(suppl):227-241. Protoplast type L is a genetically stable mutant that is cultured in a cell wall-free state and exhibits highly pleiotropic changes, including the inability to form cell walls, capsules, flagella, pili, spores, and mesosomes, altered colony and cell morphology, qualitative and quantitative changes in the lipid and protein components of the cell membrane, the absence of extracellular proteolytic activity, resistance to bacteriophages, and the inability to reproduce outside laboratory conditions. Gumpert and Taubeneck (supra); and Hoischen et al., Lipid and fatty acid composition of cytoplasmic membranes from Streptomyces hygroscopic and its stable See protoplast type L-form. J Bacteriol 1997,179:3430-3436. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Hoischen et al., J Bacteriol (1997) 179:3430-3436 Summary of the Invention [Means for solving the problem]

[0010] In one aspect, a non-naturally occurring collagen produced by a host cell is provided. The non-naturally occurring collagen is jellyfish (Hydrozoa) collagen, human collagen, Chondrosia reniformis (Cornish 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 by an internal truncation of between 50 and 500 amino acids. In another embodiment, the truncation is at the C-terminus or N-terminus of the collagen polypeptide. The non-naturally occurring collagen is 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.

[0011] In another embodiment, the non-naturally occurring collagen further comprises an amino acid sequence comprising a secretion tag, a histidine tag, a green fluorescent protein tag, a protease cleavage site, a beta-lactamase and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat. When the non-naturally occurring collagen comprises one or more amino acid trimer repeats of the sequence glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK), the number of GEK and / or GDK trimer repeats can range from 2 to 50 trimer repeats. In one embodiment, the secretion tag is DsbA, PelB, OmpA, TolB, MalE, lpp, TorA or HylA, or is a hybrid secretion tag comprising a portion of one secretion tag fused to a portion of a second secretion tag. An exemplary secretion tag is DsbA.

[0012] In one aspect, a composition is provided comprising 0.005% to 30% w / w of non-naturally occurring collagen. The composition may further comprise at least one additional ingredient including a topical carrier or a preservative.

[0013] In one aspect, the composition comprising the non-naturally occurring collagen is a topical composition for application to the skin, the topical composition being used to reduce skin damage or promote the repair of damaged skin.

[0014] One aspect provides a method for reducing skin damage or promoting repair of damaged skin. The method includes applying a composition comprising elastin to the skin of a subject. The method increases the viability of fibroblasts or keratinocytes of the skin of the subject. In another aspect, application of the composition increases the synthesis of procollagen by fibroblasts of the skin of the subject. In another aspect, topical application of the composition protects the skin or keratinocytes from UV damage. In yet another embodiment, thymine-thymine (TT) dimer formation is reduced by the collagen or elastin disclosed herein.

[0015] Another aspect provided herein is a method of increasing the viability of skin cells. The method comprises applying collagen or elastin molecules to the skin or skin cells. The collagen or elastin provided is resistant to UV radiation, urban dust, and The present invention relates to a method for treating keratinocyte and / or fibroblast cell death, and / or to a method for treating keratinocyte and / or fibroblast cell death.

[0016] 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 a collagen or elastin molecule to the skin cells. The production of inflammatory cytokines, including TNFα, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18, and TL-IRA.

[0017] In another aspect, a method of protecting skin cells from the effects of exposure to urban dust is provided. The method comprises applying to skin cells collagen or elastin as disclosed herein. Exposing the skin cells to collagen or elastin increases the viability of the skin cells. In one embodiment, the skin cells are keratinocytes or fibroblasts.

[0018] In one aspect, a non-naturally occurring elastin produced by a host cell is provided. The non-naturally occurring elastin is jellyfish elastin, human elastin, Chondrosia reniformis elastin, or Rhincodon typus elastin. In one embodiment, the non-naturally occurring elastin is full-length or truncated elastin. In one embodiment, the elastin is truncated by an internal truncation of between 50 and 500 amino acids. In another embodiment, the truncation is 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.

[0019] In another embodiment, the non-naturally occurring elastin further comprises an amino acid sequence comprising a secretion tag, a histidine tag, a green fluorescent protein tag, a protease cleavage site, a beta-lactamase and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat. When the non-naturally occurring collagen comprises one or more amino acid trimer repeats of the sequence glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK), the number of GEK and / or GDK trimer repeats can range from 2 to 50 trimer repeats. In one embodiment, the secretion tag is DsbA, PelB, OmpA, TolB, MalE, lpp, TorA or HylA, or is a hybrid secretion tag comprising a portion of one secretion tag fused to a portion of a second secretion tag. An exemplary secretion tag is DsbA.

[0020] In another embodiment, a composition is provided comprising 0.005%-30% w / w non-naturally occurring elastin. The composition may further comprise at least one additional ingredient including a topical carrier or a preservative.

[0021] In one aspect, the composition comprising the non-naturally occurring elastin is a topical composition for application to the skin. The topical composition is used to reduce skin damage or promote the repair of damaged skin.

[0022] One embodiment provides a method for reducing skin damage or promoting repair of damaged skin. The method includes applying a composition comprising elastin to the skin of a subject. The method increases the viability of fibroblasts in the skin of the subject. In another aspect, application of the composition increases the synthesis of procollagen by fibroblasts in the skin of the subject. In another aspect, topical application of the composition protects the skin or keratinocytes from UV damage. In yet another embodiment, thymine-thymine (TT) dimer formation is reduced by collagen or elastin disclosed herein.

[0023] Another embodiment provides a polynucleotide encoding a non-naturally occurring collagen or a non-naturally occurring elastin. The polynucleotide encodes a collagen or elastin from jellyfish, human, Chondrosia reniformis, 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 of between 50 and 500 amino acids.

[0024] In one embodiment, a polynucleotide is provided that encodes a fusion protein comprising a secretion tag, a histidine tag, a green fluorescent protein tag, a protease cleavage site, a beta-lactamase, and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat together with collagen or elastin. The non-naturally occurring collagen or elastin may comprise one or more amino acid trimer repeats of the sequence glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK), and the number of GEK and / or GDK trimer repeats may range from 2 to 50 trimer repeats. In one aspect, the secretion tag is DsbA, PelB, OmpA, TolB, MalE, lpp, TorA, or HylA, or is a hybrid secretion tag comprising a portion of one secretion tag fused to a portion of a second secretion tag. The secretion tag of an exemplary embodiment is DsbA.

[0025] The polynucleotides and vectors can be used to transform host cells and express the polynucleotides. Polynucleotides encoding non-naturally occurring collagens are provided, and are 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. Polynucleotides encoding non-naturally occurring elastins are provided, and 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.

[0026] Host cells expressing the polynucleotides of the invention are disclosed. The host cells can be any host cell, including bacterial cells, yeast cells, fungal cells, insect cells, mammalian cells, plant cells, and any other cells used to express exogenous polynucleotides.

[0027] Bacterial host cells are provided that are modified to inhibit cell division and have increased periplasmic space. An exemplary host cell is Escherichia coli.

[0028] One embodiment provides a method of producing a non-naturally occurring collagen or non-naturally occurring elastin, the method comprising the steps of inoculating a culture medium with a recombinant host cell comprising a polynucleotide encoding a collagen or elastin, culturing the host cell, and isolating the non-naturally occurring collagen or non-naturally occurring elastin from the host cell. [Brief description of the drawings]

[0029] [Figure 1] Figure 1 shows the physiological state differences between switched and non-switched cells. A) Non-switched E. coli cells. B) The same E. coli population as in A, but which has undergone physiological switching. C) Phase contrast of switched E. coli cells containing cytoplasmic RFP and periplasmic GFP. D) Fluorescence image of the cells in C showing the localization of the targeted protein.

[0030] [Diagram 2]Figure 2 shows enhanced protein production in switched cells. A-B) The target protein for T7-induced protein production is periplasmically expressed GFP produced in E. coli BL21. The same cell population was used and induced at OD1.1. A) Protein ladder (lane 1), IPTG-induced protein production (lane 2), IPTG-induced protein production with physiological switch (lane 3). B) Two vials of cell GFP-induced culture with IPTG only (left) and IPTG + switch (right). C) Expression of 22 kDa collagen using switched cells showing protein ladder (lane 1), supernatant after protein production (lane 2), and cell pellet (lane 3).

[0031] [Diagram 3] FIG. 3 shows a time lapse of E. coli cells switching over time.

[0032] [Figure 4] Figure 4 shows other organisms undergoing physiological switches. A) Normal physiology of Agrobacterium tumefaciens. B) Switch physiology of Agrobacterium tumefaciens. C) Normal physiology of Pseudomonas aeruginosa PAO1. D) Switch physiology of Pseudomonas aeruginosa PAO1. E) Normal physiology of Brevundimonas diminuta. F) Switch physiology of Brevundimonas diminuta. G) Normal physiology of Agrobacterium tumefaciens. H) Switch physiology of Agrobacterium tumefaciens.

[0033] [Diagram 5] FIG. 5 shows the reduction of TT dimer formation by treatment of human keratinocytes with truncated collagen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details.

[0035] As used herein, the term "about" refers to ±10%.

[0036] The term "consisting of" means "including, but not limited to."

[0037] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or moieties only if the additional ingredients, steps, and / or moieties do not materially change the basic novel characteristics of the claimed composition, method, or structure.

[0038] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0039] Throughout this application, various embodiments of the present disclosure may be described in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an invariant limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have all possible subranges specifically disclosed and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0040] Whenever a numerical range is given herein, it is meant to include any recited number (fractional or integer) within the given range. The phrases "ranging between" a first given number and a second given number and "ranging from" a first given number to a second given number are used interchangeably herein and are meant to include the first and second given numbers and all fractions and integers therebetween.

[0041] As used herein, the term "method" refers to methods, means, techniques and procedures for accomplishing a given task, including but not limited to methods, means, techniques and procedures known to a practitioner in the fields of chemistry, pharmacy, biology, biochemistry and medicine, or readily developed by said practitioner from known methods, means, techniques and procedures.

[0042] As used herein, the term "collagen" or "collagen-like" 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 acid, base, or heat to prepare gelatin. The quaternary structure of native collagen is typically a triple helix composed of three polypeptides. Of the three polypeptides that form native collagen, two are usually identical and are designated as alpha chains. The third polypeptide is designated as beta chain. Thus, a typical native collagen can be designated AAB, where collagen is composed of two alpha ("A") chains and one beta ("B") chain. As used herein, the term "procollagen" refers to a polypeptide produced by a cell that can be processed into naturally occurring collagen.

[0043] As used herein, the term "elastin" refers to an elastic polypeptide that functions to stretch and return to its original shape. Elastin is found naturally in connective tissue.

[0044] As used herein, the term "expression vector" or "vector" refers to a nucleic acid assembly capable of directing the expression of an exogenous gene. An expression vector can contain a promoter operably linked to an exogenous gene, a restriction endonuclease site, a nucleic acid encoding one or more selectable markers, and other nucleic acid useful in the practice of recombinant techniques.

[0045] As used herein, the term "fibroblast" refers to a cell that synthesizes procollagen and other structural proteins. Fibroblasts are distributed widely in the body and are found in skin, connective tissue and other tissues.

[0046] The term "fluorescent protein" refers to a protein commonly used in genetic engineering technology that is used as a reporter of the expression of an exogenous polynucleotide. The protein fluoresces when exposed to ultraviolet or blue light, emitting bright visible light. The green-emitting protein is green fluorescent protein (GFP), and the red-emitting protein is red fluorescent protein (RFP).

[0047] As used herein, the term "gelatin" refers to collagen that has been further processed by exposure to acid, base, or heat. Without being bound by theory or mechanism, it is believed that treatment of collagen with acid, base, or heat denatures the collagen polypeptides. Aqueous denatured collagen solutions form reversible gels that are used in foods, cosmetics, pharmaceuticals, industrial products, medical products, laboratory culture growth media, and many other applications.

[0048] As used herein, the term "gene" refers to a polynucleotide that encodes a particular protein, and may refer to only the coding region or may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0049] The term "histidine tag" refers to a string of 2 to 30 consecutive histidine residues on a recombinant polypeptide.

[0050] The term "host cell" is a cell that has been engineered to express an introduced exogenous polynucleotide.

[0051] The term "keratinocytes" refers to keratin-producing cells found in the epidermal layer of the skin.

[0052] As used herein, the term "lactamase" refers to an enzyme that hydrolyzes antibiotics that contain a lactam (cyclic amide) moiety. A "beta-lactamase" or "β-lactamase" is an enzyme that hydrolyzes antibiotics that contain a β-lactam moiety.

[0053] 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 recombinantly prepared. Non-naturally occurring collagen or elastin is recombinant collagen or recombinant elastin. In one embodiment, non-naturally occurring collagen is truncated collagen. Other non-naturally occurring collagen polypeptides include chimeric collagen. Chimeric collagen is a polypeptide in which one portion of a collagen polypeptide is adjacent to a portion of a second collagen polypeptide. For example, a collagen molecule that includes a portion of jellyfish collagen adjacent to a portion of human collagen is a chimeric collagen. In another embodiment, non-naturally occurring collagen includes a fusion polypeptide that includes 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 beta-lactamase. In one embodiment, non-naturally occurring elastin is truncated elastin. Other non-naturally occurring elastin polypeptides include chimeric elastin. Chimeric elastin is a polypeptide in which a portion of one elastin polypeptide is adjacent to a portion of a second elastin polypeptide.For example, a collagen molecule that includes a portion of jellyfish elastin adjacent to a portion of human elastin is a chimeric elastin.In another embodiment, non-naturally occurring elastin includes a fusion polypeptide that includes additional amino acids, such as secretion tag, histidine tag, green fluorescent protein, protease cleavage site, and / or beta-lactamase.Chimeric gelatin or chimeric elastin can include additional amino acids, such as secretion tag, histidine tag, green fluorescent protein, protease cleavage site, GEK repeat, GDK repeat, and / or beta-lactamase.

[0054] The term "protease cleavage site" is an amino acid sequence that is cleaved by a particular protease.

[0055] The term "secretion tag" or "signal peptide" refers to an amino acid sequence that recruits the cellular machinery of a host cell to transport an expressed protein to a specific location or organelle within the host cell.

[0056] The term "truncated collagen" refers to a monomeric polypeptide that is smaller than full-length collagen and in which one or more portions of full-length collagen are absent. The collagen polypeptide is truncated at the C-terminus, N-terminus, or by removal of an internal portion of the full-length collagen polypeptide.

[0057] The term "truncated elastin" refers to a monomeric polypeptide that is smaller than full-length elastin and in which one or more portions of full-length elastin are absent. The elastin polypeptide is truncated at the C-terminus, N-terminus, or by removal of an internal portion of the full-length elastin polypeptide.

[0058] In co-pending application PCT / US17 / 24857, incorporated by reference, an expression system was disclosed that uses engineered bacterial cells (switch cells) in which cell division is inhibited and growth of the periplasmic space is greatly enhanced. In this expression system, the expressed protein is targeted to the periplasmic space. Recombinant protein production in these switch cells is dramatically increased compared to that in non-switch cells. Structurally, the cells contain both inner and outer membranes but lack a functional peptidoglycan cell wall, while the cell shape is spherical and increases in volume over time. Notably, while the periplasmic space typically constitutes only 10-20% of the total cell volume, the periplasmic compartment of the switch states described herein may constitute more than 20%, 30%, 40% or 50% and up to 60%, 70%, 80% or 90% of the total cell volume.

[0059] The modified bacterial cells of PCT / US17 / 24857 are derived from a Gram-negative bacterium, e.g., selected from Gammaproteobacteria and Alphaproteobacteria. In some embodiments, the bacterium is selected from Escherichia coli, Vibrio natriegens, Pseudomonas fluorescens, Caulobacter crescentus, Agrobacterium tumefaciens, and Brevundimonas diminuta. In certain embodiments, the bacterium is Escherichia coli, e.g., strain BL21(DE3).

[0060] In another aspect, the host bacterial cell has an expanded periplasmic space in a culture medium containing magnesium salts, and the concentration of magnesium ions in the medium is at least about 3, 4, 5, or 6 mM. In further embodiments, the concentration of magnesium ions in the medium is at least about 7, 8, 9, or 10 mM. In some embodiments, the concentration of magnesium ions in the 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.

[0061] In other embodiments, the culture medium further comprises an osmotic stabilizer, including, for example, a sugar (e.g., arabinose, glucose, sucrose, glycerol, sorbitol, mannitol, fructose, galactose, saccharose, maltotriose, erythritol, ribitol, pentaerythritol, arabitol, galactitol, xylitol, iditol, maltotriose, etc.), betaine (e.g., trimethylglycine), proline, sodium chloride, and the concentration of the osmotic stabilizer in the medium is at least 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 medium is about 5% to about 20% (w / v).

[0062] In some embodiments, the cell culture may further include ammonium chloride, ammonium sulfate, calcium chloride, amino acids, ferrous sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate, and yeast extract.

[0063] The host bacterial cells may be cultured continuously or discontinuously in a batch process, a fed-batch process or a repeated fed-batch process.

[0064] In some embodiments, the antibiotic is selected from β-lactam antibiotics (e.g., penicillins, cephalosporins, carbapenems, and monobactams), phosphonate antibiotics, polypeptide antibiotics, and glycopeptide antibiotics. In certain embodiments, the antibiotic is selected from alafosfalin, amoxicillin, ampicillin, aztreonam, bacitracin, carbenicillin, cefamandole, cefotaxime, cefsulodin, cephalothin, fosmidomycin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, fosfomycin, primaxine, and vancomycin.

[0065] Without wishing to be bound by theory, it is believed that the cell morphology that promotes recombinant protein production and inhibits cell division is driven by the removal of the cell wall under the above-mentioned medium conditions. In some embodiments, the method for the removal / inhibition of cell wall synthesis may be by the use of antibiotics that inhibit peptidoglycan synthesis (e.g., ampicillin, carbenicillin, penicillin, or fosfomycin) or other methods known in the art.

[0066] If it has a suitable periplasmic targeting signal sequence, recombinantly produced polypeptides can be secreted into the periplasmic space of bacterial cells. Joly, JC and Laird, MW, in The Periplasm ed. Ehrmann, M., ASM Press, Washington DC, (2007) 345-360. The chemically oxidizing environment of the periplasm promotes the formation of disulfide bonds and thus functionally correct folding of polypeptides.

[0067] In general, the signal sequence may be a component of the expression vector, or it may be part of the exogenous gene that is inserted into the vector. The signal sequence selected should be recognized and processed by the host cell (i.e., cleaved by signal peptidase). For bacterial host cells that do not recognize and process the native signal sequence of the exogenous gene, the signal sequence is replaced with any commonly known bacterial signal sequence. In some embodiments, recombinantly produced polypeptides can be targeted to the periplasmic space using the DsbA signal sequence. Dinh and Bernhardt, JBacteriol, Sept. 2011, 4984-4987.

[0068] In one aspect, non-naturally occurring collagen or elastin produced by host cell is provided.Non-naturally occurring collagen or elastin is jellyfish collagen or elastin, human collagen or elastin, Chondrosia reniformis collagen or elastin, or Rhincodon typus collagen or elastin.Non-naturally occurring collagen or elastin is truncated collagen.Truncation is internal truncation, truncation at N-terminal part of collagen or elastin, or truncation at C-terminal part of collagen or elastin. Collagen or elastin may be truncated by truncation of 50 to 1000 amino acids, 50 to 950 amino acids, 50 to 900 amino acids, 50 to 850 amino acids, 50 to 800 amino acids, 50 to 850 amino acids, 50 to 800 amino acids, 50 to 750 amino acids, 50 to 700 amino acids, 50 to 650 amino acids, 50 to 600 amino acids, 50 to 650 amino acids, 50 to 500 amino acids, 50 to 450 amino acids, 50 to 400 amino acids, 50 to 350 amino acids, 50 to 300 amino acids, 50 to 250 amino acids, 50 to 200 amino acids, 50 to 150 amino acids or 50 to 100 amino acids. In another embodiment, the collagen or elastin is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 36 shortened by 0, 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 portions of or the entire polynucleotide sequences disclosed herein.

[0069] The non-naturally occurring collagen or elastin further comprises an amino acid sequence that includes a secretion tag. The secretion tag directs the collagen or elastin to the periplasmic space of the host cell. In certain embodiments, the signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA or HylA, or from a hybrid secretion tag that includes a portion of one secretion tag fused to a portion of a second secretion tag. In one aspect, the secretion tag is attached to the non-naturally occurring collagen or elastin. In another aspect, the secretion tag is cleaved from the non-naturally occurring collagen or elastin.

[0070] The non-naturally occurring collagen or non-naturally occurring elastin further comprises a histidine tag. A histidine tag or polyhistidine tag is a sequence of 2-20 histidine residues attached to the collagen or elastin. The histidine tag comprises 2-20 histidine residues, 5-15 histidine residues, 5-18 histidine residues, 5-16 histidine residues, 5-15 histidine residues, 5-14 histidine residues, 5-13 histidine residues, 5-12 histidine residues, 5-11, 5-10 histidine residues, 6-12 histidine residues, 6-11 histidine residues or 7-10 histidine residues. The histidine tag is useful in purifying proteins by chromatographic methods utilizing 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 non-naturally occurring elastin comprises GFP and / or RFP. In one embodiment, superfolder GFP is a naturally occurring The histidine tag is attached to a non-naturally occurring collagen or elastin. Superfolder GFP is a GFP that folds properly even when fused to a poorly folded polypeptide. In one embodiment, the histidine tag is attached to a non-naturally occurring collagen or elastin. In another embodiment, the histidine tag is cleaved from a non-naturally occurring collagen or elastin.

[0071] The non-naturally occurring collagen or non-naturally occurring elastin further comprises a protease cleavage site. The protease cleavage site is useful for cleaving recombinantly produced collagen or elastin to remove a portion of the polypeptide. The portion of the polypeptide that can be removed includes secretion tags, histidine tags, fluorescent protein tags, and / or beta-lactamase. Proteases include endoproteases, exoproteases, serine proteases, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, and metalloproteases. Exemplary protease cleavage sites include amino acids that are cleaved by thrombin, TEV protease, factor Xa, enteropeptidase, and rhinovirus 3C protease. In one embodiment, the cleavage tag is attached to the non-naturally occurring collagen or elastin. In another embodiment, the cleavage tag is removed from the non-naturally occurring collagen or elastin by a suitable protease.

[0072] The non-naturally occurring collagen or non-naturally occurring elastin further comprises an enzyme that is a beta-lactamase. The beta-lactamase is useful as a selectable marker. In one embodiment, the beta-lactamase is attached to the non-naturally occurring collagen or elastin. In another embodiment, the beta-lactamase is cleaved from the non-naturally occurring collagen or elastin.

[0073] The non-naturally occurring collagen or non-naturally occurring elastin further comprises GEK amino acid trimer repeats and / or GDK amino acid trimer repeats. The GEK and GDK trimer repeats promote gelation of the collagen and / or gelatin. In one embodiment, the non-naturally occurring collagen or non-naturally occurring elastin comprises 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 2-15 GDK trimer repeats, 2-10 GEK and / or 2-10 GDK trimer repeats. and / or 2-10 GDK trimer repeats, 2-9 GEK and / or 2-9 GDK trimer repeats, 2-8 GEK and / or 2-8 GDK trimer repeats, 2-7 GEK and / or 2-7 GDK trimer repeats, 2-6 GEK and / or 2-6 GDK trimer repeats, 2-5 GEK and / or 2-5 GDK trimer repeats or 2-4 GEK and / or 2-4 GDK trimer repeats. In one embodiment, the GEK trimer repeats or GDK trimer repeats are attached to a non-naturally occurring collagen or elastin. In another embodiment, the GEK trimer repeats or GDK trimer repeats are cleaved from a non-naturally occurring collagen or elastin.

[0074] Provided herein are compositions comprising 0.005%-30% w / w non-naturally occurring collagen and / or non-naturally occurring elastin, including 0.005%-20% w / w non-naturally occurring collagen and / or elastin, 0.005%-10% w / w non-naturally occurring collagen and / or elastin, 0.005%-5% w / w non-naturally occurring collagen and / or elastin, 0.005%-2% w / w non-naturally occurring collagen and / or elastin, 0.005%-1% w / w non-naturally occurring collagen and / or elastin, 0.005%-0.5% w / w non-naturally occurring collagen and / or elastin, and 0.005%-0.2% w / w non-naturally occurring collagen and / or elastin.

[0075] The composition comprising non-naturally occurring collagen and / or non-naturally occurring elastin is a personal care product. In some embodiments, the composition is formulated for topical administration. The composition may contain other cosmetic ingredients suitable for use in humans. The personal care product is useful for preventing or treating UV damage to human skin or hair. The personal care product is applied to skin or hair. The composition may include, for example, masks, skin cleaners such as soaps, cleansing creams, cleansing lotions, cleansing milks, cleansing pads, face washes, hair shampoos, hair conditioners and body shampoos.

[0076] The compositions comprising non-naturally occurring collagen and / or non-naturally occurring elastin may further comprise at least one additional component, including a topical carrier or a preservative. Topical carriers include liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oils, triglycerides, and the like. Lido oil, silicone oil, glycerin, glycerin monostearate, alcohol, emulsifier, liquid petroleum, white petrolatum, propylene glycol, polyethylene The topical carrier is selected from the group consisting of polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane and water. The preservative is selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, cis-isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract and EDTA.

[0077] A method is provided for reducing skin damage, promoting repair of damaged skin, protecting skin from UV damage, or protecting skin cells from the effects of exposure to urban dust. The method includes applying a composition comprising non-naturally occurring collagen and / or non-naturally occurring elastin to the skin of a subject. Without being bound by a particular theory or mechanism, the collagen and / or elastin in the composition reduces skin damage by protecting against UV damage, and / or promotes repair of damaged skin by increasing cell viability and / or increasing procollagen synthesis when applied to the skin, and / or promotes skin cell viability. In one embodiment, the collagen and elastin reduce the formation of thymine-thymine (TT) dimer formation.

[0078] One embodiment provides a polynucleotide encoding a non-naturally occurring collagen or a non-naturally occurring elastin. The polynucleotide encodes a collagen or elastin from a jellyfish, a human, Chondrosia reniformis, or Rhincodon typus. The polynucleotide encodes a full-length or truncated collagen or elastin.

[0079] Another embodiment provides a polynucleotide encoding a collagen or elastin fusion protein comprising a collagen or elastin together with a secretion tag, a histidine tag, a fluorescent protein tag, a protease cleavage site, a beta-lactamase and / or a GEK amino acid trimer repeat and / or a GDK amino acid trimer repeat.

[0080] In one embodiment, the polynucleotide is a vector used to transform a host cell and express the polynucleotide. The polynucleotide further comprises a nucleic acid encoding an enzyme that allows the host organism to grow in the presence of a selection agent. Selection agents include specific sugars, including galactose-containing sugars, or antibiotics, including ampicillin, hygromycin, G418, etc. Enzymes used to confer resistance to selection agents include β-galactosidase or β-lactamase.

[0081] In one aspect, a host cell is provided that expresses the polynucleotide of the present invention.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 cells used to express exogenous polynucleotides.An exemplary gram-negative host cell is Escherichia coli.

[0082] A bacterial host cell modified to inhibit cell division and increase periplasmic space is taught. As discussed herein and taught in Example 1, beta-lactam antibiotics are useful as switches to convert wild-type bacterial cells into modified bacterial cells with inhibited cell replication and increased periplasmic space. Exemplary beta-lactam antibiotics include penicillins, cephalosporins, carbapenems and monobactams.

[0083] The bacterial switch form (L-form) is cultivated in a culture medium containing specific salts and other nutrients. The salts and medium compositions that support physiological switch physiology that have been tested are M63 salts medium, M9 salts medium, PYE medium, and Luria-Bertani (LB) medium. Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources can also be included at appropriate introduction concentrations, either alone or as a mixture with another supplement or medium, such as a complex nitrogen source. In certain embodiments, the medium further comprises one or more components selected from ammonium chloride, ammonium sulfate, calcium chloride, casamino acids, iron (II) sulfate, magnesium sulfate, peptone, potassium phosphate, sodium chloride, sodium phosphate, and yeast extract.

[0084] Beta-lactamase is an enzyme that confers resistance to lactam antibiotics in prokaryotic cells. Typically, when beta-lactamase is expressed in bacterial host cells, the expressed beta-lactamase protein also contains a targeting sequence (secretion tag) that directs the beta-lactamase protein to the periplasmic space. Beta-lactamases do not function unless they are transported to the periplasmic space. Provided are beta-lactamases that are targeted to the periplasm without the use of a separate secretion tag to target the enzyme to the periplasmic space. By creating a fusion protein with a periplasmic secretion tag added to the N-terminus of a protein such as GFP, collagen or a GFP / collagen chimera, the function of the beta-lactamase that lacks the native secretion tag can be used to select for complete translation and secretion of the N-terminal fusion protein. Using this approach, we used DsbA-GFP-collagen-beta-lactamase fusion to select for truncated products in the target collagen that favor translation and secretion.

[0085] Another embodiment provides a method of producing a non-naturally occurring collagen or non-naturally occurring elastin, comprising the steps of inoculating a culture medium with a recombinant host cell comprising a polynucleotide encoding a collagen or elastin, culturing the host cell, and isolating the non-naturally occurring collagen or non-naturally occurring elastin from the host cell.

[0086] A process for the fermentative preparation of a protein is provided, the process comprising: a) culturing a recombinant Gram-negative bacterial cell in a medium comprising a magnesium salt, wherein the concentration of magnesium ions in said medium is at least about 6 mM, and said bacterial cell comprises an exogenous gene encoding a protein; b) adding an antibiotic to the medium, wherein the antibiotic inhibits peptidoglycan biosynthesis in the bacterial cells; and c) recovering the protein from the medium. Includes.

[0087] For the purpose of producing target proteins, bacteria can be cultured continuously, for example as described in WO 05 / 021772, or discontinuously in a batch process (batch culture) or in a fed-batch or repeated fed-batch process. In some embodiments, protein production is performed on a large scale. For the production of recombinant proteins, various large-scale fermentation procedures are available. Large-scale fermentations have a capacity of at least 1,000 liters, preferably about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermenters with a volumetric capacity not exceeding about 20 liters.

[0088] For the accumulation of target protein, the host cell is cultured under conditions sufficient for the accumulation of target protein. Such conditions include, for example, temperature, nutrients and cell density conditions that allow the cell to express and accumulate protein. Also, as known to those skilled in the art, such conditions are conditions that allow the cell to carry out the basic cellular functions of transcription, translation, and in the case of secretory protein, the passage of protein from one cellular compartment to another.

[0089] The bacterial cells are cultured at a suitable temperature. For E. coli growth, for example, typical temperatures range from about 20°C to about 39°C. In one embodiment, the temperature is about 20°C to about 37°C. In another embodiment, the temperature is about 30°C. In one embodiment, the non-switched or switched host cells are cultured at one temperature and switched to a different temperature to induce protein production. First, the host cells are cultured at one temperature to grow the cells, and then the cells are 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. Cultivation at the second temperature is performed for 1 hour to 100 hours, 5 hours to 90 hours, 5 hours to 80 hours, 5 hours to 80 hours, 5 hours to 70 hours, 10 hours to 70 hours, 15 hours to 70 hours, 15 hours to 65 hours, 15 hours to 60 hours, 20 hours to 60 hours, 20 hours to 55 hours, 20 hours to 50 hours, 24 hours to 50 hours, 24 hours to 48 hours, 30 hours to 50 hours, 30 hours to 45 hours, or 30 hours to 40 hours.

[0090] The pH of the culture medium can be any pH between about 5 and 9, depending primarily on the host organism. For E. coli, the pH is between about 6.8 and about 7.4 or about 7.0.

[0091] Induction of gene expression typically involves culturing cells until a certain optical density, e.g., an OD600 of about 1.1, is reached, at which point induction is initiated (e.g., by addition of an inducer, by repressor, suppressor, or by depletion of media components) to induce expression of the exogenous gene encoding the target protein. In some embodiments, expression of the exogenous gene is inducible by an inducer selected from, e.g., isopropyl-β-d-1-thiogalactopyranoside, lactose, arabinose, maltose, tetracycline, anhydrotetracycline, bubricin, xylose, copper, zinc, and the like. Induction of gene expression can also be achieved by reducing the dissolved oxygen level during fermentation. The dissolved oxygen level of the fermentation during cell growth is 10%-30%. To induce gene expression, the dissolved oxygen level is reduced to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%. In either the physiological state or the switched state host cells, protein production can be induced by reducing the temperature of fermentation as disclosed herein.

[0092] After product accumulation, the cells are vortexed and centrifuged to induce lysis and release of the recombinant protein. The majority of the protein is found in the supernatant, but a detergent (e.g., triton X-100) can be used to release any remaining membrane-bound protein.

[0093] Subsequent steps recover the target protein as a soluble or insoluble product released from the cell matrix in a manner that minimizes co-recovery of cellular debris with the product. Recovery can be by any means, but in one embodiment can include histidine tag purification on a nickel column. See, e.g., Purification of Proteins Using Polyhistidine Affinity Tags, Methods Enzymology. 2000;326:245-254. EXAMPLES

[0094] Example 1: Expression System material and method: KK: Physiological switches and protein production tested: From E. coli BL21(DE3)-NEB, product #c2527 E. coli K12NCM3722 - From The Coli Genetic Stock Center, CGSC#12355

[0095] Physiological switches tested: Gammaproteobacteria: Vibrio natriegens - From ATCC, product #14048 Pseudomonas fluorescens - ATCC, product #31948 Pseudomonas aeruginosa PAO1-ATCC, product #BAA-47

[0096] Alphaproteobacteria: Caulobacter crescentus - ATCC, product #19089 Agrobacterium tumefaciens / Rhizobium radiobacter - From ATCC, product #33970 Brevundimonas diminuta - From ATCC, product #13184

[0097] Media composition: 1 liter 5xM63 salt: From 10g of (NH4)2SO4-P212121, product #7783-20-2 From 68g of KH2PO4-P212121, product #7778-77-0 2.5mg of FeSO4.7H2O - from Sigma Aldrich, product #F7002 Bring volume up to 1 liter with milliQ water. Adjust to pH 7 with KOH (from P212121, product #1310-58-3). The mixture is autoclaved.

[0098] 1 liter of 1M MgSO4: From 246.5g of MgSO4 7H2O-P212121, (Sigma Aldrich, product #10034-99-8) Bring volume up to 1 liter with milliQ water. The mixture is autoclaved.

[0099] Switch medium 1 for 1 liter: 133.4mL of 5xM63 salt 10mL of 1M MgSO4 38.6g of Glucose - From P212121, product #50-99-7 66.6g of sucrose - from P212121, product #57-50-1 8.33g LBmix-P212121, product #lb-miller Bring volume up to 1 liter with milliQ water. The mixture is filter sterilized through a 0.22 μM pore vacuum filter (Sigma Aldrich, product #CLS430517).

[0100] 1 liter of Switch Medium 2: 133.4mL of 5xM63 salt 10mL of 1M MgSO4 38.6g of Glucose - From P212121, product #50-99-7 66.6g of sucrose - from P212121, product #57-50-1 10g Yeast Extract - FisherSci.com, product #J60287 Al Bring volume up to 1 liter with milliQ water. The mixture is filter sterilized through a 0.22 μM pore vacuum filter (Sigma Aldrich, product #CLS430517).

[0101] For bioreactor growth: 5 liters of bioreactor medium MGZ12: 1) Autoclave 1L of 500g / L glucose in DI water. (VWR, product #97061-170). 2) Autoclave 1 L of 500 g / L sucrose in DI water. (Geneseesci.com, product #62-112). 3) In 3946 mL of DI water: 20g of (NH4)2HPO4. (VWR, product #97061-932). 66.5g KH2PO4. (VWR, product #97062-348). 22.5g of H3C6H5O7. (VWR, Product #BDH9228-2.5KG). 2.95g MgSO4.7H2O. (VWR, product #97062-134). 10mL Trace Metals (Teknova), 1000x. (Teknova, product #T1001). Autoclave the mixture. After autoclaving, 400 mL of (1) is added to (3), 65 mL of 10 M NaOH (VWR, product #97064-480) is added to (3), and 666 mL of (2) is added to (3). A feed of 500 g / L glucose may be used during the fermentation run if necessary. When induced, Add 50 mL of 1 M MgSO4.7H2O to the 5 L bioreactor, Add IPTG to a concentration of 1-10 mM. (carbosynth.com, product #EI05931). Add fosfomycin (50 μg / mL or greater) and carbenicillin (100 μg / mL or greater).

[0102] Physiological Switch: The physiological switch is optimally flipped at an OD600 of 1-1.1 for E. coli for growth in shake flasks up to 1 L volume. For other species tested, cultures were grown in switch medium and subcultured once the cultures reached maximum OD600. In all cases, the physiological switch is flipped by the addition of 100-200ug / mL carbenicillin (P212121, from product #4800-94-6) and 50-100ug / mL fosfomycin (P212121, from product #26016-99-9). The majority of the population is in the switched state within a few hours. To confirm that the cells had undergone the physiological switch, cells were imaged with a Nikon Ti-E equipped with a perfect focus system, a Nikon CFI60 Plan Apo 100X NA 1.45 objective, a Prior automated filter wheel and stage, LED-CFP / YFP / mCherry and LED-DA / FI / TX filter sets (Semrock), a Lumencor Sola II SE LED illumination system and a Hamamatsu Flash 4.0 V2 CMOS camera.

[0103] Image analysis of physiological switches: Images were analyzed using ImageJ to measure dimensions. In the switched state, the spherical contour of the outer membrane is treated as a sphere to calculate the total volume (V = (4 / 3) 7πr3). The cytoplasmic volume is calculated as an ellipsoid present within a sphere (V = (4 / 3) π * (longest radius) * (shortest radius) 2). To calculate the periplasmic volume, the cytoplasmic volume is subtracted from the total volume of the cell.

[0104] Protein Expression and Quantitation: E. coli BL21(DE3) (NEB product #c2527) containing pET28a (emd Millipore product #69864) with GFP or collagen derivatives and its derivatives were grown overnight at 37°C in switch medium containing 50mg / mL kanamycin (p212121 product #2251180) in a shaking incubator. Subcultures are then started the next day by placing a 1:10 dilution of the overnight culture into fresh switch medium containing 50mg / mL kanamycin. Cultures are then physiologically switched and protein production is simultaneously induced at an OD600 of 1-1.1 (read by Molecular Devices Spectramax M2 microplate reader). Physiological switch and protein production are switched on by the addition of 100ug / mL carbenicillin, 50ug / mL fosfomycin and 100ug / mL IPTG (p212121 product #367-93-1). Protein expression is allowed to continue for 8 hours to overnight at room temperature (approximately 22°C) in the switched state on an orbital shaker. To quantify total protein levels, a Quick Start™ Bradford protein assay was used on a mixed portion of the culture, and a standard curve was quantified with a Molecular Devices Spectramax M2 microplate reader. To quantify the relative intensity of target protein production to the rest of the protein population, a mixed portion of the culture was run on a Mini-PROTEAN® TGX™ gel and stained with Bio-Safe™ Coomassie stain.

[0105] Induction of protein production: Protein production was induced under physiological conditions according to standard procedures. We used strain BL21(DE3) containing the plasmid pET28a driving IPTG / lactose inducible production of recombinant proteins and targeting them to the periplasmic space using the DsbA signal sequence. Using the GFP protein targeted to the periplasmic space, we demonstrated the ability to increase protein production, increasing it by 5-fold when compared to a non-switched cell population induced at the same optical density for the same amount of time (Figure). Induction was optimal at an OD600 of 1.1 and induction was continued for 10 hours, at which point we measured the protein produced to be approximately 200 mg / mL.

[0106] Example 2: Production of full-length collagen Full-length jellyfish collagen was produced using the expression system discussed herein in Example 1. The wild-type full-length amino acid sequence of Podocoryna carnea (jellyfish or Hydrozoa) collagen is provided in SEQ ID NO:1. [ka]

[0107] The non-codon optimized polynucleotide sequence encoding the full-length jellyfish collagen is disclosed in SEQ ID NO:2. [ka]

[0108] Two different codon-optimized polynucleotide sequences encoding wild-type full-length jellyfish collagen were synthesized. The two polynucleotide sequences differed slightly due to slightly different codon optimization methods. In addition to the non-truncated full-length jellyfish collagen, the polynucleotides also encoded a secretion tag, a nine amino acid his tag, a short linker and a thrombin cleavage site. The DsbA secretion tag is encoded by nucleotides 1-71. The histidine tag, which contains nine histidine residues, is encoded by nucleotides 73-99 and encodes amino acids 25-33. The linker is encoded by nucleotides 100-111. The thrombin cleavage tag is encoded by nucleotides 112-135 and encodes amino acids 38-45. The truncated collagen is encoded by nucleotides 136-1422. The two polynucleotides are disclosed below in SEQ ID NOs: 3 and 4. [ka] [ka]

[0109] The amino acid sequences encoded by the polynucleotides of SEQ ID NO:3 and SEQ ID NO:4 are disclosed below in SEQ ID NO:5. 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 nine 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. [ka]

[0110] The full length jellyfish collagen without the DsbA secretion tag, histidine tag, linker and thrombin cleavage site is disclosed in SEQ ID NO:89. [ka]

[0111] Polynucleotides SEQ ID NO:3 and SEQ ID NO:4 were synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28 vector and SEQ ID NO:3 and SEQ ID NO:4 was designed to be 30-40 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 cleaved pET28a vector and insert DNA (SEQ ID NO:3 or SEQ ID NO:4) were then assembled together into the final plasmid using SGI Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc). The sequences of the plasmids were then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0112] The transformed cells were grown in minimal medium and frozen in 1.5 aliquots with glycerol at a cell to glycerol ratio of 50:50. One vial of this frozen culture was allowed to recover overnight in 50 ml of minimal medium at 37°C and 200 rpm. The cells were transferred to 300 ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

[0113] The minimal medium used in this example and throughout this application is prepared as follows: Minimal medium (Table 1) was autoclaved with several individual fractions, salt mix (diammonium phosphate, potassium dihydrogen phosphate, anhydrous citric acid, magnesium sulfate heptahydrate), 500 g / L sucrose, 55% glucose, trace metals TM5 (Table 2) and sodium hydroxide 10 M. The minimal medium was then mixed together in the hood at the above concentrations after autoclaving. [Table 1] [Table 2]

[0114] The harvested cells were disrupted twice with a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen protein along with other proteins.

[0115] Collagen was purified by acid treatment of homogenized cell broth. The pH of the homogenized slurry was reduced 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 examined in a polyacrylamide gel and found to contain relatively high amounts of collagen compared to the starting pellet. The collagen slurry thus obtained was rich in salts. To achieve volume and salt reduction, 0.1 ml of each was added. 2 The concentration and diafiltration steps were carried out using an EMD Millipore tangential flow filtration system with an ultrafiltration cassette of 0.2 m. The total filtration area was 0.2 m using two cassettes in parallel. 2 The TFF step achieved a 5-fold volume reduction and a 19-fold salt reduction. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was dried for 3 days using a multi-tray freeze dryer to yield a white fluffy collagen powder.

[0116] Analysis of the purified collagen on an SDS-PAGE gel revealed a thick, distinct band at the expected size of 42 kilodaltons, and analysis of the purified collagen by mass spectrometry confirmed that the 42 kilodalton protein was jellyfish collagen.

[0117] Fermentations were carried out at various temperatures ranging from 25°C to 28°C. In some fermentations, the temperature of the fermentation was maintained at a constant temperature and collagen was purified immediately upon completion of the fermentation (OD600 of 5-10). In other fermentations, the temperature of the fermentation was maintained for the desired period and once a cell density of OD600 of 5-10 was reached, the temperature was reduced to induce protein production. Typically, the temperature was reduced from 28°C to 25°C. Fermentation at 25°C was continued for 40-60 hours before collagen isolation.

[0118] More full-length jellyfish collagen Full length jellyfish collagen without the His tag, linker and thrombin cleavage site is disclosed below. Two codon optimized nucleotide sequences encoding this collagen are provided in SEQ ID NO:6 and SEQ ID NO:7. The differences in the nucleotide sequences are due to different codon optimization strategies, but 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. [ka] [ka] [ka]

[0119] Example 3: Production of truncated collagen A codon-optimized DNA sequence optimized for expression in E. coli encoding a jellyfish collagen with a truncation of 240 internal amino acids was synthesized and expressed. The DNA sequence is shown below in SEQ ID NO:9. 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, which contains nine 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. [ka]

[0120] The truncated collagen is approximately 54% of the full length collagen and is disclosed below in SEQ ID NO:10. [ka]

[0121] A polynucleotide encoding a truncated jellyfish collagen without the DsbA secretion tag, histidine tag, linker and thrombin cleavage site is disclosed in SEQ ID NO:85. [ka]

[0122] A truncated jellyfish collagen without the DsbA secretion tag, histidine tag, linker and thrombin cleavage site is disclosed in SEQ ID NO: 86. [ka]

[0123] The polynucleotide of SEQ ID NO:9 was codon optimized and synthesized by Gen9 DNA (currently Ginkgo Bioworks in-house synthesis). The overlap between the pET28 vector and SEQ ID NO:9 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:9) were then assembled together into the final plasmid using SGI Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc). The sequence of the plasmid was then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0124] The transformed cells were grown in minimal medium and frozen in 1.5 aliquots with glycerol at a cell to glycerol ratio of 50:50. One vial of this frozen culture was allowed to recover overnight in 50 ml of minimal medium at 37°C and 200 rpm. The cells were transferred to 300 ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

[0125] A bioreactor was prepared with 2.7 L of minimal medium + glucose and 300 ml of culture with an OD600 of 5-10 was added to bring the starting volume to 3 L. Cells were grown at 28°C, pH 7, with agitation, air and oxygen containing cascade to maintain dissolved oxygen at 20% saturation. A 28% w / w ammonium hydroxide solution was used to control pH. Once the initial bolus of 40 g / L was exhausted in approximately 13 hours, fermentation was run in fed-batch mode using a DO-stat based feeding algorithm. After 24-26 hours of initial growth, OD600 reached >100. At this point, 300 mL of 500 g / L sucrose was added and the temperature was reduced to 25°C. High density cultures were induced for protein production using 1 mM IPTG. Fermentation was continued for an additional 20-24 hours and cells were harvested using a benchtop centrifuge at 9000rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended in a pH 8 buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at a weight ratio of 2x buffer to 1x cells.

[0126] The harvested cells were disrupted twice with a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen protein along with other proteins.

[0127] Fermentations were carried out at various temperatures ranging from 25°C to 28°C. In some fermentations, the temperature of the fermentation was maintained at a constant temperature and collagen was purified immediately upon completion of the fermentation (OD600 of 5-10). In other fermentations, the temperature of the fermentation was maintained for the desired period and once a cell density of OD600 of 5-10 was reached, the temperature was reduced to induce protein production. Typically, the temperature was reduced from 28°C to 25°C. Fermentation at 25°C was continued for 40-60 hours before collagen isolation.

[0128] Collagen was purified by acid treatment of homogenized cell broth. In addition, acid treatment was also performed on non-homogenized whole cells harvested from the bioreactor after centrifugation and resuspension in the above buffer. The pH of both homogenized slurries of resuspended whole cells was reduced to 3 using 6M hydrochloric acid. The acidified cell slurries were incubated overnight at 4°C with mixing, followed by centrifugation. The supernatants of the acidified slurries were examined in polyacrylamide gels and found to contain relatively high amounts of collagen compared to the starting pellets. The collagen slurries thus obtained were salt-rich. To achieve volume and salt reduction, 0.1 ml of each was added. 2 The concentration and diafiltration steps were carried out using an EMD Millipore tangential flow filtration system with an ultrafiltration cassette of 0.2 m. The total filtration area was 0.2 m using two cassettes in parallel. 2 The TFF step achieved a 5-fold volume reduction and a 19-fold salt reduction. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was dried for 3 days using a multi-tray freeze dryer to yield a white fluffy collagen powder.

[0129] Purified truncated collagen from homogenized cell broth or non-homogenized cells was analyzed on SDS-PAGE gels, revealing a thick, distinct band at the expected size of 27 kilodaltons, and analysis of the purified collagen by mass spectrometry confirmed that the 27 kilodalton protein was jellyfish collagen.

[0130] Alternative purification methods for full-length and truncated collagen are presented below.

[0131] The fermentation broth was mixed with 0.3-0.5% w / v polyethylimine (PEI). After 15 min incubation with PEI, the fermentation broth was centrifuged at 9000 rcf for 15 min to collect the supernatant containing collagen protein. The pellet containing the cells was discarded and the PEI-treated collagen-containing supernatant was mixed with sodium bentonite (0.2% w / v final) (Wyopure®, Wyoming Bentonite) and centrifuged. The bentonite-containing pellet was discarded and the supernatant was collected.

[0132] The bentonite-treated supernatant was concentrated 3-6 times by a tangential flow filtration system (TFF) (EMD Millipore) using a 5 kDa cassette. Collagen was retained with little loss of permeate flow. The retentate from the concentration step was diafiltered using the same TFF setup to remove salts. The final conductivity of the protein solution was less than 10 millisiemens. Typical conductivities were 400 microsiemens to 1.5 millisiemens. Highly concentrated collagen solutions had higher conductivities approaching 4 millisiemens. Those skilled in the art will appreciate that conductivities above 10 millisiemens may be observed depending on the concentration of collagen. The desalted and concentrated protein was then subjected to activated carbon treatment using WL 9000 10x40 granular resin (Carbon Activated Corporation). 5% w / v of carbon resin was mixed with the collagen-containing protein feed and mixed at 45-50°C with gentle agitation. Ertel in the presence or absence of a filter aid such as diatomaceous earth (Sigma Aldrich). The carbon-treated slurry was filtered using a Buchner funnel lined with a Filter Press Pad M-953 (Ertel Alsop). After filtration, the collagen solution was filtered through a 0.2 micron filter and subsequently treated with sodium bentonite (final 0.2% w / v) (Wyopure®, Wyoming Bentonite) for one to several hours and centrifuged at 9000 rcf for 15-30 minutes to obtain a clear, particle-free collagen solution of high purity. If removal of endotoxin proteins was desired, the protein was passed through a chromatographic filter such as Sartobind-Q (Sartorius-Stedim) to specifically remove endotoxin proteins.

[0133] When purified collagen was analyzed on an SDS-PAGE gel, a thick, distinct band was observed at 30 kilodaltons. The size upshift is due to the structure of the collagen molecule and its high glycine / proline amino acid content. Analysis of the purified collagen by mass spectrometry confirmed that the 30 kilodalton protein was a truncated collagen.

[0134] The truncated collagen was further analyzed by HPLC using an Agilent 1100 series HPLC. The column was a 50 mm Agilent PLRP-S reversed phase column with an internal diameter of 4.6 mm, μM particle size and a pore size of 1000 Angstroms.

[0135] Samples were prepared by diluting 1:1 with 0.04% sodium azide solution in HPLC grade water. After dilution, the resulting mixture was filtered through a 0.45um filter to remove any large particles that may clog the HPLC column. For analysis, the sample is appropriately diluted with 20mM ammonium acetate buffer in HPLC grade water at a pH of approximately 4.5. After mixing the sample, it was transferred to a 300μl microvial, which was then placed in the autosampler. Using ChemStation, the software that operates the HPLC, analytical parameters such as sample flow rate, column temperature, mobile phase flow rate, and mobile phase composition can be varied. In one exemplary but non-limiting analysis, the parameters were: sample flow rate of 1mL / min, column temperature of 80°C, column pressure of 60-70bar, mobile phase composition of 97.9% water / 1.9% acetonitrile and 0.2% trifluoroacetic acid; analytical UV wavelength of 214.4nm, injection volume of 10μl, and sample runtime of 10min.

[0136] Under these conditions, the truncated jellyfish collagen of SEQ ID NO:91 binds approximately 5. The elution peak has an elution time of 4 minutes. The ChemStation quantifies the peak area of ​​the elution peak and calculates the protein concentration using a calibration curve that directly relates peak area to protein concentration. A calibration curve is generated using known collagen solutions serially diluted to contain a collagen concentration range of 0.06 mg / mL to 1.00 mg / mL.

[0137] His-tag linker-truncated collagen without thrombin cleavage site A truncated jellyfish collagen without the 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. [ka]

[0138] A polynucleotide encoding a truncated jellyfish collagen without the His tag, linker and thrombin cleavage site is disclosed in SEQ ID NO:90. [ka]

[0139] A truncated jellyfish collagen without the His tag, linker and thrombin cleavage site is disclosed in SEQ ID NO:91. [ka]

[0140] Truncated collagen containing GEK repeats A 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 a GEK repeat of amino acids 25-42. The truncated collagen sequence is encoded by nucleotides 127-693 and encodes amino acids 43-231. [ka]

[0141] The polynucleotide of SEQ ID NO:13 was codon optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28 vector and SEQ ID NO:13 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:13) were then codon optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28a vector and SEQ ID NO:13 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:13) were then codon optimized and synthesized by Ginkgo Bioworks in-house synthesis. They were assembled together into the final plasmid using Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc). The sequences of the plasmids were then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0142] The transformed cells were grown in minimal medium and frozen in 1.5 aliquots with glycerol at a cell to glycerol ratio of 50:50. One vial of this frozen culture was allowed to recover overnight in 50 ml of minimal medium at 37°C and 200 rpm. The cells were transferred to 300 ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

[0143] A bioreactor was prepared with 2.7 L of minimal medium + glucose and 300 ml of culture with an OD600 of 5-10 was added to bring the starting volume to 3 L. Cells were grown at 28°C, pH 7, with agitation, air and oxygen containing cascade to maintain dissolved oxygen at 20% saturation. A 28% w / w ammonium hydroxide solution was used to control pH. Once the initial bolus of 40 g / L was exhausted in approximately 13 hours, fermentation was run in fed-batch mode using a DO-stat based feeding algorithm. After 24-26 hours of initial growth, OD600 reached >100. At this point, 300 mL of 500 g / L sucrose was added and the temperature was reduced to 25°C. High density cultures were induced for protein production using 1 mM IPTG. Fermentation was continued for an additional 20-24 hours and cells were harvested using a benchtop centrifuge at 9000rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended in a pH 8 buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at a weight ratio of 2x buffer to 1x cells.

[0144] The harvested cells were disrupted twice with a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen protein along with other proteins.

[0145] Collagen was purified by acid treatment of the whole cells harvested from the bioreactor after centrifugation and resuspension in the above buffer. The pH of either the homogenized or resuspended suspension was reduced 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 examined in a polyacrylamide gel and found to contain relatively high amounts of collagen compared to the starting pellet. The collagen slurry thus obtained was rich in salts. To achieve volume and salt reduction, 0.1 ml of each was added. 2 The concentration and diafiltration steps were carried out using an EMD Millipore tangential flow filtration system with an ultrafiltration cassette of 0.2 m. The total filtration area was 0.2 m using two cassettes in parallel. 2The TFF step achieved a 5-fold volume reduction and a 19-fold salt reduction. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was dried for 3 days using a multi-tray freeze dryer to yield a white fluffy collagen powder.

[0146] The purified collagen was analyzed on SDS-PAGE gels and was observed to migrate with an apparent molecular weight of 35 kDa. The 35 kDa band does not correspond to the expected size of 22 kDa. The upshift between the expected and apparent size is likely due to the GEK repeats interacting with the gel matrix. Mass spectrometry confirmed that the 35 kDa band was the correct collagen containing GEK repeats.

[0147] Truncated collagen containing GDK repeats A jellyfish collagen with GDK repeats is disclosed below. The codon optimized nucleotide sequence encoding this collagen is shown 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 and encodes amino acids 1-24. The GDK repeats are encoded by nucleotides 73-126 and encode a GDK repeat of amino acids 25-42. The truncated collagen sequence is encoded by nucleotides 127-693 and encodes amino acids 43-231. [ka]

[0148] The polynucleotide of SEQ ID NO:15 was codon optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28 vector and SEQ ID NO:15 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:15) were then codon optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28a vector and SEQ ID NO:15 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:15) were then codon optimized and synthesized by Ginkgo Bioworks in-house synthesis. They were assembled together into the final plasmid using Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc). The sequences of the plasmids were then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0149] The transformed cells were grown in minimal medium and frozen in 1.5 aliquots with glycerol at a cell to glycerol ratio of 50:50. One vial of this frozen culture was allowed to recover overnight in 50 ml of minimal medium at 37°C and 200 rpm. The cells were transferred to 300 ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

[0150] A bioreactor was prepared with 2.7 L of minimal medium + glucose and 300 ml of culture with an OD600 of 5-10 was added to bring the starting volume to 3 L. Cells were grown at 28°C, pH 7, with agitation, air and oxygen containing cascade to maintain dissolved oxygen at 20% saturation. A 28% w / w ammonium hydroxide solution was used to control pH. Once the initial bolus of 40 g / L was exhausted in approximately 13 hours, fermentation was run in fed-batch mode using a DO-stat based feeding algorithm. After 24-26 hours of initial growth, OD600 reached >100. At this point, 300 mL of 500 g / L sucrose was added and the temperature was reduced to 25°C. High density cultures were induced for protein production using 1 mM IPTG. Fermentation was continued for an additional 20-24 hours and cells were harvested using a benchtop centrifuge at 9000rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended in a pH 8 buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at a weight ratio of 2x buffer to 1x cells.

[0151] The harvested cells were disrupted twice with a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen protein along with other proteins.

[0152] Collagen was purified by acid treatment of the whole cells harvested from the bioreactor after centrifugation and resuspension in the above buffer. The pH of both homogenized slurries was reduced to 3 using 6M hydrochloric acid. The acidified cell slurries were incubated overnight at 4°C with mixing, followed by centrifugation. The supernatant of the acidified slurry was examined in a polyacrylamide gel and found to contain relatively high amounts of collagen compared to the starting pellet. The collagen slurry thus obtained was rich in salts. To achieve volume and salt reduction, 0.1 ml of each was added. 2 The concentration and diafiltration steps were carried out using an EMD Millipore tangential flow filtration system with an ultrafiltration cassette of 0.2 m. The total filtration area was 0.2 m using two cassettes in parallel. 2The TFF step achieved a 5-fold volume reduction and a 19-fold salt reduction. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was dried for 3 days using a multi-tray freeze dryer to yield a white fluffy collagen powder.

[0153] The purified collagen was analyzed on SDS-PAGE gels and was observed to migrate with an apparent molecular weight of 35 kDa. The 35 kDa band does not correspond to the expected size of 22 kDa. The upshift between the expected and apparent size is likely due to the GDK repeats interacting with the gel matrix. Mass spectrometry confirmed that the 35 kDa band was the correct collagen containing GDK repeats.

[0154] Truncated collagen with DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP beta-lactamase fusion (version 1): A jellyfish collagen with DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP beta-lactamase fusion is disclosed below. The codon-optimized nucleotide sequence encoding this collagen is shown 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 and encodes amino acids 1-24. The His tag is encoded by nucleotides 73-99 and encodes nine histidine tags at amino acids 25-33. The linker is encoded by nucleotides 100-111 and encodes amino acids 34-37. The thrombin cleavage site is encoded by nucleotides 112-135 and encodes amino acids 38-45. The green fluorescent protein (GFP) with linker is encoded by nucleotides 136-873 and encodes amino acids 46-291. The truncated collagen sequence is encoded by nucleotides 874-1440 and encodes amino acids 292-480. The beta-lactamase with linker is encoded by nucleotides 1441-2232 and encodes amino acids 481-744. Beta-lactamase was properly targeted to the periplasmic space even when the polypeptide did not have an independent secretion tag. The DsbA secretion tag directed the entire transcript (DsbA secretion tag-His tag-linker-truncated collagen with thrombin cleavage site and GFP beta-lactamase fusion protein) to the periplasmic space and beta-lactamase functioned properly. [ka] [ka]

[0155] The polynucleotide of SEQ ID NO: 17 was constructed by assembling several DNA fragments. The collagen-containing sequence was codon-optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). GFP was also synthesized by Gen9. Beta-lactamase was cloned from the plasmid pKD46 (http: / / cgsc2.biology.yale.edu / Strain.php?ID=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 and GFP, collagen and beta-lactamase was designed to a length of 30-40 bp and added using PCR with the enzyme PrimeStar GXL polymerase. The cleaved pET28a vector and insert were then assembled together into the final plasmid using SGI Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc). The sequence of the plasmid was then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0156] The transformed cells were grown in minimal medium and frozen in 1.5 aliquots with glycerol at a cell to glycerol ratio of 50:50. One vial of this frozen culture was allowed to recover overnight in 50 ml of minimal medium at 37°C and 200 rpm. The cells were transferred to 300 ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

[0157] A bioreactor was prepared with 2.7 L of minimal medium + glucose and 300 ml of culture with an OD600 of 5-10 was added to bring the starting volume to 3 L. Cells were grown at 28°C, pH 7, with agitation, air and oxygen containing cascade to maintain dissolved oxygen at 20% saturation. A 28% w / w ammonium hydroxide solution was used to control pH. Once the initial bolus of 40 g / L was exhausted in approximately 13 hours, fermentation was run in fed-batch mode using a DO-stat based feeding algorithm. After 24-26 hours of initial growth, OD600 reached >100. At this point, 300 mL of 500 g / L sucrose was added and the temperature was reduced to 25°C. High density cultures were induced for protein production using 1 mM IPTG. Fermentation was continued for an additional 20-24 hours and cells were harvested using a benchtop centrifuge at 9000rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended in a pH 8 buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at a weight ratio of 2x buffer to 1x cells.

[0158] The harvested cells were disrupted twice with a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen protein along with other proteins.

[0159] Collagen was purified by acid treatment of non-homogenized whole cells harvested from the bioreactor after centrifugation and resuspension in the above buffer. The pH of the resuspended suspension was lowered to 3 using 6 M hydrochloric acid. The acidified cell slurry was incubated overnight at 4°C with mixing, followed by centrifugation. The pH was then raised to 9 using 10 N NaOH, and the slurry supernatant was examined in a polyacrylamide gel and found to contain relatively high amounts of collagen compared to the starting pellet. The collagen slurry thus obtained was rich in salt. To achieve volume and salt reduction, 0.1 ml of each was added. 2 The concentration and diafiltration steps were carried out using an EMD Millipore tangential flow filtration system with an ultrafiltration cassette of 0.2 m. The total filtration area was 0.2 m using two cassettes in parallel.2 The TFF step achieved a 5-fold volume reduction and a 19-fold salt reduction. The final collagen slurry was run on an SDS-PAGE gel to confirm the presence of collagen. The slurry was dried for 3 days using a multi-tray freeze dryer to yield a white fluffy collagen powder.

[0160] The purified collagen-GFP-beta-lactamase fusion protein was analyzed on an SDS-PAGE gel and was observed to migrate with an apparent molecular weight of 90 kilodaltons. The predicted size of the fusion protein is 85 kd. Mass spectrometry confirmed that the 90 kDa band was the correct collagen fusion protein.

[0161] Truncated collagen (version 2) containing DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP beta-lactamase fusion: A jellyfish collagen with DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP beta-lactamase fusion 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 and encodes amino acids 1-24. The His tag is encoded by nucleotides 73-99 and encodes a nine histidine tag at amino acids 25-33. The linker is encoded by nucleotides 100-111 and encodes amino acids 34-37. The thrombin cleavage site is encoded by nucleotides 112-135 and encodes amino acids 38-45. The green fluorescent protein (GFP) with linker is encoded by nucleotides 136-873 and encodes amino acids 46-291. The truncated collagen sequence is encoded by nucleotides 874-1440 and encodes amino acids 292-480. The linkered beta-lactamase is encoded by nucleotides 1441-2232 and encodes amino acids 481-744. [ka] [ka] [ka]

[0162] Example 4: Production of full-length elastin Full-length human elastin was expressed as described below. The wild-type full-length amino acid sequence of human elastin is provided below. [ka]

[0163] A 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. [ka] [ka]

[0164] Codon-optimized elastin with DsbA secretion tag-His tag-linker-thrombin cleavage site A codon-optimized polynucleotide sequence encoding full length human elastin with DsbA secretion tag-His tag-linker-thrombin cleavage site is disclosed below: In SEQ ID NO:23, nucleotides 1-72 encode the DsbA secretion tag and encode amino acids 1-24 of SEQ ID NO:24; nucleotides 73-99 encode the nine His tags and encode amino acids 25-33 of SEQ ID NO:24; nucleotides 100-111 encode the linker and encode amino acids 34-37 of SEQ ID NO:24; nucleotides 112-135 encode the thrombin cleavage tag and encode amino acids 38-45 of SEQ ID NO:24; nucleotides 136-2415 encode amino acids 46-805 of full length human elastin of SEQ ID NO:24. [ka] [ka] [ka]

[0165] A polynucleotide encoding full-length human elastin without a native sequence tag is disclosed in SEQ ID NO:87. [ka] [ka] [ka]

[0166] The full length human elastin sequence without the native sequence tag is disclosed in SEQ ID NO:88. [ka]

[0167] Codon-optimized elastin with a DsbA secretion tag A codon-optimized polynucleotide sequence encoding 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 full-length human elastin of SEQ ID NO: 26. [ka] [ka] [ka]

[0168] The polynucleotide of SEQ ID NO:22 was codon optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28 vector and SEQ ID NO:22 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:22) were then codon optimized and synthesized by Gen9 DNA (now Ginkgo Bioworks in-house synthesis). The overlap between the pET28a vector and SEQ ID NO:22 was designed to be 30-40 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 cleaved pET28a vector and the insert DNA (SEQ ID NO:22) were then codon optimized and synthesized by Ginkgo Bioworks in-house synthesis. They were assembled together into the final plasmid using Gibson assembly (https: / / us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-l-step-kit-synthetic-genomics-inc). The sequences of the plasmids were then verified by Sanger sequencing by Eurofins Genomics (www.eurofinsgenomics.com).

[0169] The transformed cells were grown in minimal medium and frozen in 1.5 aliquots with glycerol at a cell to glycerol ratio of 50:50. One vial of this frozen culture was allowed to recover overnight in 50 ml of minimal medium at 37°C and 200 rpm. The cells were transferred to 300 ml of minimal medium and grown for 6-9 hours to achieve an OD600 of 5-10.

[0170] A bioreactor was prepared with 2.7 L of minimal medium + glucose and 300 ml of culture with an OD600 of 5-10 was added to bring the starting volume to 3 L. Cells were grown at 28°C, pH 7, with agitation, air and oxygen containing cascade to maintain dissolved oxygen at 20% saturation. A 28% w / w ammonium hydroxide solution was used to control pH. Once the initial bolus of 40 g / L was exhausted in approximately 13 hours, fermentation was run in fed-batch mode using a DO-stat based feeding algorithm. After 24-26 hours of initial growth, OD600 reached >100. At this point, 300 mL of 500 g / L sucrose was added and the temperature was reduced to 25°C. High density cultures were induced for protein production using 1 mM IPTG. Fermentation was continued for an additional 20-24 hours and cells were harvested using a benchtop centrifuge at 9000rcf and 15°C for 60 minutes. The cell pellet recovered from centrifugation was resuspended in a pH 8 buffer containing 0.5 M NaCl and 0.1 M KH2PO4 at a weight ratio of 2x buffer to 1x cells.

[0171] Fermentations were carried out at various temperatures ranging from 25°C to 28°C. In some fermentations, the temperature of the fermentation was maintained at a constant temperature and elastin was purified immediately upon completion of the fermentation (OD600 of 5-10). In other fermentations, the temperature of the fermentation was maintained for the desired period and once a cell density of OD600 of 5-10 was reached, the temperature was reduced to induce protein production. Typically, the temperature was reduced from 28°C to 25°C. Fermentation at 25°C was continued for 40-60 hours before isolating elastin.

[0172] The harvested cells were disrupted twice with a homogenizer at a pressure of 14,000 psi. The resulting slurry contained collagen protein along with other proteins.

[0173] Supernatants from homogenized cells were analyzed on SDS-PAGE gels and a distinct band was observed at approximately 70 kilodaltons, corresponding to the predicted size of 68 kilodaltons. Purified elastin was analyzed by mass spectrometry.

[0174] Full-length elastin with DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP beta-lactamase fusion A human elastin with DsbA secretion tag-His tag-linker-thrombin cleavage site and GFP beta-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 and encodes amino acids 1-24. The His tag is encoded by nucleotides 73-99 and encodes a nine histidine tag at amino acids 25-33. The linker is encoded by nucleotides 100-111 and encodes amino acids 34-37. The thrombin cleavage site is encoded by nucleotides 112-135 and encodes amino acids 38-45. The green fluorescent protein (GFP) with linker is encoded by nucleotides 136-873 and encodes amino acids 46-291. The full length elastin sequence is encoded by nucleotides 874-3153 and encodes amino acids 292-1051. The beta-lactamase with the linker is encoded by nucleotides 3154-3945 and encodes amino acids 1052-1315. [ka] [ka] [ka] [ka]

[0175] Example 5: Production of truncated elastin Truncated human elastin is produced using the expression system described in Example 4. The full length amino acid sequence, lacking the native secretion tag, is disclosed in SEQ ID NO:29. [ka]

[0176] A codon-optimized polynucleotide sequence encoding full-length human elastin lacking the native secretion tag is disclosed in SEQ ID NO:30. [ka] [ka]

[0177] The amino acid sequence of C-terminally truncated 60.7 kDa human elastin is disclosed in SEQ ID NO: 31. The 60.7 kDa truncated elastin has amino acids 706 to 761 deleted from full-length elastin. [ka]

[0178] The codon-optimized polynucleotide sequence encoding the truncated 60.7 kDa human elastin is disclosed in SEQ ID NO:32. [ka] [ka] [ka]

[0179] The amino acid sequence of N-terminally truncated 58.8 kDa human elastin is disclosed in SEQ ID NO: 33. The 58.8 kDa truncated elastin has amino acids 2 to 85 deleted from full-length elastin. [ka]

[0180] The codon-optimized polynucleotide sequence encoding the 58.8 kDa truncated human elastin is disclosed in SEQ ID NO:34. [ka] [ka]

[0181] The amino acid sequence of C-terminally truncated 57 kDa human elastin is disclosed in SEQ ID NO: 35. The 57 kDa truncated elastin has amino acids 661 to 761 deleted from full-length elastin. [ka]

[0182] The codon-optimized polynucleotide sequence encoding the 57 kDa truncated human elastin is disclosed in SEQ ID NO:36. [ka] [ka]

[0183] The amino acid sequence of C-terminally truncated 53.9 kDa human elastin is disclosed in SEQ ID NO: 37. The 53.9 kDa truncated elastin has amino acids 624 to 761 deleted from full-length elastin. [ka]

[0184] The codon-optimized polynucleotide sequence encoding the 53.9 kDa truncated human elastin is disclosed in SEQ ID NO:38. [ka] [ka]

[0185] The amino acid sequence of C-terminally truncated 45.3 kDa human elastin is disclosed in SEQ ID NO: 39. The 45.3 kDa truncated elastin has amino acids 529 to 761 deleted from full-length elastin. [ka]

[0186] The codon-optimized polynucleotide sequence encoding the 45.3 kDa truncated human elastin is disclosed in SEQ ID NO:40. [ka]

[0187] The amino acid sequence of N-terminally truncated 44.4 kDa human elastin is disclosed in SEQ ID NO: 41. The 44.4 kDa truncated elastin has amino acids 2 to 246 deleted from full-length elastin. [ka]

[0188] The codon-optimized polynucleotide sequence encoding the 44.4 kDa truncated human elastin is disclosed in SEQ ID NO:42. [ka]

[0189] The amino acid sequence of N-terminally truncated 40.4 kDa human elastin is disclosed in SEQ ID NO: 43. The 40.4 kDa truncated elastin has amino acids 2 to 295 deleted from full-length elastin. [ka]

[0190] The codon-optimized polynucleotide sequence encoding the 40.4 kDa truncated human elastin is disclosed in SEQ ID NO:44. [ka]

[0191] The amino acid sequence of C-terminally truncated 39.8 kDa human elastin is disclosed in SEQ ID NO: 45. The 39.8 kDa truncated elastin has amino acids 462 to 761 deleted from full-length elastin. [ka]

[0192] The codon-optimized polynucleotide sequence encoding the 39.8 kDa truncated human elastin is disclosed in SEQ ID NO:46. [ka]

[0193] The amino acid sequence of C-terminally truncated 36.1 kDa human elastin is disclosed in SEQ ID NO: 47. The 36.1 kDa truncated elastin has amino acids 418 to 761 deleted from full-length elastin. [ka]

[0194] The codon-optimized polynucleotide sequence encoding the 36.1 kDa truncated human elastin is disclosed in SEQ ID NO:48. [ka]

[0195] The amino acid sequence of N-terminally truncated 34.9 kDa human elastin is disclosed in SEQ ID NO: 49. The 34.9 kDa truncated elastin has amino acids 2 to 360 deleted from full-length elastin. [ka]

[0196] The codon-optimized polynucleotide sequence encoding the 34.9 kDa truncated human elastin is disclosed in SEQ ID NO:50. [ka]

[0197] The amino acid sequence of C-terminally truncated 32 kDa human elastin is disclosed in SEQ ID NO: 51. The 32 kDa truncated elastin has amino acids 373 to 761 deleted from full-length elastin. [ka]

[0198] The codon-optimized polynucleotide sequence encoding the 32 kDa truncated human elastin is disclosed in SEQ ID NO:52. [ka]

[0199] The amino acid sequence of C-terminally truncated 29.9 kDa human elastin is disclosed in SEQ ID NO: 53. The 60.7 kDa truncated elastin has amino acids 347 to 761 deleted from full-length elastin. [ka]

[0200] The codon-optimized polynucleotide sequence encoding the 29.9 kDa truncated human elastin is disclosed in SEQ ID NO:54. [ka]

[0201] The amino acid sequence of N-terminally truncated 29.4 kDa human elastin is disclosed in SEQ ID NO: 55. The 29.4 kDa truncated elastin has amino acids 2 to 425 deleted from full-length elastin. [ka]

[0202] The codon-optimized polynucleotide sequence encoding the 29.4 kDa truncated human elastin is disclosed in SEQ ID NO:56. [ka]

[0203] The amino acid sequence of N-terminally truncated 25.3 kDa human elastin is disclosed in SEQ ID NO: 57. The 25.3 kDa truncated elastin has amino acids 2 to 473 deleted from full-length elastin. [ka]

[0204] The codon-optimized polynucleotide sequence encoding the 25.3 kDa truncated human elastin is disclosed in SEQ ID NO:58. [ka]

[0205] The amino acid sequence of C-terminally truncated 24.1 kDa human elastin is disclosed in SEQ ID NO: 59. The 24.1 kDa truncated elastin has amino acids 277 to 761 deleted from full-length elastin. [ka]

[0206] The codon-optimized polynucleotide sequence encoding the 24.1 kDa truncated human elastin is disclosed in SEQ ID NO:60. [ka]

[0207] The amino acid sequence of C-terminally truncated 20.3 kDa human elastin is disclosed in SEQ ID NO: 61. The 20.3 kDa truncated elastin has amino acids 229 to 761 deleted from full-length elastin. [ka]

[0208] The codon-optimized polynucleotide sequence encoding the 20.3 kDa truncated human elastin is disclosed in SEQ ID NO:62. [ka]

[0209] The amino acid sequence of N-terminally truncated 19.6 kDa human elastin is disclosed in SEQ ID NO: 63. The 19.6 kDa truncated elastin has amino acids 2 to 542 deleted from full-length elastin. [ka]

[0210] The codon-optimized polynucleotide sequence encoding the 19.6 kDa truncated human elastin is disclosed in SEQ ID NO:64. [ka]

[0211] The amino acid sequence of N-terminally truncated 11 kDa human elastin is disclosed in SEQ ID NO: 65. The 11 kDa truncated elastin has amino acids 2 to 635 deleted from full-length elastin. [ka]

[0212] The codon-optimized polynucleotide sequence encoding the 11 kDa truncated human elastin is disclosed in SEQ ID NO:66. [ka]

[0213] The amino acid sequence of N-terminally truncated 7.9 kDa human elastin is disclosed in SEQ ID NO: 67. The 7.9 kDa truncated elastin has amino acids 2 to 674 deleted from full-length elastin. [ka]

[0214] The codon-optimized polynucleotide sequence encoding the 7.9 kDa truncated human elastin is disclosed in SEQ ID NO:68. [ka]

[0215] The amino acid sequence of C-terminally truncated 6.3 kDa human elastin is disclosed in SEQ ID NO: 69. The 6.3 kDa truncated elastin has amino acids 74 to 761 deleted from full-length elastin. [ka]

[0216] The codon-optimized polynucleotide sequence encoding the 6.3 kDa truncated human elastin is disclosed in SEQ ID NO:70: [ka]

[0217] The amino acid sequence of N-terminally truncated 4.3 kDa human elastin is disclosed in SEQ ID NO: 71. The 4.3 kDa truncated elastin has amino acids 2 to 717 deleted from full-length elastin. [ka]

[0218] The codon-optimized polynucleotide sequence encoding the 4.3 kDa truncated human elastin is disclosed in SEQ ID NO:72. [ka]

[0219] DsbA secretion and truncated human elastin 1 with a FLAG tag The amino acid sequence of truncated human elastin1 with DsbA secretion and FLAG tags 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. [ka]

[0220] The nucleic acid sequence of a truncated human elastin 1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:99. [ka]

[0221] The polynucleotide of SEQ ID NO:99 was subcloned into vector pET28a and expressed in host E. coli cells, and the truncated elastin was purified as described herein. The purified elastin produced a clear band on SDS-PAGE, and anti-FLAG Western was observed at about 25 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0222] DsbA secretion and truncated human elastin 2 with a FLAG tag The amino acid sequence of truncated human elastin type 2 with DsbA secretion and FLAG tags 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. [ka]

[0223] The nucleic acid sequence of a truncated human elastin 2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:101. [ka]

[0224] The polynucleotide of SEQ ID NO: 101 was subcloned into vector pET28a, expressed in host E. coli cells, and the truncated elastin was purified as described herein. The purified elastin produced a clear band on SDS-PAGE, and anti-FLAG Western was observed at about 25 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0225] Example 6: Effects of truncated collagens on fibroblast viability, procollagen synthesis and elastin synthesis Human fibroblast cultures were used to evaluate the ability of the truncated jellyfish collagen molecule of Example 2 to determine its effect on procollagen and elastin synthesis, and to determine the increase in viability of human fibroblasts following exposure to the truncated jellyfish collagen.

[0226] A stock solution of 2% w / w truncated collagen was prepared from the histidine-tagged truncated collagen of Example 3. Aliquots from the 2% stock truncated collagen solution were then used in the experiments described below.

[0227] Preparation of fibroblast cells Fibroblasts were seeded into individual wells of a 24-well plate in 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 remove any non-adherent cells and replaced with 0.5 ml of fresh FGM. Cells were grown to confluence and the medium was changed every 48–72 h. Once confluency was reached, cells were treated with DMEM supplemented with 1.5% FBS for 24 h to wash out any effects from growth factors included in the regular culture medium. After the 24-h washout period, cells were treated with specific concentrations of truncated jellyfish collagen dissolved in FGM containing 1.5% FBS. Transforming growth factor beta (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. The cells were incubated for 48 hours and at the end of the incubation period the cell culture medium was collected and either stored frozen (-75°C) or assayed immediately. Materials were tested in triplicate.

[0228] MTT assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetrazole) assay is a colorimetric assay used to measure the metabolic activity of cells. The MTT assay assessed changes in cell number. When cells are exposed to MTT, the reduction of MTT by mitochondria in viable cells leads to the formation of purple insoluble formazin crystals, which are extracted from the cells with isopropanol and quantified spectrophotometrically. Non-viable cells cannot reduce MTT and therefore cannot produce purple formazin crystals. The intensity of the purple color is directly proportional to the number of viable cells (metabolically active cells). The intensity of the purple color is directly proportional to the metabolic activity of the cells and inversely proportional to the toxicity of the test material.

[0229] After the two-day incubation, the cell culture medium was removed (see above) and the fibroblasts were washed twice with PBS to remove any remaining 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 for 1 hour at 37±2° C. and 5±1% CO2. After incubation, the DMEM / MTT solution was removed, the cells were washed once again with PBS, and then 0.5 ml of isopropyl alcohol was added to the wells to extract the purple formazin crystals. 200 microliters of the isopropyl extract was transferred to a 96-well plate and the plate was read at 540 nm using isopropyl alcohol as a blank.

[0230] The mean MTT absorbance value of the negative control cells was calculated and used to represent 100% cell viability. Individual MTT absorbance values ​​from cells receiving various treatments were then divided by the mean value of the negative control cells and expressed as a percentage to determine the change in cell viability caused by each treatment.

[0231] In Tables 1, 2 and 3 of this example, the experiments were performed by using the indicated aliquots of 2% stock truncated collagen solution in the assay. For example, in samples that tested "10% collagen solution", a sufficient aliquot of 2% truncated collagen was used to provide 10% of the assay volume. For a total assay volume of 1.0 ml, 100 μl of 2% stock truncated collagen 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.

[0232] The results of the MTT assay are shown in Table 3. Values ​​are presented as the mean % viability ± deviation from the mean.

[0233] [Table 3]

[0234] Histidine-tagged truncated jellyfish collagen demonstrated a protective effect by increasing cell viability of human fibroblasts. As can be seen in Table 3, the highest values ​​of the MTT assay were observed when fibroblasts were exposed to 0.02% to 0.2% truncated jellyfish collagen.

[0235] Procollagen synthesis Fibroblasts are the main source of extracellular matrix peptides, including the structural proteins collagen and elastin. Procollagen is found in the dermal layer of skin. It is a large peptide synthesized by fibroblasts in the endothelium and is the precursor of collagen. The peptide is processed to form mature collagen protein and the propeptide portion is cleaved (type IC peptide). Both mature collagen protein and type IC peptide fragments are then released into the extracellular environment. As collagen is synthesized, type IC peptide fragments accumulate in tissue culture medium. Since the stoichiometric ratio between the two parts of procollagen peptide is 1:1, assay of type IC peptide will reflect the amount of collagen synthesized. Type 1 C peptide can be assayed by ELISA-based method.

[0236] A series of type IC peptide standards were prepared ranging from 0 ng / ml to 640 ng / ml. ELISA microplates were then prepared by removing any unnecessary strips from the plate frame followed by the addition of 100 μl of peroxidase-labeled anti-procollagen IC peptide antibody to each well used in the assay. 20 μl of either sample (harvested tissue culture medium) or standard was then added to the appropriate wells, the microplate was covered and incubated at 37°C for 3 ± 0.25 hours. After incubation, the wells were aspirated and washed three times with 400 μl of wash buffer. After removing the final wash, 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 minutes. 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.

[0237] To quantify the amount of each substance present, a standard curve was generated using known concentrations of each substance. A regression analysis was performed to establish a line of best fit through these data points. The absorbance values ​​of the test materials and untreated samples were used to estimate the amount of each substance present in each sample.

[0238] The results of the ELISA assay are shown in Table 4.

[0239] [Table 4]

[0240] The truncated histidine-tagged jellyfish collagen was observed to have a biphasic effect on collagen synthesis. At 1%, 5% and 10% levels, collagen synthesis was increased. At the 5% concentration, the truncated jellyfish collagen significantly increased collagen synthesis with a p-value of less than 0.05.

[0241] Elastin synthesis Elastin is the main component of the network of elastic fibers, giving tissues the ability to recoil after transient stretch. This protein is released into the extracellular space by fibroblasts (soluble elastin), where it is then crosslinked with other elastin proteins to form an extensive network of fibers and sheets (insoluble elastin). Soluble elastin can be easily measured from cell culture medium by ELISA-based methods.

[0242] 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 for 1 hour at 37° C. and then washed twice with PBS containing 0.05% Tween 20.

[0243] A set of α-elastin standards was generated ranging from 0 to 100 ng / ml. 180 μl of standard or truncated jellyfish collagen was then transferred to a 650 μl microcentrifuge tube. An anti-elastin antibody solution was prepared (antibody was diluted 1:100 in PBS containing 0.25% BSA and 0.05% Tween 20) and 20 μl of the solution was added to the tube. The tube was then incubated overnight at 4 ± 2 °C. The next 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 h. The plate was then washed three times with PBS containing 0.05% Tween 20. After washing, 200 μl of a solution containing peroxidase-linked secondary antibody diluted in PBS containing 0.25% BSA and 0.05% Tween 20 was added and the plate was incubated at room temperature for 1 h. After washing the plate three times, 200 μl of substrate solution was added and the plate was incubated in the dark at room temperature for 10-30 minutes. After this final incubation, the plate was read at 460 nm using a plate reader.

[0244] [Table 5]

[0245] As shown in Table 5, when used at a concentration of 0.5%, truncated his-tagged jellyfish collagen significantly increased elastin production.

[0246] Example 7: Effect of truncated collagen on keratinocyte proliferation and UVB protection A human keratinocyte cell culture model was used to evaluate the ability of test materials to exert an effect on cell proliferation, as well as the effect of test materials on cell viability following exposure to UVB.

[0247] A stock solution of 2% w / w truncated collagen was prepared from the truncated collagen of Example 1. Aliquots from the 2% stock truncated collagen solution were then used in the experiments described below.

[0248] The study was conducted in two parts. In the first part, cultured keratinocytes were incubated with the test materials for 48 hours and then the change in the number of viable cells was assessed using the MTT assay. In the second part of the study, cultured keratinocytes were irradiated with UVB and then treated with the test materials for 48 hours. At the end of the 48 hours, the number of viable cells was again assessed by the MTT assay.

[0249] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetrazole) assay may be used to determine changes in cell number of viable cells. The MTT assay is a colorimetric analysis of cellular metabolic activity that reflects the number of viable cells. Reduction of MTT by mitochondria in viable cells results in the formation of purple insoluble formazin crystals, which are 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.

[0250] Proliferation assay For proliferation assays, keratinocytes were seeded in 96-well plates without growth factors and incubated for 24 hours at 37±2° C. and 5±1% CO2. 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. After addition of the test material, cells were cultured for 48 hours as described above. At the end of the incubation period, the change in the number of viable cells was determined using the MTT assay.

[0251] UVB protection assay For the UVB protection assay, keratinocytes were seeded in 96-well plates using normal medium and incubated for 24 hours at 37±2°C and 5±1% CO2. After this initial incubation, the medium was replaced with 100 μl of phosphate-buffered saline (PBS) and the cells were exposed to UVB (40 mJ / cm2). After exposure to UVB, the PBS was replaced with fresh medium supplemented with the test material (100 μg / ml ascorbic acid served as a positive control) and the cells were cultured for 48 hours at 37±2°C and 5±1% CO2. At the end of the 48-hour incubation, cell viability was determined using the MTT assay.

[0252] MTT assay After 48 hours of incubation, the cell culture medium was removed and replaced with 200 μl of culture medium supplemented with 0.5 μg / ml MTT. The well plate was incubated for 1 hour at 37±2°C and 5±1% CO2. After incubation, the MTT solution was removed, the cells were washed once with phosphate-buffered saline, and then 200 μl of isopropyl alcohol was added to the wells to extract the purple formazin crystals. The 96-well plate was read at 540 nm using isopropyl alcohol as a blank.

[0253] The average absorbance value of cells not treated with test materials (proliferation assay: untreated group) or exposed to UVB (UVB protection assay: non-UVB exposed group) was calculated and used to represent 100% cell viability. Individual absorbance values ​​from cells receiving various treatments were then divided by the average absorbance value representing 100% cell viability and expressed as a percentage to determine the change in cell viability caused by each treatment.

[0254] The results of the proliferation assay using his-tagged truncated jellyfish collagen are shown in Table 6. The results of the UVB protection assay using his-tagged truncated jellyfish collagen are shown in Table 7. Values ​​for both assays are shown as mean viability ± standard deviation.

[0255] [Table 6]

[0256] [Table 7]

[0257] For the proliferation assay, an untreated group was used to represent 100% cell viability. Values ​​greater than 100% reflect an increase in the number of viable cells, and are therefore indicative of cell proliferation. In this study, the test materials were not observed to promote cell proliferation.

[0258] In addition, a keratinocyte proliferation assay was performed using the truncated collagen of SEQ ID NO: 91. 1% and 0.5% collagen solutions of a 5% stock solution were prepared and tested according to Example 8. The truncated collagen of SEQ ID NO: 91 had keratinocyte cell viability assay values ​​of 102±2.9 and 102±2.0, respectively. The observed values ​​were statistically significant (p<0.05).

[0259] In addition to its effect on cell proliferation, the test material was also screened to determine if it had an effect on cell recovery after UVB exposure. In this study, it was observed that exposure to UVB resulted in a significant decrease in the number of viable cells 48 hours after exposure. However, treatment with the test material prevented this decrease in cell viability. The effect was evident for the test material within the concentration range of 0.05% to 5%, with the effect being optimal at a concentration of 0.05%. Within this concentration range, cell viability was significantly higher than the untreated group (with one exception at the 0.01% concentration), demonstrating that the material has a UVB protective effect. Since the material was added after UVB exposure, it may act to reduce the damaging effects of UVB radiation or it may help damaged cells recover at a more rapid rate. Regarding the latter, truncated collagen is beneficial when applied topically to the skin and has a regenerative effect on skin cells damaged by UVB.

[0260] In addition, a UVB protection assay was performed using the truncated collagen of SEQ ID NO: 91. The 1% and 0.5% collagen solutions of SEQ ID NO: 91 had keratinocyte cell viability assay values ​​of 80±4.6 and 78±2.5, respectively. The observed values ​​were statistically significant (p<0.05).

[0261] Example 8: Effect of truncated collagen on thymine dimer formation Exposure to ultraviolet light increases the content of thymine dimers (TT dimers) in DNA present in cells. Increased TT dimer formation correlates with skin damage and certain types of cell proliferative disorders, including skin cancer.

[0262] 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 contains a DsbA secretion tag. The DsbA tag (amino acids 1-24 of SEQ ID NO:12) is cleaved by the host cell as the polypeptide is processed through the secretory pathway. A truncated collagen without the DsbA secretion tag is provided in SEQ ID NO:91.

[0263] The truncated collagen of SEQ ID NO:91 was tested to determine whether it can reduce TT dimer formation in human epidermal keratinocytes. In this study, cells were exposed to UVB (25mJ / cm2). After exposure, cells were treated with test material or Trolox (100ug / ml) and incubated overnight. The next day, cellular DNA was extracted and assayed for thymine dimer content using an ELISA-based method.

[0264] Human keratinocytes were seeded in 12-well plates using normal medium and incubated for 24 hours at 37±2°C and 5±1% CO2. After this initial incubation, the medium was replaced with 100 μl of phosphate-buffered saline (PBS) and the cells were exposed to UVB (25 mJ / cm2). After exposure to UVB, the PBS was replaced with fresh medium supplemented with the test material or Trolox (100 μg / ml, which served as a positive control) and the cells were cultured overnight at 37±2°C and 5±1% CO2. At the end of the incubation, cellular DNA was extracted.

[0265] After overnight incubation, the cell culture medium was removed from the wells and replaced with 200 μl PBS and 20 μl Proteinase K. The plate was swirled to mix the PBS and Proteinase K, and then 200 μl Buffer AL was added to each well. The plate was swirled again to mix the reagents, and then the plate was incubated at 55 ± 2 °C for 10 minutes. After cooling the plate to room temperature, the DNA was precipitated by the addition of 200 μl of 100% ethanol. The precipitated DNA mixture was then transferred to a DNEasy spin column in a 2 ml collection tube and centrifuged at 8,000 RPM for 1 minute. The flow-through and collection tube were discarded, and 500 μl of Wash Buffer 1 was added to the spin column, and the column was placed in a new collection tube and centrifuged at 8,000 RPM for 1 minute. The flow-through and collection tube were again discarded, 500 μl of Wash Buffer 2 was added to the spin column, the column was placed in a new collection tube and centrifuged at 14,000 RPM for 3 minutes. The spin column was then placed in a new 1.5 ml centrifuge tube and 110 μl of ultrapure water was added to the column. The column was incubated at room temperature for 1 minute and then centrifuged at 8,000 RPM for 1 minute.

[0266] Extracted DNA was quantified by fluorometric assay. 2 μl aliquots of DNA samples were mixed with 100 μl of TE buffer in a 96-well plate. A series of DNA standards were also transferred (in duplicate) to wells of the 96-well plate. 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.

[0267] Thymine dimer detection was determined using the OxiSelect™ UV-Induced DNA Damage ELISA Kit.

[0268] Aliquots of genomic DNA samples or standards were converted to single-stranded DNA by incubating the samples at 95°C for 10 minutes and cooled on ice. 100 μl of each sample or standard was transferred to a DNA-binding ELISA plate and incubated overnight at 4°C. The next day, the wells were rinsed once with 100 μl of PBS and then blocked with 150 μl of assay diluent for 1 hour at room temperature. 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 reagent was added to the plate. The plate was blocked again for 1 hour at room temperature and then washed three times as before. 100 μl of secondary antibody was then 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 in the plate. The color reaction was stopped by the addition of 100 μl of stop solution and the plate was read at 460 nm using a plate reader.

[0269] To quantify the amount of DNA present, a standard curve was constructed using known concentrations of DNA and their respective fluorescence intensities (measured in RFU or relative fluorescence units). A regression analysis was performed to establish a line of best fit to these data points. The relative fluorescence units (RFU) of each unknown sample were then used to estimate the amount of DNA.

[0270] A standard curve was generated using a series of DNA standards with known thymine dimer content. This standard curve was used to determine the amount of DNA damage in the sample DNA. The means for each treatment group were calculated and compared using ANOVA. In Table 8 and Figure 5, the standard 5% collagen solution (5 g of truncated collagen in 95 ml of deionized water) was further diluted to the indicated % solution with phosphate buffered saline (PBS).

[0271] [Table 8]

[0272] Table 8 shows that 5% and 1% truncated collagen solutions statistically significantly reduced TT dimer formation (p<0.05). The data is presented graphically in FIG.

[0273] The experiment was repeated using a different lot of truncated collagen (SEQ ID NO: 91). The amount of TT dimer (in ng / ml) in non-UVB exposed cells was 1.3±1.2, untreated cells was 18.1±0.4, 100 μg / ml Trolox treated cells was 7.9±0.3, 5% collagen was 13.1±0.2, and 1% collagen was 17.4±0.7. The reduction in TT dimer formation in non-UVB exposed, Trolox treated, 5% collagen treated and 1% collagen treated cells was statistically significant (p<0.05) compared to untreated cells.

[0274] Example 9: Human Collagen Truncated human collagen type 21 alpha 1 A truncated human collagen type 21 alpha 1 without the 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 NOs: 73 and 74, the DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24. In SEQ ID NOs: 73 and 74, the truncated collagen sequence is encoded by nucleotides 73-633 and encodes amino acids 25-211.

[0275] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:73. [ka]

[0276] The amino acid sequence is disclosed in SEQ ID NO:74. [ka]

[0277] The codon-optimized nucleotide sequence encoding the truncated human collagen type 21 alpha 1 without the DsbA secretion tag collagen is provided in SEQ ID NO:75. [ka]

[0278] The amino acid sequence of truncated human collagen type 21 alpha 1 without the DsbA secretion tag is disclosed in SEQ ID NO:76. [ka]

[0279] Truncated human collagen type 1 alpha 2(1) A truncated human collagen type 1 alpha 2 without the His tag, linker and thrombin cleavage site is disclosed below. The codon optimized nucleotide and amino acid sequences are disclosed below. In SEQ ID NOs: 78 and 79, the DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24. The truncated collagen sequence is encoded by nucleotides 73-636 and encodes amino acids 25-212.

[0280] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:77. [ka]

[0281] The amino acid sequence is disclosed in SEQ ID NO:78. [ka]

[0282] The nucleic acid sequence of truncated human collagen type 1 alpha 2(1) without the DsbA secretion tag is disclosed in SEQ ID NO:79. [ka]

[0283] The amino acid sequence of truncated human collagen type 1 alpha 2(1) without the DsbA secretion tag is disclosed in SEQ ID NO:80. [ka]

[0284] Truncated human collagen type 1 alpha 2(2) A truncated human collagen type 1 alpha 2 without the His tag, linker and thrombin cleavage site is disclosed below. The codon optimized nucleotide and amino acid sequences are disclosed below. In SEQ ID NOs: 82 and 83, the DsbA secretion tag is encoded by nucleotides 1-72 and encodes amino acids 1-24. The truncated collagen sequence is encoded by nucleotides 73-609 and encodes amino acids 25-203.

[0285] The codon-optimized nucleotide sequence encoding this collagen is provided in SEQ ID NO:81. [ka]

[0286] The amino acid sequence is disclosed in SEQ ID NO:82. [ka]

[0287] The nucleic acid sequence of truncated human collagen type 1 alpha 2 (2) without the DsbA secretion tag is disclosed in SEQ ID NO:83. [ka]

[0288] The amino acid sequence of truncated human collagen type 1 alpha 2 (2) without the DsbA secretion tag is disclosed in SEQ ID NO:84. [ka]

[0289] A polynucleotide of SEQ ID NO: 73, 77 or 81 was subcloned into vector pET28a to prepare a transformation vector, as described herein. A host cell was transformed with the vector and the polynucleotide was expressed, as described in Example 2.

[0290] After fermentation was completed, the truncated human collagen was purified from the fermentation broth using the procedures disclosed in Example 3. The purified truncated human collagen was analyzed using SDS-PAGE and HPLC as disclosed in Example 3.

[0291] In SDS-PAGE analysis, all three truncated human collagens migrated at the expected molecular weight. Analysis of the truncated human collagens using HPLC utilized a standard curve using jellyfish collagen from Example 3. The retention times of the human collagens were slightly different from those of the jellyfish collagen. SEQ ID NO:76 had a retention time of 5.645 minutes, SEQ ID NO:80 had a retention time of 5.631 minutes, and SEQ ID NO:84 migrated in two peaks with retention times of 5.531 and 5.7 minutes.

[0292] DsbA secretion and truncated human collagen type 1 alpha 2 truncation 5 with a FLAG tag The amino acid sequence of truncated human collagen type 1 alpha 2 truncated 5 with DsbA secretion and FLAG tags 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. [ka]

[0293] The nucleic acid sequence of truncated human collagen type 1 alpha 2 truncated 5 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:93. [ka]

[0294] The polynucleotide of SEQ ID NO:93 was subcloned into vector pET28a, expressed in host E. coli cells, and the truncated collagen was purified as described herein. The purified collagen produced a clear band on SDS-PAGE, and anti-FLAG Western was observed at about 100 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0295] DsbA secretion and truncated human collagen type 1 alpha 2 truncation 6 with a FLAG tag The amino acid sequence of truncated human collagen type 1 alpha 2 truncated 6 with DsbA secretion and FLAG tags 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. [ka]

[0296] The nucleic acid sequence of truncated human collagen type 1 alpha 2 truncated 6 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:95. [ka]

[0297] The polynucleotide of SEQ ID NO:94 was subcloned into vector pET28a and expressed in host E. coli cells, and the truncated collagen was purified as described herein. The purified collagen produced a clear band on SDS-PAGE, and anti-FLAG Western was observed at about 25 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0298] DsbA secretion and truncated human collagen type 1 alpha 2 truncation 7 with a FLAG tag The amino acid sequence of truncated human collagen type 1 alpha 2 truncated 7 with DsbA secretion and FLAG tags 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. [ka]

[0299] The nucleic acid sequence of truncated human collagen type 1 alpha 2 truncated 7 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO: 97: [ka]

[0300] The polynucleotide of SEQ ID NO:97 was subcloned into vector pET28a, expressed in host E. coli cells, and the truncated collagen was purified as described herein. The purified collagen produced a clear band on SDS-PAGE, and anti-FLAG Western was observed at about 30 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0301] Example 10: Protective effect of truncated human collagen on fibroblasts The method of Example 6 is followed to determine the effect of truncated human collagen on fibroblast viability, procollagen synthesis and elastin synthesis.

[0302] The method of Example 7 is followed to determine the effect of truncated human collagen on keratinocyte proliferation and UVB protection.

[0303] The method of Example 8 is followed to determine the effect of truncated collagen on thymine dimer formation following exposure to UV radiation.

[0304] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0305] Example 11: Effect of truncated collagen on inflammatory cytokines Keratinocytes and dermal fibroblasts play an important role in the immune response of the skin. In response, keratinocytes can release a wide range of cytokines that are thought to support immune cell involvement at the site of inflammation. Cytokines released by keratinocytes include TNFα, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, IL-10, IL-18, and IL-1RA.

[0306] The test model used in this study was MatTek EpiDerm. This skin model consists of normal human-derived epidermal keratinocytes that have been cultured to form a highly differentiated, multi-layered model of the human epidermis. Ultrastructural analysis revealed the presence of a multi-layered stratum corneum containing keratohyalin granules, tonofilament bundles, desmosomes, and intercellular stratified lipid layers arranged in a pattern characteristic of the in vivo epidermis. Markers of mature epidermis-specific differentiation, such as profilaggrin, the K1 / K10 cytokeratin pair, capsule, and type I epithelial transglutaminase, are localized in this model. MatTek EpiDerm is also mitotically and metabolically active.

[0307] MatTek EpiDerm tissue was used to evaluate the ability of various test materials to inhibit the release of the inflammatory mediator IL-1a. Test materials were compared to a commercial topical hydrocortisone preparation (positive control) as well as untreated tissue (negative control 1) and non-inflamed untreated tissue (negative control 2). The test was also used to evaluate tissue viability following exposure to the test materials.

[0308] IL-1α, IL-6 and IL-8 are synthesized and stored in keratinocytes and have been identified as mediators of skin irritation and inflammation. The release of these cytokines can be measured directly in tissue culture medium by a colorimetric-based enzyme-linked immunosorbent assay (ELISA). Briefly, an antibody covalently linked to a solid support will bind to IL-1a, IL-6 or IL-8 present in the spent culture medium sample. A secondary antibody covalently attached to an acetylcholinesterase enzyme will then detect the specifically bound cytokine. Upon addition of an appropriate color substrate, the acetylcholinesterase enzyme will generate a colored end product that can be measured spectrophotometrically.

[0309] MatTek EpiDerm tissues were purchased from MatTek and stored at 4°C until use. Prior to use, the tissues to be used were removed from the agarose transport trays and placed into 6-well plates containing 0.9 ml of hydrocortisone-free assay medium (37±2°C). The tissues were incubated 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). For each test material, triplicate tissues were prepared.

[0310] An inflammatory response in tissues was initiated by UV irradiation (UVB). A UV lamp was used to deliver a dose of 300 mJ / cm. 2 The tissues were exposed to 100 μl of UVB radiation. Immediately after application of the inflammatory stimulus, 50 μl or mg of test material was applied directly to the surface of the tissue. A commercially available hydrocortisone cream was used as a positive control. For the negative control, the tissues were exposed to the inflammatory stimulus but were not treated with any type of anti-inflammatory substance. One additional set of tissues was left unexposed to the inflammatory stimulus to provide baseline measurements of cytokines. After exposure to the inflammatory stimulus, the tissues were incubated at 37±2°C and 5±1% CO2 for 24 hours. After the 24-hour incubation, the cell culture medium was collected and stored at -75°C until analysis of cytokines.

[0311] ELISA plates were prepared by diluting the appropriate capture antibody in PBS. 100 μl of the diluted capture antibody was then added to the wells 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 (0.05% Tween 20 in PBS) and then blocked by adding 300 μl of blocking buffer (1% BSA in PBS) to each well. The plate was incubated with blocking buffer for at least 1 hour. After incubation, the blocking buffer was removed and the plate was washed three times as above.

[0312] A series of standards was prepared and 100 μl of each of these standards was dispensed into two wells (in duplicate) of an appropriate 96-well plate. 100 μl of each sample was then added to an additional well and the plate was incubated at room temperature for 2 hours. After incubation, the plate was washed three times as described above. Once the final wash was removed, 100 μl of biotin-conjugated detection antibody was added. After the plate was incubated at room temperature for 2 hours, the plate was washed again as described above. 100 μl of HRP-streptavidin was then added to each well and the plate was incubated at room temperature for 20 minutes. Once the final wash was removed, 100 μl of substrate solution (hydrogen peroxide + tetramethylbenzidine as chromogen) was added to each well. Once a sufficient level of color development had occurred, 50 μl of stop solution (2N sulfuric acid) was added to each well and the plate was read at 460 nm.

[0313] After 24 hours of incubation, the tissue was rinsed twice with at least 100 μl of phosphate buffered saline to remove test material, then transferred to a 6-well plate containing 1.0 ml of assay medium supplemented with MTT (1 mg / ml) and incubated at 37±2° C. and 5±1% CO2 for 3±0.25 hours. After incubation, the tissue was rinsed at least twice with 100 μl of phosphate buffered saline, blotted dry, and then placed into a 24-well plate containing 2 ml of isopropanol per well. The 24-well plate was covered and incubated at room temperature on a rocking platform for at least 2 hours to extract reduced MTT from the tissue. After extraction, a 200 μl sample of the isopropanol / MTT mixture was transferred to a 96-well plate, and the absorbance of the sample was read at 540 nm using a plate reader with 200 μl of isopropanol as a blank. The MTT assay is described in Example 6 herein. The cell viability results of the MTT assay were similar to those obtained in Example 6.

[0314] The results of the IL-1a assay are shown in Table 9 below. Sample 4 is a 2% stock solution of jellyfish collagen of SEQ ID NO: 91, and Sample 3 is a 2% stock solution of truncated jellyfish collagen of SEQ ID NO: 10. In Table 9 below, the percentages shown are the dilution % of the stock solution used in the test. For example, 1% sample 4 treatment is a 1% solution of the 2% stock truncated collagen solution. Untreated cells produced 18.2 pg / ml of II-1a. All samples showed a decrease in Il-1a production upon treatment with truncated collagen. 1% sample 4 treatment reduced IL-1A production to 13.4 pg / ml, which is significant at a p-value of less than 0.05. The decrease in IL-1a production indicates that truncated collagen has an anti-inflammatory effect. [Table 9]

[0315] Example 12: Urban dust protection with truncated collagen A keratinocyte cell culture model was used to evaluate the ability of truncated collagen to exert a protective effect by promoting cell survival following exposure to urban dust.

[0316] Human epidermal keratinocytes were pretreated with the test materials and then exposed to urban dust, and changes in cell viability were then determined by MTT assay at the end of the treatment period.

[0317] Keratinocytes were seeded into individual wells of a 96-well plate in 100 μl of medium and incubated overnight at 37 ± 2 °C and 5 ± 1% CO2. The following day, the medium was removed by aspiration to eliminate any non-adherent cells and replaced with 100 μl of fresh medium. Cells were grown to confluence and medium was changed every 48-72 hours.

[0318] Pretreatment with test materials followed by urban dust treatment Test materials were prepared in cell culture medium at 2x final desired concentration. Urban dust (NIST1649B from Sigma Chemicals) was also prepared in 2x solution. For pretreatment, 50μl of 2x test materials were combined with 50μl of culture medium and cells were incubated for 24 hours. At the end of the pretreatment period, the culture medium containing test materials was removed and replaced with 50μl of 2x urban dust and 50μl of medium. Another set of cells was treated with medium only (non-dust exposure) and used as a reference control to represent 100% cell viability. The cells were then incubated for 24 hours and then subjected to MTT assay to determine the change in cell viability.

[0319] At the end of the treatment period, the cell culture medium was removed and the cells were washed with PBS. After washing, 100 μl of cell culture medium supplemented with 0.5 mg / ml MTT was added to each well and the cells were incubated for 30 minutes at 37+2° C. and 5+1% CO2. After incubation, the medium / MTT solution was removed and the cells were washed once again with PBS, and then 100 μl of isopropyl alcohol was added to the wells to extract the purple formazin crystals. The 96-well plate was then read at 540 nm using isopropyl alcohol as a blank.

[0320] The mean MTT absorbance value of non-dust exposed cells was calculated and used to represent the 100% value of cell number. Individual MTT values ​​from cells receiving various treatments were then divided by the mean value of non-dust exposed cells and expressed as a percentage to determine the change in cell number caused by each treatment.

[0321] The MTT results of pretreatment with test materials followed by dust treatment are shown in Table 10. Table 10 shows that treatment of cells with increasing amounts of collagen increased cell viability upon pretreatment with truncated collagen followed by exposure to urban dust. These results indicate that truncated collagen protects against the loss of cell viability associated with urban dust exposure. [Table 10]

[0322] Example 12: Truncated Chondrosia reniformis collagen DsbA secretion and truncated Chondrosia reniformis fibrillar collagen 1 with a FLAG tag The amino acid sequence of truncated Chondrosia reniformis fibrillar collagen 1 with DsbA secretion and 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 fibrillar collagen nucleotide sequence 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. [ka]

[0323] The nucleic acid sequence of a truncated Chondrosia reniformis fibrillar collagen 1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:103. [ka]

[0324] The polynucleotide of SEQ ID NO: 103 was subcloned into vector pET28a and expressed in host E. coli cells to purify truncated Chondrosia reniformis fibrillar collagen 1 as described herein. The purified fibrillar collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 40 kilodaltons. In the absence of expression of this protein, no band appeared at that position on the gel.

[0325] DsbA secretion and truncated Chondrosia reniformis fibrillar collagen 2 with a FLAG tag The amino acid sequence of truncated Chondrosia reniformis fibrillar collagen 2 with DsbA secretion and 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 fibrillar collagen nucleotide sequence 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:105. [ka]

[0326] The nucleic acid sequence of a truncated Chondrosia reniformis fibrillar collagen 2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:105. [ka]

[0327] The polynucleotide of SEQ ID NO: 105 was subcloned into vector pET28a and expressed in host E. coli cells to purify truncated Chondrosia reniformis fibrillar collagen 2 as described herein. The purified fibrillar collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 55 kilodaltons. In the absence of expression of this protein, no band appeared at that position on the gel.

[0328] DsbA secretion and truncated Chondrosia reniformis nonfibrillar collagen 1 with a FLAG tag The amino acid sequence of truncated Chondrosia reniformis nonfibrillar collagen 1 with DsbA secretion and 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 nonfibrillar collagen nucleotide sequence 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. [ka]

[0329] The nucleic acid sequence of a truncated Chondrosia reniformis nonfibrillar collagen 1 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:107. [ka]

[0330] The polynucleotide of SEQ ID NO: 107 was subcloned into vector pET28a and expressed in host E. coli cells to purify the truncated Chondrosia reniformis nonfibrillar collagen 1 as described herein. The purified nonfibrillar collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 30 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0331] DsbA secretion and truncated Chondrosia reniformis nonfibrillar collagen 2 with a FLAG tag The amino acid sequence of truncated Chondrosia reniformis nonfibrillar collagen 2 with DsbA secretion and 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 nonfibrillar collagen nucleotide sequence 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. [ka]

[0332] The nucleic acid sequence of a truncated Chondrosia reniformis nonfibrillar collagen 2 with DsbA secretion and a FLAG tag is disclosed in SEQ ID NO:109. [ka]

[0333] The polynucleotide of SEQ ID NO: 109 was subcloned into vector pET28a and expressed in host E. coli cells to purify truncated Chondrosia reniformis nonfibrillar collagen 2 as described herein. The purified fibrillar collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 60 kilodaltons. In the absence of expression of this protein, no band appeared at that position on the gel.

[0334] Example 13: Truncated Rhincodon typus (whale shark) collagen DsbA secretion and truncated Rhincodon typus (whale shark) collagen type 1 alpha 1 truncation 1 with a FLAG tag The amino acid sequence of truncated Rhincodon typus collagen type 1 truncated 1 with DsbA secretion and 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. [ka]

[0335] The nucleic acid sequence of truncated Rhincodon typus collagen type 1 truncated 1 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:111. [ka]

[0336] The polynucleotide of SEQ ID NO:111 was subcloned into vector pET28a and expressed in host E. coli cells to purify truncated Rhincodon typus collagen type 1 truncated 1 as described herein. The purified collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 25 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0337] DsbA secretion and truncated Rhincodon typus (whale shark) collagen type 6 alpha 1 truncation 2 with a FLAG tag The amino acid sequence of truncated Rhincodon typus collagen type 6 truncated 2 with DsbA secretion and 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. [ka]

[0338] The nucleic acid sequence of truncated Rhincodon typus collagen type 6 truncated 2 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:113. [ka]

[0339] The polynucleotide of SEQ ID NO: 113 was subcloned into vector pET28a and expressed in host E. coli cells to purify truncated Rhincodon typus collagen type 6 truncated 2 as described herein. The purified collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 35 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel.

[0340] DsbA secretion and truncated Rhincodon typus (whale shark) collagen type 6 alpha 1 truncation 3 with a FLAG tag The amino acid sequence of truncated Rhincodon typus collagen type 6 alpha 1 truncated 3 with DsbA secretion and 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. [ka]

[0341] The nucleic acid sequence of truncated Rhincodon typus collagen type 6 alpha 1 truncated 3 with DsbA secretion and FLAG tag is disclosed in SEQ ID NO:115. [ka]

[0342] The polynucleotide of SEQ ID NO: 115 was subcloned into vector pET28a and expressed in host E. coli cells to purify truncated Rhincodon typus collagen type 1 truncated 1 as described herein. The purified collagen gave rise to a distinct band on SDS-PAGE and anti-FLAG Western was observed at approximately 25 kilodaltons. In the absence of expression of this protein, no band was present at that position on the gel. The present invention provides, for example, the following items. (Item 1) A non-naturally occurring collagen selected from the group consisting of jellyfish collagen, human, Chondrosia reniformis (whale sponge) and Rhincodon typus (whale shark). (Item 2) 2. The non-naturally occurring collagen according to item 1, which is truncated. (Item 3) 3. The non-naturally occurring truncated collagen according to item 2, which is truncated by an internal truncation of 50 to 300 amino acids. (Item 4) 4. The non-naturally occurring collagen according to any one of items 1 to 3, wherein the amino acid sequence of the collagen is selected from the group consisting of 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. (Item 5) 5. The non-naturally occurring collagen according to any of items 1 to 4, further comprising an amino acid sequence selected from the group consisting of a secretion tag, a histidine tag, a green fluorescent protein, a protease cleavage site and a beta-lactamase protein. (Item 6) 6. The non-naturally occurring collagen of item 5, wherein the secretion tag is DsbA. (Item 7) 7. The non-naturally occurring collagen according to any of items 1 to 6, further comprising one or more amino acid trimer repeats of the sequences glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK). (Item 8) 8. The non-naturally occurring collagen according to item 7, comprising 2 to 50 amino acid trimer repeats. (Item 9) 9. The non-naturally occurring collagen according to any of items 1 to 8, which is a jellyfish collagen. (Item 10) 10. A composition comprising 0.005% to 30% w / w of the non-naturally occurring collagen according to any one of items 1 to 9, which stimulates fibroblast growth and / or stimulates the synthesis of procollagen and / or reduces the formation of thymine-thymine (TT) dimer formation. (Item 11) 11. The composition according to item 10, comprising 0.005% to 1% of non-naturally occurring collagen. (Item 12) 11. The composition according to item 10, comprising 0.01% to 1% of non-naturally occurring collagen. (Item 13) 11. The composition according to item 10, comprising 0.02% to 0.5% non-naturally occurring collagen. (Item 14) 14. The composition according to any of items 10 to 13, which is a topical composition further comprising at least one additional ingredient comprising a topical carrier and / or a preservative. (Item 15) Item 15. The composition according to item 14, wherein the topical composition is a mask. (Item 16) 16. The topical composition according to item 14 or 15, wherein the topical carrier is selected from the group consisting of liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifiers, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane and water. (Item 17) 17. The topical composition according to any of items 14 to 16, wherein the preservative is selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, cis-isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract and EDTA. (Item 18) 18. A method for reducing skin damage, promoting repair of damaged skin, protecting skin from UV damage, or increasing viability of skin cells, comprising applying a composition according to any one of items 1 to 17 to the skin or skin cells of a subject. (Item 19) 20. The method of claim 18, further comprising increasing the viability of fibroblasts present in the skin of the subject. (Item 20) 20. The method of claim 18, further comprising increasing the synthesis of procollagen by fibroblasts present in the skin of the subject. (Item 21) A non-naturally occurring elastin selected from the group consisting of jellyfish elastin, human elastin, Chondrosia reniformis elastin, or Rhincodon typus elastin. (Item 22) 22. The non-naturally occurring elastin according to item 21, which is truncated. (Item 23) 23. The non-naturally occurring truncated elastin according to item 22, which is truncated by a truncation of at least 50 amino acids. (Item 24) 24. The non-naturally occurring elastin according to any one of items 21 to 23, wherein the amino acid sequence of the elastin is selected from the group consisting of 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. (Item 25) 25. The non-naturally occurring elastin according to any one of items 21 to 24, further comprising an amino acid sequence selected from the group consisting of a secretion tag, a histidine tag, a green fluorescent protein, a protease cleavage site and a beta-lactamase protein. (Item 26) 26. The non-naturally occurring elastin of item 25, wherein the secretion tag is DsbA. (Item 27) 27. The non-naturally occurring elastin according to any of items 21 to 26, further comprising one or more amino acid trimer repeats of the sequences glycine-glutamic acid-lysine (GEK) and / or glycine-aspartic acid-lysine (GDK). (Item 28) 28. The non-naturally occurring elastin according to item 27, comprising 2 to 50 amino acid trimer repeats. (Item 29) 29. The non-naturally occurring elastin according to any of items 21 to 28, which is human. (Item 30) 30. A composition comprising 0.005% to 30% w / w of the non-naturally occurring elastin according to any of items 21 to 29, which stimulates fibroblast growth and / or stimulates procollagen synthesis. (Item 31) 31. The composition according to item 30, comprising 0.005% to 1% of non-naturally occurring elastin. (Item 32) 31. The composition according to item 30, comprising 0.01% to 1% of non-naturally occurring elastin. (Item 33) 31. The composition according to item 30, comprising 0.02% to 0.5% non-naturally occurring elastin. (Item 34) 34. The composition according to any of items 30 to 33, which is a topical composition further comprising at least one additional ingredient comprising a topical carrier and / or a preservative. (Item 35) 35. The composition according to item 34, wherein the topical composition is a mask. (Item 36) 36. The topical composition according to item 34 or 35, wherein the topical carrier is selected from the group consisting of liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifiers, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbates, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane and water. (Item 37) 37. The topical composition according to any of items 34 to 36, wherein the preservative is selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA. (Item 38) A method for reducing skin damage, promoting repair of damaged skin, or protecting skin from UV damage, comprising applying the composition according to any one of items 21 to 37 to the skin of a subject. (Item 39) 39. The method of claim 38, wherein the viability of fibroblasts or keratinocytes present in the skin of the subject is increased. (Item 40) 39. The method of claim 38, further comprising increasing the synthesis of procollagen by fibroblasts present in the skin of the subject. (Item 41) 1. A polynucleotide encoding a non-naturally occurring collagen, the polynucleotide being selected from the group consisting of 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. (Item 42) 42. The polynucleotide according to item 41, which is a vector. (Item 43) 43. The polynucleotide of claim 41 or 42, further comprising a nucleic acid selected from the group consisting of a nucleic acid encoding a secretion tag, a nucleic acid encoding a histidine tag, a nucleic acid encoding a green fluorescent protein, a nucleic acid encoding a protease cleavage site, and a nucleic acid encoding a beta-lactamase protein. (Item 44) 44. The polynucleotide according to any of items 41 to 43, further comprising 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). (Item 45) 46. ​​A host cell comprising a polynucleotide according to any of items 41 or 45. (Item 46) 50. The host cell of item 48, wherein the non-naturally occurring collagen is transported into the periplasmic space of the host cell. (Item 47) 47. The host cell according to item 45 or 46, which produces jellyfish collagen. (Item 48) 48. The host cell according to any one of items 45 to 47, which is a gram-negative bacterium. (Item 49) 49. The method according to item 48, wherein the bacterium is Escherichia coli. (Item 50) 50. The method of claim 49, wherein the E. coli is a switch E. coli. (Item 51) 1. A method for producing non-naturally occurring collagen, comprising: a. culturing a host cell according to any one of items 45 to 50 in a culture medium; and b. isolating the non-naturally occurring collagen from the host cell. A method comprising: (Item 52) A polynucleotide encoding a non-naturally occurring elastin, the polynucleotide being selected from the group consisting of 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. (Item 53) 53. The polynucleotide according to item 52, which is a vector. (Item 54) 54. The polynucleotide of claim 52 or 53, further comprising a nucleic acid selected from the group consisting of a nucleic acid encoding a secretion tag, a nucleic acid encoding a histidine tag, a nucleic acid encoding a green fluorescent protein, a nucleic acid encoding a protease cleavage site, and a nucleic acid encoding a beta-lactamase protein. (Item 55) 55. The polynucleotide according to any of items 52 to 54, further comprising 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). (Item 56) 56. A host cell comprising the polynucleotide according to any one of items 52 to 55. (Item 57) 57. The host cell of item 56, wherein the non-naturally occurring collagen is transported into the periplasmic space of the host cell. (Item 58) 58. The host cell according to item 56 or 57, which produces jellyfish collagen. (Item 59) 59. The host cell according to any one of items 56 to 58, which is a gram-negative bacterium. (Item 60) 60. The method according to item 59, wherein the bacterium is Escherichia coli. (Item 61) Item 61. The method of item 60, wherein the E. coli is a switch E. coli. (Item 62) 1. A method for producing non-naturally occurring elastin, comprising: a. culturing a host cell according to any one of items 56 to 61 in a culture medium; and b. Isolating the non-naturally occurring elastin from the host cell. A method comprising: (Item 63) 18. A method for reducing the production of inflammatory cytokines by skin cells, comprising the step of applying to said skin cells a composition according to any one of items 1 to 17. (Item 64) 64. The method of item 63, wherein the inflammatory cytokine is IL-1a. (Item 65) Item 64. The method of item 63, wherein the skin cells are keratinocytes. (Item 66) 18. A method for increasing the viability of skin cells, comprising applying the composition according to any one of items 1 to 17 to the skin or skin cells of a subject. (Item 67) 67. The method of claim 66, wherein the viability of keratinocytes present in the skin of the subject is increased. (Item 68) Item 67. The method of item 66, wherein the viability of fibroblasts present in the skin of the subject is increased. (Item 69) 18. A method for protecting skin cells against the effects of exposure to urban dust, comprising applying to skin cells a composition according to any of items 1 to 17, wherein the viability of said skin cells is increased. (Item 70) 70. The method of claim 69, wherein the skin cells are keratinocytes or fibroblasts.

Claims

1. A recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:91, which is capable of increasing the viability of keratinocytes and / or fibroblasts.

2. 2. The recombinant polypeptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:

91.

3. 3. The recombinant polypeptide of claim 1 or 2, further comprising at least one of a secretion tag, a histidine tag, a fluorescent protein tag, a protease cleavage site and / or a beta-lactamase protein.

4. The recombinant polypeptide of claim 3, wherein the secretion tag is DsbA.

5. 5. The recombinant polypeptide of any one of claims 1 to 4, further comprising one or more GEK amino acid trimer repeats, one or more GDK amino acid trimer repeats, or a combination thereof.

6. A composition comprising 0.005% w / w to 30% w / w of a recombinant polypeptide according to any one of claims 1 to 5.

7. The composition of any one of claims 1 to 6, which is formulated for topical application.

8. The composition of claim 7 comprising a topical carrier, a preservative, or both.

9. 9. The composition of claim 8, wherein the topical carrier is selected from the group consisting of liposomes, biodegradable microcapsules, lotions, sprays, aerosols, dusting powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifiers, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, water, and any combination thereof.

10. 9. The composition of claim 8, wherein the preservative is selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, cis-isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, rosemary extract, ethylenediaminetetraacetic acid (EDTA), and any combination thereof.

11. A personal care product comprising the composition of any one of claims 6 to 10.

12. 12. The personal care product of claim 11 formulated for application to skin and / or hair.

13. 13. The personal care product of claim 11 or 12 which is a cosmetic product.

14. 14. The personal care product of any one of claims 11 to 13, selected from the group consisting of a mask, a skin cleaner, a cleansing cream, a cleansing lotion, a cleansing milk, a cleansing pad, a facial cleanser, a hair shampoo, a hair conditioner, a body shampoo, and any combination thereof.

15. A composition comprising a recombinant polypeptide according to any one of claims 1 to 5, a composition according to any one of claims 6 to 10, or a personal care product according to any one of claims 11 to 14 for providing a cosmetic benefit to the skin of a subject, wherein the composition or personal care product is administered to the skin of the subject, thereby providing a cosmetic benefit to the skin of the subject.

16. 16. The composition or personal care product of claim 15, which results in increased viability of keratinocytes, fibroblasts, or both in the subject after exposure to UV radiation.

17. 17. The composition or personal care product of claim 15 or 16, which results in increased viability of keratinocytes, fibroblasts, or both in the subject following exposure to urban dust.

18. A composition or personal care product according to any one of claims 15 to 17, which results in a reduction in the production of pro-inflammatory cytokines.

19. 19. The composition or personal care product of any one of claims 15 to 18, wherein the cosmetic benefit is selected from the group consisting of reducing skin damage, promoting repair of damaged skin, protecting skin from UV damage, protecting skin cells against the effects of exposure to urban dust, increasing skin cell viability, increasing fibroblast cell viability, increasing keratinocyte cell viability, increasing procollagen synthesis, reducing the production of inflammatory cytokines, and any combination thereof.

20. The composition or personal care product of any one of claims 15 to 19, wherein the subject is a human.

21. A recombinant cell comprising at least one copy of a heterologous nucleic acid sequence encoding a recombinant polypeptide according to any one of claims 1 to 5.

22. 22. The recombinant cell of claim 21 which is a bacterial cell.

23. The recombinant cell of claim 22, which is Escherichia coli.

24. A recombinant cell described in any one of claims 21 to 23, wherein the heterologous nucleic acid sequence comprises SEQ ID NO:

90.

25. A recombinant cell according to any one of claims 21 to 24, wherein the heterologous nucleic acid sequence is codon-optimized for expression in the recombinant cell.

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