Caviar polypeptide

Abstract

The present invention relates to recombinant or synthetic caviar polypeptides and methods of production thereof. The invention also relates to cosmetics comprising the recombinant or synthetic polypeptides of the invention.

Description

Field of the Invention

[0001] The present invention relates to a recombinant or synthetic caviar polypeptide and a method for producing the same. The present invention also relates to cosmetics containing the recombinant or synthetic polypeptide of the present invention. Background Art

[0002] Caviar is not only a luxury food, but its cosmetic benefits are also widely recognized. Desirable properties associated with caviar include enhanced cellular metabolism, stimulation of collagen production by fibroblasts, protection against oxidative stress (and the resulting improved maintenance of cell membranes), and skin hydration. These desirable properties have led to a wide range of applications for caviar and its extracts, extending far beyond its traditional use as a luxury food. This is particularly evident in the cosmetic and nutritional product sectors, where caviar and its extracts are now used in skin creams, as well as in supplements for pet food and vodka.

[0003] The term "caviar" is used here only in its traditional sense, referring to the salt-treated, unfertilized eggs (oocytes) or roe removed from the ovaries of female sturgeons. Due to its difficulty in production and perishability, caviar is a luxury food, prized for its flavor, shape, color, and texture. "Sturgeon" is a generic name for numerous fish species in the Acipenseridae family, including the genera Acipenser, Sturgeon, Acipenser, and Pseudoscaphe (for more information, see www.sturgeonweb.co.uk). The term "caviar" is used more specifically to refer to the two species commonly associated with caviar production: Acipenser and Sturgeon. For example, Beluga caviar, sourced from wild European sturgeons (Huso huso), has traditionally been highly prized, but other sturgeon species are also well-known for their caviar production and are increasingly farmed. These include species such as the Siberian sturgeon (Acipenserbaerii), which was first farmed for caviar production by Exmoor Caviar in the UK, and other Acipenser species.

[0004] Despite the considerable and growing demand for caviar and its extracts, caviar production remains labor-intensive and slow. A key limiting factor in caviar production is the many years it takes female sturgeons to reach sexual maturity. Female Siberian sturgeons typically take eight to ten years to mature and spawn; females of some species, such as the beluga sturgeon, take much longer, around 20 years or even longer. To shorten this time, some hybridization has been achieved, such as crossing male European sturgeons with faster-maturing species such as the sterlet (also known as the Russian sturgeon, scientific name Acipenserruthenus) or the Siberian sturgeon to create hybrids that can be used in aquaculture, but even hybrids take several years to reach sexual maturity.

[0005] While sturgeon farming addresses the ecological challenges of relying on wild sturgeon for caviar production and has made caviar production more widely available, challenges remain for caviar producers. Harvesting the eggs requires careful timing, ideally shortly before spawning, and once the egg sacs are removed, they must be processed quickly to prevent spoilage. This remains a delicate task, often performed manually. The egg sacs are typically gently rubbed against a sieve, either nylon or stainless steel mesh, to separate the eggs from the sac membrane and collect them through the mesh. Cold water can be used to aid in the release and separation of the eggs. Salt is added to the separated eggs, depending on taste and to aid preservation. A stabilizer, such as E285, may also be added. The now caviar-like eggs are then sealed in sealed containers, often under vacuum, for refrigerated storage. The caviar may then be repackaged into smaller portions.

[0006] Ultimately, despite some progress in the caviar industry, production remains slow and prices remain relatively high. Existing caviar production methods are also limited by the requirement to sacrifice sturgeons when harvesting the caviar, which also limits the potential commercial market for products containing caviar and its extracts.

[0007] There is a need in the art for products (e.g., cosmetics) that possess the highly desirable properties associated with caviar but that can be produced more quickly, cheaply, and ethically, and in sufficient quantities to meet rapidly growing demand. There is also a need in the art for a method of producing such products.

[0008] The present invention is based on the surprising realization that many of the requirements of caviar as a luxury food (such as flavor, shape, color and texture) are largely unrelated to its use in other applications (such as nutrition or cosmetics). In fact, the appearance and texture of caviar (loved by caviar lovers) are often destroyed during the preparation process (such as nutrition or cosmetics) and may even be undesirable (such as in face creams). Despite this, caviar and its extracts remain the gold standard in many applications. Therefore, existing products containing caviar and its extracts (but not caviar itself) are expensive, which is largely driven by the requirements of the luxury food industry, even though many of these requirements are unrelated to other applications. In addition, the current production of products containing caviar and its extracts often requires the sacrifice of sturgeons, which raises ethical issues and limits their potential commercial market.

[0009] Despite caviar’s numerous challenges and shortcomings, the market remains focused on caviar, even in applications unrelated to its consumption as a luxury food. In fact, the market’s focus on caviar is so intense that it fails to consider alternatives that fail to meet the requirements of caviar as a luxury food. Summary of the Invention

[0010] The present invention is based on the surprising discovery that key polypeptides are present in caviar, which possess highly desirable cellular functions, particularly for use in cosmetics. Significant technical challenges were overcome to identify these key caviar polypeptides; their successful identification now enables their "animal-free" production (e.g., through recombinant or synthetic means) without the ethical, economic, or time burdens associated with caviar. Furthermore, the present invention enables the production of compositions containing these key caviar polypeptides at concentrations significantly higher than those achievable using caviar, resulting in compositions of the present invention having superior efficacy compared to prior art caviar-containing compositions.

[0011] By providing key caviar polypeptides, the present invention advantageously "decouples" (A) the inherently highly desirable properties of caviar itself (e.g., for cosmetics) from (B) the demands placed on caviar as a luxury food. Compared to caviar:

[0012] i) the composition of the present invention can be produced faster than caviar (without waiting for the sturgeons to reach sexual maturity, including without waiting for the oocytes to reach at least stage 3);

[0013] ii) the composition of the invention can be produced at a lower cost than caviar (the costs associated with in vitro production are much lower than the costs of catching or farming sturgeon);

[0014] iii) the composition of the present invention avoids ethical issues and offers a larger commercial market (its production process does not involve sturgeons);

[0015] iv) The compositions of the present invention can be produced with concentrations of key polypeptides that are much higher than those that can be achieved when these polypeptides are provided by caviar itself. Therefore, the compositions of the present invention exhibit superior efficacy.

[0016] The present invention provides a composition comprising a recombinant or synthetic caviar polypeptide, wherein the caviar polypeptide is selected from the group consisting of: a superoxide dismutase (SOD3) polypeptide, wherein the SOD3 polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1; or (ii) at least 50 consecutive amino acids of SEQ ID NO: 1; a superoxide dismutase (SOD1) polypeptide, wherein the SOD1 polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2; or (ii) at least 50 consecutive amino acids of SEQ ID NO: 2; a tissue inhibitor of metalloproteinases 1 (TIMP1) polypeptide, wherein the TIMP1 polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3; or (ii) at least 50 consecutive amino acids of SEQ ID NO: 3; an acidic fibroblast growth factor (aFGF) polypeptide, wherein the aFGF polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3; or (ii) at least 50 consecutive amino acids of SEQ ID NO: 3; NO:4; or (ii) at least 50 contiguous amino acids of SEQ ID NO:4; a basic fibroblast growth factor (bFGF) polypeptide, wherein the bFGF polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO:5; or (ii) at least 50 contiguous amino acids of SEQ ID NO:5; an insulin-like growth factor 2 (IGF-2) polypeptide, wherein the IGF-2 polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO:6; or (ii) at least 50 contiguous amino acids of SEQ ID NO:6; and a laminin polypeptide, wherein the laminin polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO:7; or (ii) at least 50 contiguous amino acids of SEQ ID NO:7.

[0017] In some embodiments, the composition comprises two or more recombinant or synthetic caviar polypeptides. In some embodiments, the composition comprises two or more recombinant or synthetic caviar polypeptides selected from the group consisting of SOD3, SOD1, and TIMP1. In some embodiments, the composition comprises SOD3, SOD1, and TIMP1. In some embodiments, the composition comprises SOD3, SOD1, TIMP1, and stem cell factor (SCF).

[0018] In some embodiments, the composition further comprises a recombinant or synthetic stem cell factor (SCF) polypeptide, wherein the SCF polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8; or (ii) at least 50 contiguous amino acids of SEQ ID NO: 8.

[0019] In some embodiments, the composition comprises two or more recombinant or synthetic caviar polypeptides selected from the group consisting of SOD3, SOD1, and SCF. In some embodiments, the composition comprises SOD3 and SOD1. In some embodiments, the composition comprises SOD3 and SOD1. In some embodiments, the composition comprises SOD3 and SCF. In some embodiments, the composition comprises SOD1 and SCF. In some embodiments, the composition comprises SOD3, SOD1, and SCF.

[0020] In some embodiments, the composition comprises SOD3, SOD1, and SCF, and a sodium phosphate buffer.

[0021] In some embodiments, the composition further comprises collagen.

[0022] In some embodiments, the composition is a cosmetic composition. In some embodiments, the cosmetic composition is selected from the group consisting of: skin moisturizers, perfumes, lipsticks, nail polishes, eye and / or facial makeup preparations, shampoos, hair dye preparations, toothpastes, and deodorants.

[0023] The invention also provides an isolated nucleic acid encoding one or more (eg, 1, 2, 3, 4, 5, 6 or 7) caviar polypeptides as defined above.

[0024] The present invention also provides an isolated nucleic acid encoding SCF as defined above.

[0025] In some embodiments, the nucleic acid is codon-optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments: the nucleic acid encoding SOD3 has at least 70% sequence identity to SEQ ID NO: 9; the nucleic acid encoding SOD1 has at least 70% sequence identity to SEQ ID NO: 10; the nucleic acid encoding TIMP1 has at least 70% sequence identity to SEQ ID NO: 11; and the nucleic acid encoding SCF has at least 70% sequence identity to SEQ ID NO: 12.

[0026] The present invention also provides a vector comprising the isolated nucleic acid as described above. In some embodiments, the vector further comprises: (a) an origin of replication; (b) a promoter sequence operably linked to the nucleic acid; and / or (c) a reporter gene.

[0027] The present invention also provides a recombinant cell engineered to express a recombinant or synthetic caviar polypeptide as defined above.

[0028] The present invention also provides a recombinant cell transformed with the vector described above. In some embodiments, the recombinant cell is a Pichia pastoris cell.

[0029] The present invention also provides a method for producing one or more recombinant caviar polypeptides as described above, the method comprising: (a) culturing the recombinant cell as described above in a suitable culture medium; and (b) expressing the recombinant caviar polypeptide.

[0030] The present invention also provides a method for producing one or more synthetic caviar polypeptides as described above, wherein the synthetic caviar polypeptides are produced by: (a) cell-free protein synthesis; or (b) solid phase chemical synthesis.

[0031] The present invention also provides a method for producing a cosmetic, comprising combining: (a) one or more recombinant or synthetic caviar polypeptides as described above, optionally in combination with a recombinant or synthetic SCF as described above; and (b) a cosmetic ingredient.

[0032] The present invention also provides the use of a composition as described above for improving the appearance of skin and / or hair.

[0033] The present invention also provides a method of improving the appearance of skin, comprising applying the composition described above to the skin of a subject.

[0034] The present invention also provides a method of improving the appearance of a subject's hair, the method comprising applying to the subject's hair a composition as defined above.

[0035] The present invention also provides a cosmetic method comprising applying a composition as described above to the skin and / or hair of a subject. The subject is typically a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, such as a domestic pet, such as a dog, cat, or horse.

[0036] The compositions of the present invention are preferably free of animal ingredients. In some embodiments, the compositions of the present invention are suitable for vegetarians.

[0037] "Cosmetics" means products that are intended to be spread, poured, sprinkled, sprayed, introduced into, or otherwise applied to the human body for cleansing, beautification, increased attractiveness, or altered appearance. This definition includes products such as skin moisturizers, perfumes, lipsticks, nail polishes, eye and facial makeup preparations, shampoos, hair dyes, toothpastes, and deodorants, as well as any material intended to be used as an ingredient in a cosmetic. In one embodiment, the cosmetic is a finished cosmetic product.

[0038] The cosmetics of the present invention comprise one or more recombinant or synthetic caviar polypeptides of the present invention and typically comprise one or more cosmetic ingredients. Typical ingredients of cosmetics are selected from the group consisting of: water; emulsifiers such as laureth-4, polysorbate, and potassium cetyl sulfate; preservatives; thickeners such as xanthan gum, gelatin, silica, or cetyl alcohol; emollients such as waxes, oils, or silicones; pigments such as iron oxide, manganese, mica flakes, or beet powder; shimmers such as mica or bismuth oxychloride; and / or fragrance.

[0039] In some embodiments of the present invention, the cosmetic comprises a buffer. In some embodiments, the buffer is a phosphate buffer, such as a sodium phosphate buffer. The pH of the buffer is generally selected to be compatible with the skin, such as pH 4 to pH 7.5; pH 5 to pH 7.5; pH 5.5 to pH 7.5; pH 6 to pH 7.5; pH 4 to pH 7; pH 5 to pH 7; pH 5.5 to pH 7; pH 6 to pH 7; pH 4 to pH 6.5; pH 5 to pH 6.5; pH 5.5 to pH 6.5; pH 6 to pH 6.5; pH 4 to pH 6; pH 5 to pH 6; or pH 5.5 to pH 6. In some embodiments, the pH of the buffer is pH 5.5 to pH 6.5, such as pH 5.7 to pH 6.3; or pH 5.9 to pH 6.1. In some embodiments, the cosmetic comprises a buffer salt (e.g., sodium phosphate) at a concentration of 1 mM to 10 mM, for example, 1 mM to 9 mM; 1 mM to 8 mM; 1 mM to 7 mM; 1 mM to 6 mM; 1 mM to 5 mM; 2 mM to 9 mM; 2 mM to 8 mM; 2 mM to 7 mM; 2 mM to 6 mM; 2 mM to 5 mM; 3 mM to 9 mM; 3 mM to 8 mM; 3 mM to 7 mM; 3 mM to 6 mM; 3 mM to 5 mM; 4 mM to 9 mM; 4 mM to 8 mM; 4 mM to 7 mM; 4 mM to 6 mM; or 4 mM to 5 mM. In some embodiments, the cosmetic comprises a buffer salt (e.g., sodium phosphate) at a concentration of 4 mM to 6 mM, for example, 4.5 mM to 5.5 mM; or 4.7 mM to 5.3 mM. In some embodiments, the cosmetic comprises a sodium phosphate buffer having a concentration of 4 mM to 6 mM and a pH of pH 5.5 to pH 6.5.

[0040] In some embodiments, the composition comprises 100 μg / L to 1500 μg / L of a polypeptide. In some embodiments, the composition comprises 200 μg / L to 1500 μg / L, 300 μg / L to 1500 μg / L, 400 μg / L to 1500 μg / L, 500 μg / L to 1500 μg / L, 600 μg / L to 1500 μg / L, 700 μg / L to 1500 μg / L, 800 μg / L to 1500 μg / L, 900 μg / L to 1500 μg / L, 1000 μg / L to 1500 μg / L, 1100 μg / L to 1500 μg / L, 1200 μg / L to 1500 μg / L, 1300 μg / L to 1500 μg / L, 1400 μg / L to 1500 μg / L, g / L to 1500 μg / L, 100 μg / L to 1400 μg / L, 100 μg / L to 1300 μg / L, 100 μg / L to 1200 μg / L, 100 μg / L to 1100 μg / L, 100 μg / L to 1000 μg / L, 100 μg / L to 900 μg / L, 100 μg / L to 800 μg / L, 100 μg / L to 700 μg / L, 100 μg / L to 600 μg / L, 100 μg / L to 500 μg / L, 100 μg / L to 400 μg / L, 100 μg / L to 300 μg / L, or 100 μg / L to 200 μg / L of a polypeptide. When the composition comprises two or more polypeptides, the total concentration of the polypeptides in the composition may be 100 μg / L to 1500 μg / L. Alternatively, when the composition comprises two or more polypeptides, the concentration of each polypeptide may be 100 μg / L to 1500 μg / L. For example, the composition may comprise 100 μg / L to 500 μg / L of SOD3, 100 μg / L to 500 μg / L of SOD1, and 100 μg / L to 500 μg / L of SCF.

[0041] The advantages of the present invention are not limited to any particular type of polypeptide production method. The caviar polypeptides of the present invention are typically recombinant or synthetic. Recombinant methods for protein production are well known, including, for example, expression of nucleic acids in recombinant host cells. Synthetic methods for protein production are also well known, including, for example, cell-free protein synthesis or solid-phase chemical synthesis. Detailed Description of the Invention

[0043] The present invention is based on the surprising discovery that key polypeptides in caviar have highly desirable cellular functions, particularly for use in cosmetics.

[0044] To identify the peptides in caviar, the inventors analyzed Siberian sturgeon (Acipenser baerii) caviar using liquid chromatography-mass spectrometry. Significant technical challenges were encountered throughout the analytical process, requiring modifications to existing mass spectrometry techniques. The improved method involved preparing a biphasic solution of oil and water, followed by a customized ammonium sulfate precipitation, and peptide separation using urea, followed by mass spectrometry fingerprinting. After identifying the peptides in the caviar, the inventors meticulously analyzed the cellular functions associated with the identified peptides and determined which functions were ideal (and highly desirable) for cosmetic applications in caviar.

[0045] The key caviar polypeptides of the present invention include:

[0046] a) superoxide dismutase (SOD3) polypeptide;

[0047] b) superoxide dismutase (SOD1) polypeptide;

[0048] c) tissue inhibitor of metalloproteinases 1 (TIMP1) polypeptide;

[0049] d) acidic fibroblast growth factor (aFGF) polypeptide;

[0050] e) basic fibroblast growth factor (bFGF) polypeptide;

[0051] f) insulin-like growth factor 2 (IGF-2) polypeptide; and

[0052] g) Laminin polypeptide.

[0053] In a preferred embodiment, the composition of the present invention comprises one or more key caviar polypeptides of the present invention and stem cell factor (SCF). For example, the composition of the present invention may comprise SOD3 and SCF; SOD1 and SCF; or SOD1, SOD3, and SCF. Caviar superoxide dismutase (SOD3)

[0054] Superoxide dismutase (SOD3) is an antioxidant enzyme that catalyzes the dismutation of two superoxide radicals to produce hydrogen peroxide and oxygen. Cosmetic benefits of SOD3 activity include anti-aging, hair growth promotion, and antioxidant activity.

[0055] Prior to the present invention, the presence of SOD3 in caviar was unknown. In fact, automated computational analysis of the corresponding NCBI reference sequence (XP_033870136.2) predicted that SOD3 was only present in the testicles and blood.

[0056] The SOD3 polypeptides of the present invention may be recombinant or synthetic.

[0057] In some embodiments, the SOD3 polypeptide of the present invention comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the SOD3 polypeptide of the present invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the SOD3 polypeptide of the present invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 1.

[0058] In some embodiments, the SOD3 polypeptide of the present invention comprises at least 50 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the SOD3 polypeptide of the present invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 240, at least 241, or at least 242 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the SOD3 polypeptide of the present invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 240, at least 241, or at least 242 consecutive amino acids of SEQ ID NO: 1, wherein the consecutive amino acids comprise the catalytic domain of the SOD3 polypeptide.

[0059] The SOD3 polypeptide sequence is represented by SEQ ID NO: 1:

[0060] MTMSAFSFLLALAIAGTHVSHSEESPTSEENTMKNIESKVNDLWQSLLHPVA

[0061] FVAKDAELVYASCEMKPSTKLEEGKPQVTGKVLFKQAYPQGRLESIINLEGFP

[0062] KTSNQSRAIHIHEFGDLSDGCDAAGGHFNPFKVNHPRHPGDFGNFLPKNS

[0063] The catalytic domain of SOD3 comprises the following amino acid sequence: QIKTLKKNIQATMFGPNSFLSRSVVIHELKDDLGKGDNPASLLNGNAGKRLACCVIGISNKNLWEKTSQSLTSSKKKRNARGLANKQA (SEQ ID NO: 1).

[0064] HFNPFKVNHPRHPGD (SEQ ID NO: 17; underlined amino acids indicate key catalytic site residues).

[0065] The SOD3 polypeptides of the present invention may have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO: 1.

[0066] The present invention also provides a fusion protein comprising at least a portion (e.g., a fragment or domain) of a SOD3 polypeptide of the present invention linked to one or more fusion fragments, which are generally heterologous to the SOD3 polypeptide.

[0067] The SOD3 nucleic acid of the present invention encodes the SOD3 polypeptide of the present invention. The SOD3 nucleic acid is typically an isolated nucleic acid. The SOD3 nucleic acid of the present invention can be optimized for expression in recombinant cells.

[0068] In some embodiments, the SOD3 nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 9. In some embodiments, the SOD3 nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 9 over a region of at least about 10 nucleotides, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 729 nucleotides, or the catalytic domain thereof.

[0069] SEQ ID NO: 9 represents the nucleic acid sequence corresponding to the SOD3 polypeptide (SEQ ID NO: 1), having the codon preference of S. cerevisiae for optimized expression in Pichia pastoris:

[0070]

[0071] AAAATTCTCAAATTAAAACTTTGAAAAAAAATATTCAAGCTACTATGTTT

[0072] GGTCCAAATTCTTTTTTGTCTAGATCTGTTGTTATTCATGAATTGAAAGAT

[0073] GATTTGGGTAAAGGTGATAATCCAGCTTCTTTGTTGAATGGTAATGCTGG

[0074] TAAAAGATTGGCTTGTTGTGTTATTGGTATTTCTAATAAAAATTTGTGGGA

[0075] AAAAACTTCTCAATCTTTGACTTCTTCTAAAAAAAAAAGAAATGCTAGAGGTTTGGCTAATAAACAAGCT (SEQ ID NO: 9).

[0076] Caviar Superoxide Dismutase (SOD1)

[0077] Superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the decomposition of superoxide radicals. Cosmetic benefits of SOD1 activity include anti-aging, hair growth promotion, and antioxidant activity.

[0078] Prior to the present invention, the presence of SOD1 in caviar was unknown. In fact, automated computational analysis of the corresponding NCBI reference sequence (XP_033865220.2) predicted that SOD1 is only present in testicles and blood.

[0079] The SOD1 polypeptides of the present invention may be recombinant or synthetic. In some embodiments, the SOD1 polypeptides of the present invention comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the SOD1 polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the SOD1 polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 2.

[0080] In some embodiments, the SOD1 polypeptide of the present invention comprises at least 50 consecutive amino acids of SEQ ID NO: 2. In some embodiments, the SOD1 polypeptide of the present invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 151, at least 152, at least 153, or at least 154 consecutive amino acids of SEQ ID NO: 2. In some embodiments, the SOD1 polypeptide of the present invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 151, at least 152, at least 153, or at least 154 consecutive amino acids of SEQ ID NO: 2, wherein the consecutive amino acids comprise the catalytic domain of the SOD1 polypeptide.

[0081] The SOD1 polypeptide sequence is represented by SEQ ID NO: 2:

[0082] MVLKAVCVLKGTGDVCGTVHFVQEKEAGPVKLTGQITGLTPGEHGFHVHA

[0083] FGDNTNGCASAGPHFNPLGKTHGAPQDEIRHIGDLGNVIAGDDKVAIINIE

[0084] DKLITLSGAYSIIGRTTMVIHEKADDLGKGGNDESLVTGNAGGRLACGVIGIAQS (SEQ ID NO: 2).

[0085] The catalytic domain of SOD1 contains the following amino acid sequence:

[0086] GFHVHAFGDNT (SEQ ID NO: 18; underlined amino acids indicate key catalytic site residues)

[0087] The SOD1 polypeptides of the present invention may have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40 or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO: 2.

[0088] The present invention also provides a fusion protein comprising at least a portion (e.g., a fragment or domain) of a SOD1 polypeptide of the present invention, linked to one or more fusion segments, which are generally heterologous to the SOD1 polypeptide.

[0089] The SOD1 nucleic acid of the present invention encodes the SOD1 polypeptide of the present invention. The SOD1 nucleic acid is generally an isolated nucleic acid. The SOD1 nucleic acid of the present invention can be optimized for expression in recombinant cells.

[0090] In some embodiments, the SOD1 nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 10. In some embodiments, the SOD1 nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 10 over a region of at least about 10 nucleotides, for example, at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 465 nucleotides, or a catalytic domain thereof.

[0091] SEQ ID NO: 10 represents the nucleic acid sequence corresponding to the SOD1 polypeptide (SEQ ID NO: 2) with the codon preference of Saccharomyces cerevisiae for optimized expression in Pichia pastoris: ATGGTTTTGAAAGCTGTTTGTGTTTTGAAAGGTACTGGTGATGTTTGTGGTACTGTTCATTTTGTTCAAGAAAAAGAAGCTGGTCCAGTTAAATTGACTGGTCAAATTACTGGTTTGACTCCAGGTGAACATGGTTTTCATGTTCATGCTTTTGGTGATAATACTAATGGTTGTGCTTCTGCTGGTCCACATTTTAATCCATTGGGTAAAACTC ATGGTGCTCCACAAGATGAAATTAGACATATTGGTGATTTGGGTAATGTTATTGCTGGTGATGATAAAGTTGCTATTATTAATATTGAAGATAAATTGATTACTTTGTCTGGTGCTTATTCTATTAT TGGTAGAACTATGGTTATTCATGAAAAAGCTGATGATTTGGGTAAAGGTGGTAATGATGAATCTTTGGTTACTGGTAATGCTGGTGGTAGATTGGCTTGTGGTGTTATTGGTATTGCTCAATCT(SEQ ID NO: 10).

[0092] Caviar tissue inhibitor of metalloproteinases 1 (TIMP1)

[0093] Tissue inhibitor of metalloproteinases 1 (TIMP1) is involved in the degradation of the extracellular matrix, can promote cell proliferation in various cell types, and is thought to have anti-apoptotic functions. Cosmetic benefits of TIMP1 activity include anti-aging, hair growth promotion, and wound healing.

[0094] Prior to the present invention, the presence of TIMP1 in caviar was unknown. In fact, automated computational analysis of the corresponding NCBI reference sequence (XP_034775618.1) predicted that TIMP1 is only present in testicles and blood.

[0095] The TIMP1 polypeptides of the present invention can be recombinant or synthetic. In some embodiments, the TIMP1 polypeptides of the present invention comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the TIMP1 polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the TIMP1 polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 3.

[0096] In some embodiments, the TIMP1 polypeptide of the invention comprises at least 50 contiguous amino acids of SEQ ID NO: 3. In some embodiments, the TIMP1 polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 204, at least 205, or at least 206 contiguous amino acids of SEQ ID NO: 3. In some embodiments, the TIMP1 polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 204, at least 205, or at least 206 consecutive amino acids of SEQ ID NO: 3, wherein the consecutive amino acids comprise the catalytic domain of the TIMP1 polypeptide.

[0097] The TIMP1 polypeptide sequence is represented by SEQ ID NO: 3:

[0098] MAPLAPLASCILLLLWLAAPSRACTCAPLHPQTAFCSSDFIIRAKFVGTAEVN

[0099] QTALSQRYEIKMTKMFKGFSALGDASDIRFVYTPTAESVCGYFHRSQNRSEE

[0100] FLIAGKLRNGHLHINTCSYVVPWNSLSSSSQRRGFTKTYAAGCEECTVFSCSSI

[0101] PCKLQNDTHCLWTDQFLTGTDKGFQSRHLACLPREPGICTWQSLRTRMA (SEQ ID NO: 3).

[0102] The TIMP1 polypeptides of the present invention may have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO: 3.

[0103] The present invention also provides a fusion protein comprising at least a portion (eg, a fragment or domain) of a TIMP1 polypeptide of the present invention linked to one or more fusion segments, which are typically heterologous to the TIMP1 polypeptide.

[0104] The TIMP1 nucleic acid of the present invention encodes the TIMP1 polypeptide of the present invention. The TIMP1 nucleic acid is typically an isolated nucleic acid. The TIMP1 nucleic acid of the present invention can be optimized for expression in recombinant cells.

[0105] In some embodiments, the TIMP1 nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 11. In some embodiments, the TIMP1 nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 11 over a region of at least about 10 nucleotides, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 621 nucleotides, or the catalytic domain thereof.

[0106] SEQ ID NO: 11 represents the nucleic acid sequence corresponding to the TIMP1 polypeptide (SEQ ID NO: 3), having the codon preference of S. cerevisiae for optimized expression in Pichia pastoris:

[0107]

[0108] CTTGTTCTATTCCATGTAAATTGCAAAATGATACTCATTGTTTGTGGA

[0109] CTGATCAATTTTTGACTGGTACTGATAAAGGTTTTCAATCTAGACATTTGG

[0110] CTGTTTGCCAAGAGAACCAGGTATTTGTACTTGGCAATCTTTGAGAACTAGAATGGCT (SEQ ID NO: 11).

[0111] Caviar acidic fibroblast growth factor (aFGF)

[0112] Acidic fibroblast growth factor (aFGF), also known as fibroblast growth factor 1, is a fibroblast activation protein. The cosmetic benefits of aFGF activity include anti-aging, anti-wrinkle, wound healing, prevention of hair loss, and promotion of hair growth.

[0113] Before the present invention, people did not know that aFGF existed in caviar.In fact, the corresponding U.S. National Center for Biotechnology Information (GenBank) reference sequence (RXM90924.1) was predicted by automatic computational analysis that aFGF only exists in blood.

[0114] The aFGF polypeptides of the present invention can be recombinant or synthetic. In some embodiments, the aFGF polypeptides of the present invention comprise an amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the aFGF polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the aFGF polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 4.

[0115] In some embodiments, the aFGF polypeptide of the invention comprises at least 50 contiguous amino acids of SEQ ID NO: 4. In some embodiments, the aFGF polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 151, at least 152, at least 153, or at least 154 contiguous amino acids of SEQ ID NO: 4. In some embodiments, the aFGF polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 151, at least 152, at least 153, or at least 154 contiguous amino acids of SEQ ID NO: 4, wherein the contiguous amino acids comprise the catalytic domain of the aFGF polypeptide.

[0116] The aFGF polypeptide sequence is represented by SEQ ID NO: 4:

[0117] MAEGKVTVLTILPEKFNLHLENYKKPNLLYCYNGGYFLRVLPNGAVDGIRDR

[0118] SDKHIQLQVTAENVGVVSIKGLEAGRYLAMSTDGQLCGSQTLTDECFFLETL

[0119] EENHYATYKAQKYQDRNWYVGIKKNGRCKSGEKTHIGQKAILFLLPLSASSD (SEQ ID NO: 4).

[0120] The aFGF polypeptides of the invention can have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO:4.

[0121] The present invention also provides a fusion protein comprising at least a portion (eg, a fragment or domain) of an aFGF polypeptide of the present invention linked to one or more fusion segments, which are typically heterologous to the aFGF polypeptide.

[0122] The aFGF nucleic acid of the present invention encodes the aFGF polypeptide of the present invention. The aFGF nucleic acid is typically an isolated nucleic acid. The aFGF nucleic acid of the present invention can be optimized for expression in recombinant cells.

[0123] In some embodiments, the aFGF nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 13. In some embodiments, the aFGF nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 13 over a region of at least about 10 nucleotides, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 465 nucleotides, or the catalytic domain thereof.

[0124] SEQ ID NO: 13 represents the nucleic acid sequence corresponding to the aFGF polypeptide (SEQ ID NO: 1), with codon preference for S. cerevisiae for optimized expression in Pichia pastoris:

[0125]

[0126]

[0127] Caviar basic fibroblast growth factor (bFGF)

[0128] Basic fibroblast growth factor (bFGF), also known as fibroblast growth factor 2, is a fibroblast activation protein. The cosmetic benefits of bFGF activity include anti-aging, anti-wrinkle, wound healing, prevention of hair loss, and promotion of hair growth.

[0129] Before the present invention, people did not know that bFGF existed in caviar.In fact, the corresponding U.S. National Center for Biotechnology Information (GenBank) reference sequence (RXM97423.1) was predicted by automatic computational analysis that bFGF only exists in blood.

[0130] The bFGF polypeptides of the present invention can be recombinant or synthetic. In some embodiments, the bFGF polypeptides of the present invention comprise an amino acid sequence that has at least 70% sequence identity to SEQ ID NO:5. In some embodiments, the bFGF polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5. In some embodiments, the bFGF polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO:5.

[0131] In some embodiments, a bFGF polypeptide of the invention comprises at least 50 contiguous amino acids of SEQ ID NO: 5. In some embodiments, a bFGF polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 151, at least 152, at least 153, or at least 154 contiguous amino acids of SEQ ID NO: 5. In some embodiments, a bFGF polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 151, at least 152, at least 153, or at least 154 contiguous amino acids of SEQ ID NO: 5, wherein the contiguous amino acids comprise the catalytic domain of the bFGF polypeptide.

[0132] The bFGF polypeptide sequence is represented by SEQ ID NO: 5:

[0133] MAAGGITTFPTVPDDGGSSTFPPGNFKEPKRLYCKNGGYFLRINPDGRVDGI

[0134] REKDDPRIKLQLQAESIGVVSIKGVSANRYLAMNEDGRLFGSKCTTDECFFFE RLESNNYNTYRSQKYPDWYVALKRTGQFKSGSKTGPGQKAILFLPMSAKS (SEQ ID NO: 5).

[0135] The bFGF polypeptides of the present invention can have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO:5.

[0136] The present invention also provides a fusion protein comprising at least a portion (eg, a fragment or domain) of a bFGF polypeptide of the present invention linked to one or more fusion segments, which are typically heterologous to the bFGF polypeptide.

[0137] The bFGF nucleic acid of the present invention encodes the bFGF polypeptide of the present invention. The bFGF nucleic acid is typically an isolated nucleic acid. The bFGF nucleic acid of the present invention can be optimized for expression in recombinant cells.

[0138] In some embodiments, the bFGF nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 14. In some embodiments, the bFGF nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 14 over a region of at least about 10 nucleotides, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 465 nucleotides, or the catalytic domain thereof.

[0139] SEQ ID NO: 14 represents the nucleic acid sequence corresponding to the bFGF polypeptide (SEQ ID NO: 5) with codon preference of S. cerevisiae for optimized expression in Pichia pastoris:

[0140]

[0141] Caviar Insulin-like Growth Factor 2 (IGF-2)

[0142] Insulin-like growth factor 2 (IGF-2) promotes cell growth and proliferation. The cosmetic benefits of IGF-2 activity include anti-aging, anti-wrinkle, wound healing, prevention of hair loss, and promotion of hair growth.

[0143] Prior to the present invention, the presence of IGF-2 in caviar was unknown. In fact, automated computational analysis of the corresponding GenBank reference sequence (RXM35556.1) predicted that IGF-2 was only present in blood.

[0144] The IGF-2 polypeptides of the present invention can be recombinant or synthetic. In some embodiments, the IGF-2 polypeptides of the present invention comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the IGF-2 polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the IGF-2 polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 6.

[0145] In some embodiments, the IGF-2 polypeptides of the invention comprise at least 50 contiguous amino acids of SEQ ID NO: 6. In some embodiments, the IGF-2 polypeptides of the invention comprise at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 215, at least 216, at least 217, or at least 218 contiguous amino acids of SEQ ID NO: 6. In some embodiments, an IGF-2 polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 215, at least 216, at least 217, or at least 218 consecutive amino acids of SEQ ID NO: 6, wherein the consecutive amino acids comprise the catalytic domain of the IGF-2 polypeptide.

[0146] The IGF-2 polypeptide sequence is represented by SEQ ID NO: 6:

[0147] MEDHLSYNKHQAYCHNCIESGNSSSNSIKVRKMSTSRQLLVFTIALTVYIVDV

[0148] ANSIASAETLCGGELVDTLQFVCGDRGFYFSKPPSRSNIRRSQKGIVEVCCYS

[0149] SCDLRLLEMYCAKPAKSERDVSSTQFQVIPLALNKDVSKKPIIAKYSKYELWQ

[0150] KKAAQRLRRGVPSILKARKFRRHAEEIKALEQTRFHRPLITLPSKQPVTVKPLTENYASKK(SEQ IDNO:6).

[0151] The IGF-2 polypeptides of the invention can have one or more (e.g., up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO: 15.

[0152] The present invention also provides a fusion protein comprising at least a portion (eg, a fragment or domain) of an IGF-2 polypeptide of the present invention linked to one or more fusion segments, which are typically heterologous to the IGF-2 polypeptide.

[0153] The IGF-2 nucleic acids of the invention encode the IGF-2 polypeptides of the invention. The IGF-2 nucleic acid is typically an isolated nucleic acid. The IGF-2 nucleic acids of the invention can be optimized for expression in recombinant cells.

[0154] In some embodiments, the IGF-2 nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 15. In some embodiments, the IGF-2 nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 15 over a region of at least about 10 nucleotides, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 657 nucleotides, or the catalytic domain thereof.

[0155] SEQ ID NO: 15 represents the nucleic acid sequence corresponding to the IGF-2 polypeptide (SEQ ID NO: 6) with codon preference of S. cerevisiae for optimized expression in Pichia pastoris:

[0156]

[0157] Caviar Laminin

[0158] Laminin is an important, biologically active component of the basement membrane, influencing cell differentiation, migration, and adhesion. Cosmetic benefits of laminin activity include anti-aging effects and hair growth promotion.

[0159] Prior to the present invention, the presence of laminin in caviar was unknown. In fact, automated computational analysis of the corresponding GenBank reference sequence (RXN01125.1) predicted that laminin was only present in blood.

[0160] The laminin polypeptides of the present invention can be recombinant or synthetic. In some embodiments, the laminin polypeptides of the present invention comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7. In some embodiments, the laminin polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the laminin polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 7.

[0161] In some embodiments, the laminin polypeptide of the present invention comprises SEQ ID NO:

[0162] In some embodiments, the laminin polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2280, at least 2281, at least 2282, at least 2283, or at least 2284 consecutive amino acids of SEQ ID NO:7. In some embodiments, the laminin polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2280, at least 2281, at least 2282, at least 2283, or at least 2284 contiguous amino acids of SEQ ID NO:7.

[0163] The laminin polypeptide sequence is represented by SEQ ID NO: 7:

[0164]

[0165]

[0166]

[0167] The laminin polypeptides of the invention can have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO:7.

[0168] The present invention also provides a fusion protein comprising at least a portion (eg, a fragment or domain) of a laminin polypeptide of the present invention linked to one or more fusion segments, which are typically heterologous to the laminin polypeptide.

[0169] The laminin nucleic acid of the present invention encodes the laminin polypeptide of the present invention. The laminin nucleic acid is generally an isolated nucleic acid. The laminin nucleic acid of the present invention can be optimized for expression in recombinant cells.

[0170] In some embodiments, the laminin nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 16. In some embodiments, the laminin nucleic acid sequence is over a region of at least about 10 nucleotides, such as at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 10 : 16 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 16 over a region of at least 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 6855 nucleotides.

[0171] SEQ ID NO: 16 represents the nucleic acid sequence corresponding to the laminin polypeptide (SEQ ID NO: 7) with codon preference of S. cerevisiae for optimized expression in Pichia pastoris:

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] GAAACTAAAGAAGGTACTACTTCTGGTTCTGCTTCTGATTTGAATAG

[0180] ATCTGATAAAGATCCAATTTATATTGGTGGTTTGCCAAGATCTAGACC

[0181] AGTTAGAAGACAATTGGTTACTAGATCTTATGTTGGTTGTATTAAAAA

[0182] TTTGGAAATTGCTAGATCTAATTTTGATTTGTTGAAAGAATCTTATGG

[0183] TGTTAAAAAAGGTTGTGTTTTGGAACATGGTACTGAACCATGGTAT

[0184] (SEQ ID NO:16).

[0185] Stem Cell Factor (SCF) Peptide

[0186] In some embodiments, the compositions of the present invention comprise a stem cell factor (SCF) polypeptide. SCF is sometimes referred to in the literature as "Kit ligand." The cosmetic benefits of SCF activity are believed to include restoration of skin health, skin regeneration, and slowing the aging of skin cells.

[0187] The corresponding National Center for Biotechnology Information (GenBank) reference sequence (RXN01125.1) annotated that SCF is present in blood.

[0188] The SCF polypeptides of the present invention can be recombinant or synthetic. In some embodiments, the SCF polypeptides of the present invention comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8. In some embodiments, the SCF polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the SCF polypeptides of the present invention have at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: 8.

[0189] In some embodiments, the SCF polypeptide of the invention comprises at least 50 contiguous amino acids of SEQ ID NO: 8. In some embodiments, the SCF polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 310, at least 311, at least 312, at least 313, or at least 314 contiguous amino acids of SEQ ID NO: 8. In some embodiments, an SCF polypeptide of the invention comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 310, at least 311, at least 312, at least 313, or at least 314 contiguous amino acids of SEQ ID NO:8, wherein the contiguous amino acids comprise the catalytic domain of the SCF polypeptide.

[0190] The SCF polypeptide sequence is represented by SEQ ID NO: 8:

[0191]

[0192] The SCF polypeptides of the invention can have one or more (eg, up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO:8.

[0193] The present invention also provides a fusion protein comprising at least a portion (eg, a fragment or domain) of an SCF polypeptide of the present invention linked to one or more fusion segments, which are typically heterologous to the SCF polypeptide.

[0194] The SCF nucleic acids of the invention encode the SCF polypeptides of the invention. The SCF nucleic acids are typically isolated nucleic acids. The SCF nucleic acids of the invention can be optimized for expression in recombinant cells.

[0195] In some embodiments, the SCF nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 12. In some embodiments, the SCF nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 12 over a region of at least about 10 nucleotides, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 945 nucleotides, or a catalytic domain thereof.

[0196] SEQ ID NO: 12 represents the nucleic acid sequence corresponding to the SCF polypeptide (SEQ ID NO: 8) with codon preferences of S. cerevisiae for optimized expression in Pichia pastoris:

[0197] ATGTATTTGTTTCCACAAATTTGGATTACTGCTTTTATTTATCCATGTTTGT

[0198] TGTTTTGTTTTACTTTTGTTGAACATTCTTGTGGTTTGGGTAATGTTGTTAC

[0199] TGATGATGTTAATAAAATTCCAATTTTGAAAGGTAATATTCCAAATGATT

[0200]

[0201] An example of an algorithm suitable for determining sequence similarity is the BLAST algorithm, described by Altshull et al. in J. Mol. Biol., Vol. 215, pp. 403-410, 1990. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These short words either match or satisfy a positive threshold score T when aligned with words of the same length in a database sequence. These initial local word hits serve as starting points for searching for longer high-scoring sequence pairs containing them. The word hits are extended in each direction between the two compared sequences for as long as the cumulative alignment score can be increased. Extension of the word hits is terminated when: the cumulative alignment score drops by an amount X from the maximum achieved; the cumulative score reaches zero or lower; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses by default a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1992, Proc. Nat'l. Acad. Sci. USA, vol. 89, pp. 10915-10919), the number of alignments (B) of 50, the expectation (E) of 10, the M value of 5, the N value of -4, and a comparison of both strands.

[0202] Any amino acid sequence described herein can be generated with at least 1 (e.g., at least (or at most) 2, 3, 5, 10, or 20) heterologous amino acids flanking each C-terminus and / or N-terminus of the specified amino acid sequence, or with at least 1 (e.g., at least (or at most) 2, 3, 5, 10, or 20) amino acids deleted from the C-terminus and / or N-terminus.

[0203] Conservative substitutions can be selected from a group of amino acids with similar side chains to the reference amino acid. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxy side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Thus, for each naturally occurring amino acid, exemplary conservative substitutions are as follows: alanine for serine; arginine for lysine; asparagine for glutamine or histidine; aspartic acid for glutamic acid; cysteine ​​for serine or alanine; glutamine for asparagine; glutamic acid for aspartic acid; glycine for proline; histidine for asparagine or glutamine; isoleucine for leucine or valine; leucine for isoleucine or valine; lysine for arginine, glutamine, or glutamic acid; methionine for leucine or isoleucine; phenylalanine for methionine, leucine, or tyrosine; serine for threonine; threonine for serine; tryptophan for tyrosine; tyrosine for tryptophan or phenylalanine; valine for isoleucine or leucine.

[0204] Suitable fusion fragments include, but are not limited to, fragments that can provide other desired biological activities or facilitate purification of the polypeptides of the invention (e.g., by affinity chromatography). The fusion fragment can be attached to the amino terminus or carboxyl terminus of the polypeptides of the invention. The fusion fragment can be cleavable. In some embodiments, the fusion fragment is a tag, such as a purification tag. In some embodiments, the purification tag is a histidine tag (e.g., a 6x histidine tag or an 8x histidine tag).

[0205] An "optimized" nucleic acid sequence encodes an amino acid sequence using codons that are preferred in recombinant cells. The optimized nucleic acid sequence is typically designed to retain, in full or as much as possible, the amino acid sequence originally encoded by the starting nucleic acid sequence (also referred to as the "parental" sequence). Several codon optimization methods are known in the art. Preferably, the codon-optimized sequence is prepared by adjusting settings in commercial software or manually modifying the sequence to avoid nucleotide repeats and restriction enzyme sites used when cloning the nucleic acids of the present invention, for example, by replacing codons that introduce unwanted sequences with codons that are highly used in the target heterologous organism.

[0206] In some embodiments, nucleic acid is carried out codon optimized to express in prokaryotic cell.In some embodiments, nucleic acid is carried out codon optimized to express in acinetobacter, Agrobacterium, Escherichia coli, Cuprum Pseudomonas, Clostridium, Rhodobacter, Marinobacter, Bacillus, Klebsiella, Tatum Pseudomonas, Lars, Rhodococcus, Methylobacterium, Methylophilus, Methylococcus, Methylmicrobacterium, Methylomonas, Pantoea, Streptomyces, Parachlorella, Synechococcus, Synechocystis and Thermosynechococcus.In some embodiments, nucleic acid is carried out codon optimized to express in intestinal bacteria.

[0207] In some embodiments, nucleic acid is codon optimized to express in eukaryotic cells. In some embodiments, nucleic acid is codon optimized to express in yeast cells, fungal cells, algae cells and plant cells. In some embodiments, nucleic acid is codon optimized to express in Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces, Candida, Schizosaccharomyces, Schefflera, Rhodosporidium, Hansenula, Klerkera, Schwann yeast, Ithamisaccharomyces, Yarrowia or Rhodotorula. In some embodiments, nucleic acid is codon optimized to express in Saccharomyces cerevisiae, Yarrowia lipolytica, Rhodotorula glutinosus, Sporobolomyces buleri, Barnett's yeast, microzyme, grape yeast, diastatic yeast, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces fragilis, Pichia kudriavzevii, Pichia stipitis or Ithamisaccharomyces orientalis.

[0208] In some embodiments (e.g., where codon optimization is not well established for a particular cell type), the nucleic acid is codon-optimized for expression in a similar cell type. For example, in some embodiments, the nucleic acid sequence is optimized with the codon bias of Saccharomyces cerevisiae for optimal expression in Pichia pastoris.

[0209] In some embodiments, nucleic acid is codon optimized for expression in filamentous fungal cells. In some embodiments, nucleic acid is codon optimized for expression in Aspergillus, Penicillium, Rhizopus, Chrysosporium, Myceliophthora thermophila, Trichoderma, Humicola, Cephalosporium acremonium or Fusarium. In some embodiments, nucleic acid is codon optimized for expression in Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, Myceliophthora thermophila or Rhizopus oryzae.

[0210] In some embodiments, the nucleic acid is codon optimized for expression in Botrytis cinerea, Nannochloropsis sp., Chlorella sp., Chlamydomonas sp., Dunaliella salina, Chaetoceros sp., Porphyridium sp., Scenedesmus sp., or Pseudochlorococcus sp. In some embodiments, the nucleic acid is codon optimized for expression in Botrytis cinerea or Nannochloropsis gadirii.

[0211] carrier

[0212] The vector of the present invention comprises a nucleic acid of the present invention. The vector may comprise an origin of replication, a promoter sequence operably linked to the nucleic acid of the present invention, and one or more of a reporter gene or a selectable marker. Any suitable vector may be used, for example, a pPIC9K vector.

[0213] The promoter can be homologous or heterologous. The promoter can be constitutive or inducible.

[0214] In one embodiment, the promoter is inducible and is activated in the presence of an inducer. Inducers include, but are not limited to, carbohydrates, metal salts, and antibiotics. In some embodiments, the inducible promoter is the AOX1 promoter. In some cases, the promoter allows constitutive expression of the polypeptide of the present invention.

[0215] In one embodiment, promoters that are active at different stages of growth may be used.The promoter sequence may be functional in prokaryotic cells (eg, E. coli cells).

[0216] The promoter sequence may be functional in eukaryotic cells, such as yeast cells, fungal cells, algal cells, or plant cells.

[0217] When the recombinant cell is a fungal cell, the promoter can be a fungal promoter (including but not limited to a filamentous fungal promoter), a promoter that works in a plant cell, or a promoter that works in a mammalian cell. When the 5' untranslated region (UTR) sequence (i.e., the sequence that starts from the transcription start site and ends at one nucleotide before the start codon) that is usually associated with a mammalian or mammalian viral promoter is replaced by a fungal 5'UTR sequence, mammals, mammalian viruses, plants, and plant viral promoters can drive particularly high expression. The source of the 5'UTR can vary as long as it works in filamentous fungal cells. In various embodiments, the 5'UTR can be derived from a yeast gene or a filamentous fungal gene. The 5'UTR can be from the same species as the recombinant cell, or from a different species.

[0218] Promoters for recombinant expression in yeast are known in the art. Suitable promoters for use in Saccharomyces cerevisiae include, but are not limited to, the MFα1 promoter, galactose-inducible promoters (such as GAL1, GAL7, and GAL10 promoters), glycolytic enzyme promoters (including TPI and PGK promoters), TDH3 promoter, TEF1 promoter, TRP1 promoter, CYC1 promoter, CUP1 promoter, PHO5 promoter, ADH1 promoter, and HDP promoter. A suitable promoter in Pichia pastoris is the AOX1 promoter.

[0219] Suitable reporter genes or selectable markers include, but are not limited to, drug resistance genes, metabolic enzymes, factors required for the survival of the recombinant cells, fluorescent markers, or enzymes that produce a detectable product. Cells transformed with the vector can be selected based on their ability to grow in the presence of inhibitors (e.g., antibiotics) or under conditions where untransformed cells cannot grow.

[0220] The vector may be a high copy number vector, a medium copy number vector or a low copy number vector.

[0221] recombinant cells

[0222] The present invention provides recombinant cells that have been genetically engineered to express the polypeptides of the present invention. The present invention also provides recombinant cells transformed with the nucleic acids of the present invention. The nucleic acids can be extrachromosomal and present on a vector (usually a plasmid). In some embodiments, the recombinant cells are transformed with the vectors of the present invention. In some embodiments, the recombinant cells are transiently transformed. Alternatively, the recombinant cells can be stably transformed, wherein the nucleic acids are integrated into the cell genome in one or more copies. Nucleic acids can be randomly integrated into the cell genome by non-homologous recombination, but preferably, as is well known in the art, the nucleic acid constructs can be integrated into the cell genome by homologous recombination.

[0223] In some embodiments, the recombinant cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is selected from acinetobacter, Agrobacterium, Escherichia coli, Cuprum Pseudomonas, Clostridium, Rhodobacter, Marinobacter, Bacillus, Klebsiella, Tatum Pseudomonas, Lars, Rhodococcus, Methylobacterium, Methylophilus, Methylococcus, Methylmicrobacterium, Methylomonas, Pantoea, Streptomyces, Parachlorella, Synechococcus, Synechocystis and Thermosynechococcus. In some embodiments, the prokaryotic cell is Escherichia coli. Suitable cells in the genus Bacteria include but are not limited to cells of Lactobacillus, Pseudomonas and Streptomyces. Suitable cells in the bacterial species include but are not limited to cells of bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis, Pseudomonas aeruginosa and Streptomyces lividans.

[0224] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is selected from yeast cells, fungal cells, algae cells and plant cells. In some embodiments, the yeast cell is selected from Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces, Candida, Schizosaccharomyces, Schefflera, Rhodosporidium, Hansenula, Klerkera, Schwannella, Issaffron, Yarrowia and Rhodotorula. In some embodiments, the yeast cell is selected from Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Rhodotorula glutinosus, Sporobolomyces bulleri, Barnett's yeast, Microzyme, Utricularia saccharomyces, Saccharomyces diastaticus, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces fragilis, Pichia kudriavzevii, Pichia stipitis and Issaffron orientalis. Suitable yeast cells include but are not limited to Candida albicans, Schizosaccharomyces pombe, Hansenula polymorpha, Candida canadensis or Phaffia rhodozyma.

[0225] In some embodiments, the fungal cell is a filamentous fungal cell. In some embodiments, the filamentous fungal cell is selected from Aspergillus, Penicillium, Rhizopus, Chrysosporium, Myceliophthora thermophila, Trichoderma, Humicola, Cephalosporium acremonium and Fusarium. In some embodiments, the filamentous fungal cell is selected from Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, Myceliophthora thermophila and Rhizopus oryzae. Suitable cells in the filamentous fungi include but are not limited to Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus (Coprinus), Coriolus (Coriolus), Corynascus, Chaetomium (Chaetomium), Cryptococcus (Cryptococcus), Filobasidium, Gibberella (Gibberella), Hypocrea (Hypocrea), Magnaporthe oryzae (Magnaporthe), Mucor (Mucor), Neocallimastix (Neocallimastix), Neurospora (Neocallimastix), eurospora), Paecilomyces, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldium, Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia, tolypocladium, and Trametes. In certain aspects, the recombinant cell is a cell of the genus Trichoderma sp., Penicillium sp., Humicola sp., such as Humicola insolens, Aspergillus sp., Chrysosporium sp., Fusarium sp., or Hypocreas sp. Suitable cells may also include cells of various asexual and teleomorphs of these filamentous fungi.

[0226] Suitable cells of filamentous fungal species include, but are not limited to, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium neem, and Fusarium tumefaciens. negundi), Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkanderaadusta, Ceriporiopsis aneurinina, Ceriporiopsis aneurinina, Ceriporiopsis caregiea, Ceriporiopsis flavosum

[0227] (Ceriporiopsisgilvescens), Ceriporiopsis

[0228] (Ceriporiopsis pannocinta), stream Ceriporiopsis

[0229] Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitarius solitum), Penicillium funiculosum, Phanerochae techrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0230] In some embodiments, the algal cell is selected from the genera Botrytis cinerea, Nannochloropsis spp., Chlorella spp., Chlamydomonas spp., Dunaliella salina, Chaetoceros spp., Porphyridium spp., Scenedesmus spp., and Pseudochlorococcus spp. In some embodiments, the algal cell is selected from the genera Botrytis cinerea and Nannochloropsis spp.

[0231] The recombinant cells can be cultured in conventional nutrient media, which can be modified as needed to activate the promoter (if an inducible promoter is present), screen for transformants, or amplify the nucleic acid sequence encoding the polypeptide of the present invention. The culture conditions, such as temperature, pH, etc., are those previously used for the selected expressing recombinant cells and will be apparent to those skilled in the art. Preferred culture conditions for a given recombinant cell can be found in the scientific literature and / or obtained from the source of the recombinant cell, such as the American Type Culture Collection (ATCC).

[0232] BRIEF DESCRIPTION OF THE DRAWINGS

[0233] Figure 1 .pPIC9K vector map.

[0234] Figure 2 .Procedure for protein purification by histidine affinity chromatography. Example

[0235] The invention will now be described with reference to the following non-limiting examples.

[0236] To identify the peptides in caviar, the inventors performed liquid chromatography-mass spectrometry (LC-MS) analysis on caviar (fish eggs), fish egg sacs, and caviar oil. The samples analyzed were from the Siberian sturgeon (Acipenserbaerii).

[0237] Significant technical challenges were encountered throughout the LC-MS analysis, which were overcome by developing a custom sample preparation technique. The ammonium sulfate precipitation method involves preparing a biphasic solution of oil and water (equal volumes) to which solid ammonium sulfate is added to reach 90% (w / v) saturation (approximately 0.6 g / ml). This is stirred in a cold room for 1 hour. The solution is then centrifuged at 20,000 × g for 30 minutes at 4°C, the upper oil layer removed, and the lower aqueous layer carefully removed, retaining the precipitated protein. The precipitate is resuspended in a minimal volume of lysis buffer and stored on ice or at 4°C for subsequent analysis by SDS-PAGE. The urea protein separation method involves preparing a 5 M urea solution and adding it to the oil to achieve a 1:3 aqueous:oil ratio. The emulsion is stirred overnight at 4°C and then centrifuged at 20,000 × g for 30 minutes at 4°C. The upper oil layer is removed, and the lower aqueous layer (5 M urea) containing the precipitate and denatured proteins is removed. A minimum volume of lysis buffer was added and the samples were stored on ice or at 4°C for subsequent analysis by SDS-PAGE. The samples were electrophoresed on a protein gel. Once on the separating gel, the electrophoresis was stopped, the samples were stained, and the single band containing all protein material was subjected to LC-MS analysis and mass spectrometry fingerprinting. Since there is no extensive published genome data for the Siberian sturgeon (Acipenserbaerii), this analysis confirmed a high match with the protein sequence of the Russian sturgeon (Acipenserbaerii).

[0238] After identifying the types of peptides in caviar, the inventors analyzed the cellular functions associated with each detected protein and determined which proteins had desirable functions in cosmetic applications. Those key caviar peptides included: superoxide dismutase 3 (SOD3), superoxide dismutase 1 (SOD1), tissue inhibitor of metalloproteinases 1 (TIMP1), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGF-2), and laminin (the details of which are described above).

[0239] Superoxide dismutase 1 (SOD1, SEQ ID NO: 2), superoxide dismutase 3 (SOD3, SEQ ID NO: 1), and stem cell factor (SCF, SEQ ID NO: 8) were expressed in Pichia pastoris. Briefly, codon-optimized genes were subcloned into the pPIC9K vector using EcoRI and NotI restriction sites. Genes were expressed under the control of the AOX1 promoter. The expressed proteins contained a C-terminal 8x histidine tag to aid purification.

[0240] The SOD3 polypeptide of SEQ ID NO: 1 comprising a C-terminal 8x histidine tag is represented by SEQ ID NO: 19:

[0241]

[0242] The superoxide dismutase 1 (SOD1) polypeptide of sequence number 2 has an 8-histidine tag at its C-terminus, represented by sequence number 20:

[0243]

[0244] A stem cell factor (SCF) polypeptide having a sequence identifier of SEQ ID NO:8, comprising a C-terminal 8x histidine tag, represented by the sequence having a sequence identifier of SEQ ID NO:21:

[0245]

[0246] VRYVPQSEELNDICWLMLNIYELQLSLTSLAVKFAETSSNKENITVLIDMVMK

[0247] MRRPFQYEEEVIDDYHCHYQDGNFNTSVYFDYLKKMIETYELHERQFISPDC

[0248] VSPPCPTSETTTLVAGSITIATELLIMNTTACVSASDCNTQETRRDKNTEAKT

[0249] NTQGAEKLHIPLYLLLIPVFGILLVLTWKGHITSCCRTIVLKGVDVASNTWVTL

[0250] YMLLPDEAVLYKSTYSSFIWSEGQKLDSCSVELLSETRVCKTSTPEIQHHHHHHHH(SEQ ID NO:21).

[0251] Figure 1 The pPIC9K vector map is shown.

[0252] Positive clones were identified based on resistance to G418 and subsequently expressed on a small scale in Pichia pastoris. Figure 2 Summarized protocol for protein purification by histidine affinity chromatography.

[0253] To prepare an exemplary cosmetic composition, a stock solution of a protein mixture (comprising superoxide dismutase 1 (SOD1), superoxide dismutase 3 (SOD3), and stem cell factor (SCF)) was prepared in 5 mM sodium phosphate buffer at pH 6.0. The concentration of each protein was 0.04 mg / mL. This stock solution was diluted in 5 mM sodium phosphate buffer at pH 6.0 to a final concentration of 400 μg / L.

Claims

1. A composition comprising a recombinant or synthetic caviar polypeptide, wherein the caviar polypeptide is selected from: a) a superoxide dismutase (SOD3) polypeptide, wherein the SOD3 polypeptide comprises a polypeptide sequence having the following characteristics: (i) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; or (ii) comprising at least 50 consecutive amino acids of SEQ ID NO: 1; b) a superoxide dismutase (SOD1) polypeptide, wherein the SOD1 polypeptide comprises a polypeptide sequence having the following characteristics: (i) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; or (ii) comprising at least 50 consecutive amino acids of SEQ ID NO: 2; c) a tissue inhibitor of metalloproteinases 1 (TIMP1) polypeptide, wherein the TIMP1 polypeptide comprises a polypeptide sequence having the following characteristics: (i) having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 3; or (ii) comprising at least 50 consecutive amino acids of SEQ ID NO: 3; d) an acidic fibroblast growth factor (aFGF) polypeptide, wherein the aFGF polypeptide comprises a polypeptide sequence having the following characteristics: (i) having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4; or (ii) comprising at least 50 consecutive amino acids of SEQ ID NO: 4; e) a basic fibroblast growth factor (bFGF) polypeptide, wherein the bFGF polypeptide comprises a polypeptide sequence having the following characteristics: (i) having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 5; or (ii) comprising at least 50 consecutive amino acids of SEQ ID NO: 5; f) an insulin-like growth factor 2 (IGF-2) polypeptide, wherein the IGF-2 polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 6; or (ii) at least 50 consecutive amino acids of SEQ ID NO: 6; and g) a laminin polypeptide, wherein the laminin polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 7; or (ii) at least 50 consecutive amino acids of SEQ ID NO:

7.

2. The composition of claim 1, wherein the composition comprises two or more recombinant or synthetic caviar polypeptides.

3. The composition of claim 1 or claim 2, wherein the composition comprises two or more recombinant or synthetic caviar polypeptides selected from the group consisting of SOD3, SOD1 and TIMP1.

4. The composition according to any one of the preceding claims, wherein the composition comprises a recombinant or synthetic SOD3 polypeptide and a recombinant or synthetic SOD1 polypeptide.

5. The composition of any of the preceding claims, wherein the composition further comprises a recombinant or synthetic stem cell factor (SCF) polypeptide, wherein the SCF polypeptide comprises a polypeptide sequence having the following characteristics: (i) at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8; or (ii) comprising at least 50 contiguous amino acids of SEQ ID NO:

8.

6. The composition according to any one of the preceding claims, wherein the composition comprises a recombinant or synthetic SOD3 polypeptide, a recombinant or synthetic SOD1 polypeptide and a recombinant or synthetic SCF polypeptide.

7. A composition according to any one of the preceding claims, wherein one or more of the recombinant or synthetic polypeptides comprises a purification tag, such as a histidine tag.

8. The composition of any preceding claim, wherein the composition further comprises collagen.

9. The composition according to any one of the preceding claims, wherein the composition is a cosmetic composition.

10. The composition according to claim 9, wherein the cosmetic composition is selected from the group consisting of skin moisturizers, perfumes, lipsticks, nail polishes, eye and / or face makeup preparations, shampoos, hair dye preparations, toothpastes and deodorants.

11. An isolated nucleic acid encoding one or more caviar polypeptides as defined in claim 1.

12. An isolated nucleic acid encoding the SCF as defined in claim 5.

13. The isolated nucleic acid of claim 11 or claim 12, wherein the nucleic acid is codon-optimized for expression in a eukaryotic cell.

14. The isolated nucleic acid of claim 13, wherein the eukaryotic cell is a yeast cell.

15. The isolated nucleic acid of claim 14, wherein: a) a nucleic acid encoding SOD3 having at least 70% sequence identity to SEQ ID NO: 9; b) a nucleic acid encoding SOD1 having at least 70% sequence identity to SEQ ID NO: 10; c) the nucleic acid encoding TIMP1 has at least 70% sequence identity to SEQ ID NO: 11; and d) the nucleic acid encoding SCF has at least 70% sequence identity to SEQ ID NO:

12.

16. A vector comprising the isolated nucleic acid according to any one of claims 11 to 15.

17. The vector according to claim 16, further comprising: (a) Origin of replication; (b) a promoter sequence operably linked to the nucleic acid; and / or (c) Reporter gene.

18. A recombinant cell genetically engineered to express one or more recombinant or synthetic caviar polypeptides according to any one of claims 1 to 4, 6 or 7.

19. A recombinant cell transformed with the vector according to claim 16 or claim 17.

20. The recombinant cell of claim 18 or claim 19, wherein the recombinant cell is a P. pastoris cell.

21. A method for producing one or more recombinant caviar polypeptides according to any one of claims 1 to 4, 6 or 7, the method comprising: (a) cultivating the recombinant cell according to any one of claims 18 to 20 in a suitable culture medium; as well as (b) expressing the recombinant caviar polypeptide.

22. A method of producing one or more synthetic caviar polypeptides according to any one of claims 1 to 4, 6 or 7, wherein the synthetic caviar polypeptides are produced by: (a) cell-free protein synthesis; or (b) Solid-phase chemical synthesis.

23. A method for producing a cosmetic, the method comprising combining: (a) one or more recombinant or synthetic caviar polypeptides according to any one of claims 1 to 4, 6 or 7, optionally in combination with a recombinant or synthetic SCF according to claim 5 or 7; and (b) Cosmetic ingredients.

24. Use of a composition according to any one of claims 1 to 10 for improving the appearance of skin and / or hair.

25. A method of improving the appearance of skin, the method comprising applying a composition according to any one of claims 1 to 10 to the skin of a subject.

26. A method of improving the appearance of a subject's hair, the method comprising applying a composition according to any one of claims 1 to 10 to the subject's hair.

27. A cosmetic method comprising applying a composition according to any one of claims 1 to 10 to the skin and / or hair of a subject.

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