Animal-free dietary collagen

Abstract

Provided herein are non-naturally occurring polypeptides comprising the sequence of a collagen fragment, and recombinant cells comprising heterologous nucleic acid sequences encoding the non-naturally occurring polypeptides. Also provided herein are animal-free methods for producing and purifying such non-naturally occurring polypeptides using microorganisms, preferably from bacterial cells. Collagen is one of the most abundant proteins found in various connective tissues in the body, including tendons, ligaments, skin, and hair.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application Nos. 62 / 965,700, filed January 24, 2020, and 63 / 117,243, filed November 23, 2020, which applications are incorporated herein by reference in their entireties. [Background technology]

[0002] background Collagen is one of the most abundant proteins found in various connective tissues in the body, including tendons, ligaments, skin, and hair. Collagen or collagen supplements are popular for medical, cosmetic, and / or health purposes (e.g., stimulating skin growth, promoting wound healing, strengthening nails or joints, etc.). The collagen for most collagen supplements is derived from animals as a by-product of the animal processing industry. However, such animal-derived collagen may increase the risk of disease transmission and allergies. Furthermore, certain consumers are widely interested in animal-free products for various other reasons. Therefore, there remains a need for improved compositions and methods for collagen derived from non-animal sources. Summary of the Invention [Means for solving the problem]

[0003] Abstract In one embodiment, a non-naturally occurring polypeptide is provided that comprises an amino acid sequence that has (i) at least 80% sequence identity to SEQ ID NO: 31, together with the N-terminal truncation, the C-terminal truncation, or both; or (ii) at least 80% sequence identity to SEQ ID NO: 32, together with the N-terminal truncation, the C-terminal truncation, or both. In some cases, the non-naturally occurring polypeptide comprises an amino acid sequence that has (i) at least 85% sequence identity to SEQ ID NO: 31, together with the N-terminal truncation, the C-terminal truncation, or both; or (ii) at least 85% sequence identity to SEQ ID NO: 32, together with the N-terminal truncation, the C-terminal truncation, or both. In some cases, the non-naturally occurring polypeptide comprises (i) at least 90% sequence identity to SEQ ID NO: 31, together with the N-terminal truncation, the C-terminal truncation, or both; or (ii) at least 90% sequence identity to SEQ ID NO: 32, together with the N-terminal truncation, the C-terminal truncation, or both. In some cases, the non-naturally occurring polypeptide comprises an amino acid sequence having (i) at least 95% sequence identity to SEQ ID NO: 31, together with the N-terminal truncation, the C-terminal truncation, or both; or (ii) at least 95% sequence identity to SEQ ID NO: 32, together with the N-terminal truncation, the C-terminal truncation, or both. In some cases, the non-naturally occurring polypeptide comprises (i) at least 98% sequence identity to SEQ ID NO: 31, together with the N-terminal truncation, the C-terminal truncation, or both; or (ii) at least 98% sequence identity to SEQ ID NO: 32, together with the N-terminal truncation, the C-terminal truncation, or both. In some cases, the non-naturally occurring polypeptide comprises (i) the amino acid sequence of SEQ ID NO: 31, together with the N-terminal truncation, the C-terminal truncation, or both; or (ii) the amino acid sequence of SEQ ID NO: 32, together with the N-terminal truncation, the C-terminal truncation, or both. In some cases, the non-naturally occurring polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31, together with the N-terminal truncation.In some cases, the N-terminal truncation is an N-terminal truncation of between 50 and 600 amino acids. In some cases, the non-naturally occurring polypeptide, together with the C-terminal truncation, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31. In some cases, the C-terminal truncation is a C-terminal truncation of between 50 and 250 amino acids. In some cases, the non-naturally occurring polypeptide, together with both the N-terminal truncation and the C-terminal truncation, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31. In some cases, the N-terminal truncation is an N-terminal truncation of between 50 and 600 amino acids, and the C-terminal truncation is a C-terminal truncation of between 50 and 250 amino acids. In some cases, the non-naturally occurring polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In some cases, the non-naturally occurring polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In some cases, the non-naturally occurring polypeptide, together with the N-terminal truncation, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32. In some cases, the N-terminal truncation is an N-terminal truncation of between 50 and 750 amino acids. In some cases, the non-naturally occurring polypeptide, together with the C-terminal truncation, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32. In some cases, the C-terminal truncation is a C-terminal truncation of between 50 and 250 amino acids. In some cases, the non-naturally occurring polypeptide, together with both the N-terminal truncation and the C-terminal truncation, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32. In some cases, the N-terminal truncation is an N-terminal truncation of between 50 and 750 amino acids, and the C-terminal truncation is a C-terminal truncation of between 50 and 250 amino acids. In some cases, the non-naturally occurring polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In some cases, the non-naturally occurring polypeptide consists of the amino acid sequence of SEQ ID NO: 8.In some cases, the non-naturally occurring polypeptide has a total truncation of 50 amino acids to 900 amino acids. In some cases, the non-naturally occurring polypeptide is 50 amino acids to 250 amino acids in length. In some cases, the non-naturally occurring polypeptide does not include one or more of a laminin G domain, a von Willebrand factor type A (vWA) domain, and a fibrillar collagen C-terminal domain. In some cases, the non-naturally occurring polypeptide includes one or more collagen triple helix repeats. In some cases, the non-naturally occurring polypeptide is a monomer. In some cases, the non-naturally occurring polypeptide does not form a stable triple helix structure of naturally occurring collagen. In some cases, the non-naturally occurring polypeptide is substantially free of other collagen chains. In some cases, the non-naturally occurring polypeptide has a non-naturally occurring level of hydroxylation compared to naturally occurring collagen. In some cases, less than 10% of the prolines present in the non-naturally occurring polypeptide are hydroxylated. In some cases, the non-naturally occurring polypeptide is not hydroxylated. In some cases, the non-naturally occurring polypeptide has a non-naturally occurring level of glycosylation compared to naturally occurring collagen, hi some cases, the non-naturally occurring polypeptide protein contains less than 5 wt.% glycosylation.

[0004] In another aspect, a composition is provided comprising 0.001% to 30% w / w of any one of the foregoing non-naturally occurring polypeptides. In some cases, the composition is formulated for consumption by an individual. In some cases, the composition is a dietary supplement. In some cases, the individual is a human.

[0005] In another aspect, a method is provided for improving the appearance of a subject's skin, hair, and / or nails, and / or improving the health of a subject's bones, muscles, and / or joints, comprising administering to the subject any one of the foregoing compositions. In some cases, the administering comprises orally administering to the subject.

[0006] In yet another aspect, a recombinant cell is provided that includes at least one copy of a heterologous nucleic acid sequence encoding any one of the foregoing non-naturally occurring polypeptides. In some cases, the recombinant cell is a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is a cell of the species Escherichia coli. In some cases, the recombinant cell lacks an enzyme that hydroxylates one or more amino acids of the non-naturally occurring polypeptide. In some cases, the recombinant cell lacks prolyl 4-hydroxylase and / or prolyl 3-hydroxylase. In some cases, the heterologous nucleic acid sequence includes a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In some cases, the heterologous nucleic acid sequence includes a nucleic acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In some cases, the heterologous nucleic acid sequence comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In some cases, the heterologous nucleic acid sequence comprises a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In some cases, the heterologous nucleic acid sequence comprises a nucleic acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30. In some cases, the non-naturally occurring polypeptide further comprises a secretion signal. In some cases, the recombinant cell secretes the non-naturally occurring polypeptide into the periplasm, culture medium, or extracellularly. In some cases, the heterologous nucleic acid sequence is codon-optimized for expression in the recombinant cell. In some cases, the heterologous nucleic acid sequence is operably linked to an inducible promoter or a constitutive promoter. In some cases, the heterologous nucleic acid is a plasmid or is contained in a plasmid.In some cases, the heterologous nucleic acid sequence is stably integrated into a chromosome of the recombinant cell.

[0007] In yet another aspect, a culture medium comprising any one of the foregoing recombinant cells is provided. In some cases, the culture medium further comprises any one of the foregoing non-naturally occurring polypeptides secreted from the recombinant cell.

[0008] The present disclosure further provides a recombinant cell comprising at least one copy of a heterologous nucleic acid sequence encoding a collagen selected from the group consisting of Gallus gallus collagen or Acipenser schrenckii (Amur sturgeon) collagen. In some embodiments, the recombinant cell is a microbial cell. In some embodiments, the microbial cell is a bacterial cell. In some embodiments, the bacterial cell is a cell of the species Escherichia coli. In some embodiments, the heterologous nucleic acid sequence comprises any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 25-30.

[0009] In some embodiments, the collagen is Gallus gallus type 21 collagen. In some embodiments, the collagen is Acipenser schrenckii type 2 alpha 1 collagen. In some embodiments, the collagen is a non-naturally occurring collagen. In some embodiments, the collagen is a truncated collagen. In some embodiments, the collagen comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8.

[0010] In some embodiments, the collagen further comprises a secretory signal sequence. In some cases, the secretory signal sequence comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments, the recombinant cells secrete the collagen into the culture medium. In some embodiments, the recombinant cells secrete the collagen into the periplasm. In some embodiments, the recombinant cells secrete the collagen into the extracellular space.

[0011] In some embodiments, the heterologous nucleic acid sequence is codon-optimized for expression in the recombinant cell. In some embodiments, the heterologous nucleic acid sequence is operably linked to an inducible promoter or a constitutive promoter. In some embodiments, the heterologous nucleic acid is a plasmid or is contained within a plasmid. In some embodiments, the heterologous nucleic acid sequence is stably integrated into the chromosome of the recombinant cell.

[0012] The present disclosure also provides a culture medium comprising the recombinant cells described herein. In some embodiments, the culture medium further comprises recombinant collagen secreted from the recombinant cells.

[0013] The present disclosure also provides a recombinant protein comprising a sequence having at least 90% sequence identity to a fragment of a collagen selected from the group consisting of Gallus gallus collagen and Acipenser schrenckii collagen. In some embodiments, the collagen is Gallus gallus type 21 collagen. In some embodiments, the collagen is Acipenser schrenckii type 2 alpha 1 collagen.

[0014] In some embodiments, the collagen is a non-naturally occurring collagen or a fragment thereof. In some embodiments, the protein has a non-naturally occurring level of glycosylation (e.g., compared to the corresponding native collagen). In some embodiments, the protein contains less than 5 wt.% glycosylation (e.g., less than 3 wt.%, less than 1 wt.%, less than 0.5 wt.%, or less than 0.1 wt.%). In some embodiments, the protein is a truncated collagen. In some embodiments, the protein contains the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8 (or an amino acid sequence having at least 90%, at least 95%, at least 98%, etc., sequence identity thereto).

[0015] In some embodiments, the collagen further comprises a secretory signal sequence. In some embodiments, the secretory signal sequence comprises the amino acid sequence set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, and 24.

[0016] The present disclosure also provides compositions comprising a recombinant protein as disclosed herein. In some embodiments, the composition further comprises a culture medium. Additionally and / or alternatively, the composition further comprises a recombinant cell as disclosed herein. In some embodiments, the recombinant cell is a microbial cell. In some embodiments, the microbial cell is a bacterial cell. In some embodiments, the bacterial cell is a cell of the species Escherichia coli. In some embodiments, the recombinant cell comprises an integrated heterologous nucleic acid sequence encoding a collagen, truncated collagen, or a fragment thereof. In some embodiments, the heterologous nucleic acid sequence comprises any one of SEQ ID NOs: 1, 3, 5, 7, and 25-30.

[0017] The present disclosure also provides a process for purifying recombinant collagen, the process comprising incubating recombinant cells described herein in a culture medium, wherein the recombinant cells secrete recombinant collagen into the culture medium, recovering the culture medium containing the secreted recombinant collagen, and purifying the recombinant collagen from the culture medium.

[0018] The present disclosure also provides recombinant collagen purified from the culture medium of the processes disclosed herein, in some embodiments, the recombinant collagen has a purity of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0019] The present disclosure also provides an expression vector comprising a nucleic acid sequence encoding a non-naturally occurring truncated collagen operably linked to a promoter, wherein the non-naturally occurring truncated collagen is selected from the group consisting of Gallus gallus collagen and Acipenser schrenckii collagen. In some embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs: 1, 3, 5, 7, and 25-30. In some embodiments, the Gallus gallus collagen is type 21 collagen. In some embodiments, the Acipenser schrenckii collagen is type 2 alpha 1 collagen.

[0020] In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a secretory signal sequence. In some embodiments, the nucleic acid sequence encoding the secretory signal sequence comprises any one of SEQ ID NOs: 11, 13, 15, 17, 19, 21, and 23. In some embodiments, the nucleic acid sequence is codon-optimized for expression in a cell.

[0021] The present disclosure also provides compositions comprising the recombinant collagen disclosed herein formulated for consumption by an individual. In some embodiments, the composition is a dietary supplement. In some embodiments, the individual is a human. In some embodiments, the composition comprises 0.1% to 10% recombinant collagen. In some embodiments, the composition comprises at least 50% recombinant collagen. In some embodiments, the composition comprises 70% to 99% recombinant collagen. In some embodiments, the composition further comprises at least one of a carrier and a preservative.

[0022] The present disclosure also provides methods of improving the appearance of a subject's skin, hair, and / or nails by administering to the subject a composition disclosed herein. In some embodiments, the administering step comprises orally administering to the subject.

[0023] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein merely illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0024] The novel features of the subject matter disclosed herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the subject matter disclosed herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, which utilize the principles of the subject matter disclosed herein, and the accompanying drawings in which: [Brief explanation of the drawings]

[0025] [Figure 1]Figure 1 shows two SDS-PAGE gel images showing collagen protein bands in a supernatant sample from a microbial cell culture. The identity of each protein is indicated above each band.

[0026] [Figure 2] 2A-2C show images of SDS-PAGE gels showing bands for non-naturally occurring polypeptides of the present disclosure before and after pH 3.0 treatment.

[0027] [Figure 3] FIG. 3 shows increased cell viability of human dermal fibroblasts when treated with a non-naturally occurring polypeptide of the present disclosure (comprising the amino acid sequence set forth in SEQ ID NO:2).

[0028] [Figure 4] FIG. 4 shows increased type I collagen production in human dermal fibroblasts when treated with a non-naturally occurring polypeptide of the present disclosure (comprising the amino acid sequence set forth in SEQ ID NO:2).

[0029] [Figure 5] FIG. 5 shows increased type I collagen production in tenocytes when treated with a non-naturally occurring polypeptide of the present disclosure (comprising the amino acid sequence set forth in SEQ ID NO:2).

[0030] [Figure 6] Figure 6 shows an alignment of the non-naturally occurring polypeptides of the present disclosure with the corresponding naturally occurring collagens. Figure 6 discloses SEQ ID NOs: 33 and 34, respectively, in order of appearance.

[0031] [Figure 7A] FIG. 7A shows the effect of pH on the viscosity of an exemplary non-naturally occurring polypeptide solution of the present disclosure.

[0032] [Figure 7B]FIG. 7B shows a comparison of the viscosity of solutions of exemplary non-naturally occurring polypeptides of the present disclosure against a benchmark.

[0033] [Figure 8] FIG. 8 shows the viscosity of various blends of exemplary non-naturally occurring polypeptides of the present disclosure with xanthan.

[0034] [Figure 9] FIG. 9 shows the gel hardness of exemplary non-naturally occurring polypeptide solutions of the present disclosure.

[0035] [Figure 10A] 10A and 10B show the gel hardness of various lots of exemplary non-naturally occurring polypeptide solutions of the present disclosure. [Figure 10B] 10A and 10B show the gel hardness of various lots of exemplary non-naturally occurring polypeptide solutions of the present disclosure.

[0036] [Figure 11] FIG. 11 shows the effect of compression and lecithin aggregation on an exemplary non-naturally occurring polypeptide of the disclosure.

[0037] [Figure 12] FIG. 12 shows the effect of pH and oil type on gel hardness of gels containing exemplary non-naturally occurring polypeptides of the disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description definition The terms used herein are for descriptive purposes only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0039] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to convention for a given value. When a particular value is described in this application and claims, unless otherwise stated, it should be assumed that the term "about" means an acceptable error range for that particular value.

[0040] The terms "individual," "patient," or "subject" are used interchangeably herein. None of these terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent supervision) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, hospital worker, or hospice worker).

[0041] As used herein, the term "comprise" or variations thereof, such as "comprises" or "comprising," should be interpreted as indicating the inclusion of any recited feature, and not the exclusion of any other feature. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited features. In some embodiments of any of the compositions and methods provided herein, "comprising" can be replaced with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" is used herein to require certain features, as well as features that do not materially affect the nature or function of the claimed disclosure. As used herein, the term "consisting of" is used to indicate the presence of only the recited features.

[0042] Throughout this disclosure, various embodiments are presented in 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 inflexible limitation on the scope of any embodiment. Thus, unless the context clearly dictates otherwise, the description of a range should be deemed to have expressly disclosed all possible subranges, as well as any individual numerical values ​​within that range, to the tenth of the unit of the lower limit. For example, the description of a range such as 1 to 6 should be deemed to have expressly disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as any individual values ​​within that range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are encompassed within the disclosure, subject to any explicitly excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0043] The terms "treatment of," "treat," "apply," "alleviate," or "ameliorate" are used interchangeably herein. These terms refer to an approach to obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. A "therapeutic benefit" refers to the eradication or amelioration of the underlying disorder being treated. A therapeutic benefit is also achieved by the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient despite the patient still suffering from the underlying disorder. Regarding prophylactic benefit, in some embodiments, the composition is administered to a patient at risk of developing a particular disease or condition, or to a patient who reports one or more physiological symptoms of a disease even if the patient has not been diagnosed with the disease.

[0044] The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" can be a biological entity that contains expressed genetic material. The biological entity can be a plant, animal, or microorganism (including, for example, bacteria, viruses, fungi, and protozoa). A subject can be tissues, cells, and progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. A mammal can be a human. A subject can be diagnosed with a disease or suspected of being at high risk for a disease. In some cases, a subject is not necessarily diagnosed with or suspected of being at high risk for the disease.

[0045] As used herein, the term "truncated collagen" generally refers to a polypeptide smaller than full-length (e.g., native) collagen, in which one or more portions of full-length (e.g., native) collagen are not present. The non-naturally occurring polypeptides provided herein may be truncated at the C-terminus, the N-terminus, truncated by removal of an internal portion of the full-length collagen sequence (e.g., internal truncation), truncated at both the C-terminus and the N-terminus, or have one or both of a C-terminal truncation and an N-terminal truncation as well as an internal truncation. In a non-limiting embodiment, the truncated collagen may comprise the amino acid sequence set forth in SEQ ID NO: 2, or a homolog thereof. In another non-limiting embodiment, the truncated collagen may comprise the amino acid sequence set forth in SEQ ID NO: 8, or a homolog thereof.

[0046] "Truncation," when used in reference to an amino acid position, includes that amino acid position. For example, an N-terminal truncation at amino acid 100 of a full-length protein refers to a truncation of 100 amino acids from the N-terminus of the full-length protein (i.e., the truncated protein is missing amino acids 1 through 100 of the full-length protein). Similarly, a C-terminal truncation at amino acid 901 of a full-length protein (assuming a 1000 amino acid full-length protein) refers to a truncation of 100 amino acids from the C-terminus (i.e., the truncated protein is missing amino acids 901 through 1000 of the full-length protein). Similarly, internal truncations at amino acids 101 and 200 refer to 100 amino acid internal truncations of the full-length protein (i.e., the truncated protein is missing amino acids 101 through 200 of the full-length protein).

[0047] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0048] Compositions, methods, and systems for producing non-naturally occurring polypeptides (e.g., animal-free collagen or collagen-like polypeptides, and collagen fragments and / or truncated collagens (e.g., those expressed in and / or by genetically engineered microorganisms)) are provided herein, by way of non-limiting example, in certain embodiments. Accordingly, in various aspects of the present disclosure, the non-naturally occurring polypeptides provided herein include collagen or collagen-like polypeptides, recombinant collagens, collagen fragments, or truncated collagens. In certain embodiments, the non-naturally occurring polypeptides described herein (e.g., recombinant collagens, collagen fragments, or truncated collagens) are derived from any suitable source, e.g., mammalian or non-mammalian. For example, in some embodiments, the non-naturally occurring polypeptides described herein (e.g., recombinant collagens, collagen fragments, or truncated collagens), or at least a portion thereof, are derived from (e.g., modified, truncated, fragments, etc.) avian or avian collagen (e.g., Gallus gallus collagen), freshwater or marine fish collagen (e.g., Acipenser schrenckii collagen), or any combination thereof.

[0049] The non-naturally occurring polypeptides provided herein are not normally found in nature. Generally, the non-naturally occurring polypeptides described herein exhibit one or more differences from naturally occurring collagen. In certain embodiments, the non-naturally occurring polypeptides provided herein may have a different amino acid sequence from naturally occurring polypeptides (e.g., truncated collagen). In some cases, the non-naturally occurring polypeptides may have a different structure from naturally occurring collagen. The quaternary structure of natural collagen is usually a triple helix composed of three polypeptides. In some embodiments, the non-naturally occurring polypeptides described herein may not have the quaternary structure of natural collagen or may not form the quaternary structure of natural collagen. For example, in some cases, the non-naturally occurring polypeptides described herein may not form the stable triple helix structure of naturally occurring collagen. In certain cases, of the three polypeptides that form natural collagen, two are usually identical and are called alpha chains. The third polypeptide is called beta chain. In certain instances, normal, naturally occurring collagen may be designated AAB, which is composed of two alpha ("A") chains and one beta ("B") chain. In some embodiments, the non-naturally occurring polypeptides described herein do not have the AAB structure of naturally occurring collagen. In some cases, the non-naturally occurring polypeptides described herein are free of, or substantially free of, different collagen chains (e.g., the non-naturally occurring polypeptides described herein may comprise alpha chain collagen and free of, or substantially free of, beta chain collagen). In some embodiments, the non-naturally occurring polypeptides described herein are monomeric (e.g., do not form multimeric structures). In other embodiments, the non-naturally occurring polypeptides described herein may, in some cases, form multimeric structures (e.g., homodimers, homotrimers, etc.) of identical monomers.

[0050] In some embodiments, the non-naturally occurring polypeptide is a recombinant polypeptide (e.g., recombinantly prepared in a host cell). In one embodiment, the non-naturally occurring collagen is a truncated collagen. Other non-naturally occurring collagen polypeptides include chimeric collagens. Chimeric collagens are polypeptides in which a portion of a collagen polypeptide is contiguous with a portion of a second collagen polypeptide. For example, a collagen molecule in which a portion of a collagen from one species is contiguous with a portion of a collagen from another species is a chimeric collagen. In another embodiment, the non-naturally occurring collagen includes a fusion polypeptide containing additional amino acids such as a secretion tag, a histidine tag, a green fluorescent protein, a protease cleavage site, a GEK repeat, a GDK repeat, and / or a beta-lactamase.

[0051] In some embodiments, the non-naturally occurring polypeptides (e.g., recombinant polypeptides) provided herein have a non-naturally occurring level of glycosylation, e.g., compared to the corresponding native or naturally occurring collagen. For example, in some embodiments, the non-naturally occurring polypeptides (e.g., recombinant polypeptides) contain less than 10 wt.%, less than 9 wt.%, less than 8 wt.%, less than 7 wt.%, less than 6 wt.%, less than 5 wt.%, less than 4 wt.%, less than 3 wt.%, less than 2 wt.%, less than 1 wt.%, less than 0.9 wt.%, less than 0.8 wt.%, less than 0.7 wt.%, less than 0.6 wt.%, less than 0.5 wt.%, less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, or less than 0.1 wt.% glycosylation. Alternatively and / or additionally, a non-naturally occurring polypeptide (e.g., a recombinant polypeptide) comprises less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total glycosylation of the corresponding native or naturally occurring collagen. For example, if a naturally occurring collagen ABC from species XYZ has 20 glycosylations (over the entire length of the collagen ABC or portion thereof), it is contemplated that a non-naturally occurring polypeptide (e.g., a recombinant polypeptide) comprises less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glycosylation. In some embodiments, these lower levels of glycosylation may be specific to one or more types of glycosylation (e.g., O-glycosylation or N-glycosylation, etc.) and / or glycosylated residues (e.g., galactosylhydroxylysine (Gal-Hyl), glucosylgalactosylhydroxylsine (GlcGal-Hyl), etc.).In some cases, non-naturally occurring polypeptides that are recombinantly produced (e.g., in a recombinant host cell) may have a glycosylation level and / or glycosylation pattern that differs from naturally occurring collagen.

[0052] In some embodiments, the non-naturally occurring polypeptides provided herein have a non-naturally occurring amount of hydroxyproline. In some cases, the non-naturally occurring polypeptides provided herein lack hydroxyproline. In some cases, the non-naturally occurring polypeptides provided herein contain less hydroxyproline than naturally occurring collagen. Hydroxyproline includes, but is not limited to, 3-hydroxyproline, 4-hydroxyproline, and 5-hydroxyproline. In some cases, less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10% or less) of the prolines present in the amino acid sequence of the non-naturally occurring polypeptides provided herein are hydroxyproline. In some embodiments, the non-naturally occurring polypeptides produced recombinantly (e.g., in recombinant host cells) may have less hydroxyproline than naturally occurring collagen. In some cases, a recombinant polypeptide as provided herein is recombinantly expressed in a recombinant host cell (e.g., a bacterial cell) that lacks an enzyme that hydroxylates one or more amino acids (e.g., proline) of the recombinant polypeptide. In some cases, a recombinant polypeptide as provided herein is recombinantly expressed in a host cell (e.g., a bacterial cell) that lacks prolyl 4-hydroxylase and / or prolyl 3-hydroxylase.

[0053] In some embodiments, the non-naturally occurring polypeptides provided herein lack or substantially lack lysyl oxidation. Lysyl oxidation involves the conversion of lysine residues to highly reactive aldehydes that can form crosslinks with other proteins. Naturally occurring collagen may have some level of lysyl oxidation. Thus, non-naturally occurring polypeptides may differ from natural collagen in that they lack or substantially lack lysyl oxidation.

[0054] Generally, the non-naturally occurring polypeptides provided herein (e.g., truncated collagens) may have functions and / or provide advantages similar or substantially similar to those of native or full-length collagens (e.g., as provided herein). In some cases, the non-naturally occurring polypeptides provided herein (e.g., truncated collagens) may have improved or enhanced functions and / or advantages (e.g., as provided herein) compared to native or full-length collagens.

[0055] The non-naturally occurring polypeptides disclosed herein often have beneficial properties related to their monomeric structure and / or the absence of amino acids capable of cross-linking with other collagen chains, such as the absence of hydroxyproline residues.In addition, collagen hydrolysis products of the non-naturally occurring polypeptides disclosed herein are also produced, which have higher solubility than full-length collagen or natural collagen.In addition, the monomeric structure, in contrast to natural triple-helical collagen, is more easily digestible and bioavailable or degraded by digestive proteases.Because full-length or natural collagen or collagen chains interact to form a stronger structure that can precipitate due to the presence of hydroxyproline residues, other beneficial properties include improved physical properties in liquid compositions and purification processes.

[0056] In certain preferred embodiments, the non-naturally occurring polypeptides (e.g., truncated collagens) provided herein comprise an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least a portion of the naturally occurring mammalian or non-mammalian collagen from which they are derived. In some cases, a portion of the naturally occurring amino acid sequence is deleted, while the remainder of the sequence is substantially similar or identical to the naturally occurring amino acid sequence. In certain exemplary embodiments, the non-naturally occurring polypeptide has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to Gallus gallus type 21 alpha 1 collagen or a fragment thereof. In another example, the non-naturally occurring polypeptide has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to Acipenser schrenckii type 2 alpha 1 collagen fragment.

[0057] In some embodiments, the recombinant protein is a truncated collagen. In certain cases, a truncated collagen is a polypeptide smaller than full-length (e.g., native) collagen, in which one or more portions (e.g., internal and / or terminal portions) of full-length (e.g., native) collagen are absent. In various cases, the non-naturally occurring polypeptides (e.g., truncated collagens) provided herein are truncated at the C-terminus, the N-terminus, truncated by removal of an internal portion of the full-length collagen polypeptide (e.g., internal truncation), truncated at both the C-terminus and the N-terminus, or contain one or both of a C-terminal truncation and an N-terminal truncation as well as an internal truncation. In some cases, the non-naturally occurring polypeptide is a fragment of a naturally occurring collagen that retains at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function (e.g., the function of interest) of native collagen or the corresponding naturally occurring collagen. In some cases, the term truncated collagen is used interchangeably with the term collagen fragment. In some cases, truncated collagen includes any contiguous collagen fragment that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the full-length native collagen or the corresponding naturally occurring collagen. In some embodiments, the cleavage is an internal cleavage, a cleavage in the N-terminal portion of the collagen, a cleavage in the C-terminal portion of the collagen, a cleavage in an internal portion, or a cleavage in both the C- and N-termini.The truncated collagen provided herein may be truncated by 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 750 amino acids, 50 to 700 amino acids, 50 to 650 amino acids, 50 to 600 amino acids, 50 to 550 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 truncated collagen 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, 360 , 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 amino acids.

[0058] The non-naturally occurring polypeptides (e.g., truncated collagens) disclosed herein may comprise truncations relative to full-length (e.g., native) collagen. In some embodiments, the truncated collagens disclosed herein may comprise truncations relative to full-length (e.g., native) chicken (Gallus gallus) type 21 alpha 1 collagen (e.g., SEQ ID NO: 31). In some embodiments, the truncated collagens disclosed herein may comprise the amino acid sequence of SEQ ID NO: 31, including an N-terminal truncation, a C-terminal truncation, an internal truncation, or a combination thereof. In some embodiments, the truncated collagens disclosed herein may comprise truncations relative to full-length (e.g., native) Amur sturgeon (Acipenser schrenckii) type 2 alpha 1 collagen (e.g., SEQ ID NO: 32). In some embodiments, the truncated collagens disclosed herein may comprise the amino acid sequence of SEQ ID NO: 32, including an N-terminal truncation, a C-terminal truncation, an internal truncation, or a combination thereof. Non-limiting examples of full-length (e.g., native) collagens are provided in Table 1 below.

[0059] In other embodiments, the polypeptides may be truncated collagen polypeptides comparable to fish collagen, including those from other species of sturgeon, or other species that produce fish eggs suitable for caviar (including salmon, steelhead, trout, lumpfish, whitefish, or carp), as well as other fish such as tilapia and sharks. Suitable comparable sequences from Acipenser schrenckii (Amur sturgeon) include NCBI accession numbers BAO58965.1, BAO58966.1, BAO58967.1, BAT51012.1, BAR72360.1, BAR72359.1, BAR72358.1, BAR72357.1, and BAR72356.1.Suitable sequences from Acipenser ruthenus (sterlet) include those listed under NCBI accession numbers A0A444UGW0, A0A444TZM6, A0A444UC45, A0A444UC53, A0A662YTX1, A0A662Z270, A0A662YZ39, A0A444U1F5, A0A444UJK3, A0A444UNU0, X5HZZ7, X5IHC1, A0A444UPK8, A0A444UBS1, A0A444UYQ7, A0A444TWQ3, A0A444ULY4, and A0A444TZ23. , A0A662YS48, A0A444U4C8, A0A444UD64, A0A662YX10, A0A662YXI2, A0A444TXQ4, A0A444TZ42, A0A444U8N8, A0A444UJU3, A0A444UQ51, A0A4 44U2T2, A0A662YJ50, A0A444V1V9, A0A444V113, A0A662YWR6, A0A662YW91, A0A444U5J5, A0A662YR93, A0A444UJB0, A0A444UFS4, A0A444UVK2 , A0A444UJU1, A0A444ULY9, A0A444UKA7, A0A444U5L7, A0A444V6M4, A0A444V788, A0A444UFS9, A0A444UVP7, A0A444U4D9, A0A444UHN6, A0A6 62YJC1, A0A444V1E8, A0A444UPM0, A0A662YU87, A0A444TZS8, A0A444U200, A0A444V2E3, A0A662YXD3, A0A662YQA4, A0A444U1H9, A0A444V7I5 , A0A444UFX8, A0A444V7B8, A0A444U2K4, A0A444V762, A0A444UQ49, A0A662YMD3, A0A662YWF2, A0A444UE44, A0A444UAR6, A0A444UX46, A0A444U5P4, A0A662YRG8, A0A444USC3, A0A444UK09, A0A444UNQ7, A0A444UN69, A0A444V5D9, E6Y298, A0A444TZY1, A0A444TYS0 and E6Y299.

[0060] In other embodiments, the polypeptide can be a truncated collagen polypeptide comparable to chicken collagen or other poultry collagen (e.g., from poultry, including chicken, turkey, goose, and duck). Suitable comparable sequences from Gallus gallus (chicken) include NCBI accession numbers V9GZR2, Q9PSS5, A0A3Q2UDI3, Q90802, A0A1D5PNH7, Q4TZW6, Q90803, Q91014, A0A1D5PPI0, A0A1D5P1A5, A0A3Q2U6K2, A0A3Q2U8F9, Q90689, A0A3Q2U3U6, P13731, A0A1D5 PFE0, A0A3Q2TXZ7, Q5FY72, A0A1D5PR16, A0A1D5PKR6, F1NDF5, Q90589, P08125, F1NRH2, P32017, A0A1D5PW49 , Q90800, P12108, E1C353, Q7LZR2, P02460, A0A1L1RNI7, Q90796, P12106, F1NQ20, Q9I9K3, P20785, A0A1D5PWN 6, P15988, P12105, F1NIL4, O93419, P02467, A0A5H1ZRJ7, A0A1D5PKQ4, A0A5H1ZRK9, Q90W37, A0A1D5NY11, A0 A1D5P959, P02457, A0A1D5PYU1, A0A1D5PE57, Q90ZA0, Q90584, A0A1L1RZW7, A0A1D5NVM0, A0A1D5P8P3, F1NIP0 , F1P2Q3, A0A1D5PE74, Q9IAU4, A0A3Q2TTC1, F1NHH4, P32018, A0A1D5P0F4, R4GHP9, A0A3Q2UD12, A0A3Q2UMJ2, A0A3Q2U4U7, F1NX22, A0A1D5P8I8, A0A1L1RPW4, P13944, P15989, F1P2F0, A0A1D5PGD5 and A0A3Q3AR07. [Table 1]

[0061] In some cases, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein may comprise the amino acid sequence of SEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including N-terminal truncations at any of amino acid positions (e.g., relative to SEQ ID NO: 31) between amino acids 1-537; amino acids 1-542; amino acids 1-547; amino acids 1-552; amino acids 1-557; amino acids 1-562; amino acids 1-567; amino acids 1-572; or amino acids 1-577. In some cases, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein can comprise the amino acid sequence of SEQ ID NO:31 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including a C-terminal truncation at any of the following amino acid positions (relative to SEQ ID NO:31): amino acids 726-957; amino acids 731-957; amino acids 736-957; amino acids 741-957; amino acids 746-957; amino acids 751-957; amino acids 756-957; amino acids 761-957; amino acids 766-957; amino acids 769-957; amino acids 774-957; amino acids 779-957; or amino acids 784-957. In some cases, a non-naturally occurring polypeptide as described herein (eg, a truncated collagen) can include both an N-terminal truncated portion and a C-terminal truncated portion.For example, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein can include an N-terminal truncation at any amino acid position (e.g., relative to SEQ ID NO: 31) between amino acids 1-537; amino acids 1-542; amino acids 1-547; amino acids 1-552; amino acids 1-557; amino acids 1-562; amino acids 1-567; amino acids 1-572; or amino acids 1-577; and amino acids 726-957; amino acids 731-957; amino acids 736-957; amino acids 741-957; The amino acid sequence may comprise the amino acid sequence of SEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including a C-terminal truncation at any of the following amino acid positions (based on SEQ ID NO: 31): amino acids 751 to 957; amino acids 756 to 957; amino acids 761 to 957; amino acids 766 to 957; amino acids 769 to 957; amino acids 774 to 957; amino acids 779 to 957; or amino acids 784 to 957. In a specific embodiment, a non-naturally occurring polypeptide (e.g., a truncated collagen) disclosed herein may comprise the amino acid sequence of SEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including an N-terminal truncation at amino acid position 557 (relative to SEQ ID NO: 31); and a C-terminal truncation at amino acid position 746 (relative to SEQ ID NO: 31). In another specific embodiment, a non-naturally occurring polypeptide (e.g., a truncated collagen) disclosed herein may comprise the amino acid sequence of SEQ ID NO: 31 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including an N-terminal truncation at amino acid position 557 (relative to SEQ ID NO: 31); and a C-terminal truncation at amino acid position 769 (relative to SEQ ID NO: 31).

[0062] In some cases, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein may comprise the amino acid sequence of SEQ ID NO: 32 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including an N-terminal truncation at any amino acid position (e.g., relative to SEQ ID NO: 32) between amino acids 1-660; amino acids 1-665; amino acids 1-670; amino acids 1-675; amino acids 1-680; amino acids 1-685; amino acids 1-690; amino acids 1-695; or amino acids 1-700. In some cases, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein can include the amino acid sequence of SEQ ID NO: 32 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including a C-terminal truncation at any of the following amino acid positions (e.g., relative to SEQ ID NO: 32): amino acids 855-1420; amino acids 860-1420; amino acids 865-1420; amino acids 870-1420; amino acids 875-1420; amino acids 880-1420; amino acids 885-1420; amino acids 890-1420; amino acids 895-1420; or amino acids 900-1420. In some cases, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein can include both an N-terminal truncation and a C-terminal truncation.For example, a non-naturally occurring polypeptide (e.g., truncated collagen) as described herein can include an N-terminal truncation at any amino acid position between amino acids 1-660; amino acids 1-665; amino acids 1-670; amino acids 1-675; amino acids 1-680; amino acids 1-685; amino acids 1-690; amino acids 1-695; or amino acids 1-700 (e.g., relative to SEQ ID NO: 32); and amino acids 855-1420; amino acids 860-1420; amino acids 865-1420. amino acids from positions 870 to 1420; amino acids from positions 875 to 1420; amino acids from positions 880 to 1420; amino acids from positions 885 to 1420; amino acids from positions 890 to 1420; amino acids from positions 895 to 1420; or amino acids from positions 900 to 1420 (based on SEQ ID NO: 32). In specific embodiments, a non-naturally occurring polypeptide (e.g., a truncated collagen) disclosed herein may comprise the amino acid sequence of SEQ ID NO: 32 (or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%) sequence identity thereto), including an N-terminal truncation at amino acid position 680 (relative to SEQ ID NO: 32); and a C-terminal truncation at amino acid position 880 (relative to SEQ ID NO: 32).

[0063] In some cases, a non-naturally occurring polypeptide (e.g., a truncated collagen) can comprise any amino acid sequence provided herein. In some cases, a non-naturally occurring polypeptide (e.g., a truncated collagen) can consist of any amino acid sequence provided herein. In some cases, a non-naturally occurring polypeptide (e.g., a truncated collagen) can consist essentially of any amino acid sequence provided herein. In specific embodiments, a non-naturally occurring polypeptide has or comprises the amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In some embodiments, a non-naturally occurring polypeptide (e.g., a truncated collagen) comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In some embodiments, a non-naturally occurring polypeptide consists of or consists essentially of the amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

[0064] In some embodiments, non-naturally occurring polypeptides can include any chimeric collagen containing at least one discontinuous collagen fragment. For example, a non-naturally occurring polypeptide can be a chimeric collagen in which a portion of the N-terminal collagen is contiguous with a portion of the C-terminal collagen, where the N-terminal collagen and the C-terminal collagen are not contiguous in native collagen or the corresponding naturally occurring collagen. In another example, a non-naturally occurring polypeptide can be a chimeric collagen in which a portion of the C-terminal collagen is contiguous with a portion of the N-terminal collagen (e.g., inverted or reversed order, i.e., the C-terminal collagen is located N-terminal to the N-terminal collagen), where the C-terminal collagen and the N-terminal collagen are contiguous or discontinuous in native collagen or the corresponding naturally occurring collagen. In another example, a non-naturally occurring polypeptide can be a chimeric collagen in which a portion of a collagen polypeptide is contiguous with a portion of a second collagen polypeptide (e.g., a collagen molecule in which a portion of a collagen from a first species is contiguous with a portion of a collagen from a second species is a chimeric collagen).

[0065] Exemplary amino acid sequences of recombinant proteins or exemplary nucleic acid sequences encoding recombinant proteins are provided below:

[0066] SEQ ID NO:1 - Nucleotide sequence encoding truncated collagen type 21 alpha 1 polypeptide from Gallus gallus (chicken) [ka]

[0067] SEQ ID NO:2 - Amino acid sequence of truncated collagen type 21 alpha 1 polypeptide from Gallus gallus (chicken) [ka]

[0068] SEQ ID NO:3 - Nucleotide sequence encoding truncated collagen type 21 alpha 1 polypeptide from Gallus gallus (chicken) [ka]

[0069] SEQ ID NO: 4 - Amino acid sequence of truncated collagen type 21 alpha 1 polypeptide from Gallus gallus (chicken) [ka]

[0070] SEQ ID NO:5 - Nucleotide sequence encoding truncated collagen type 21 alpha 1 polypeptide from Gallus gallus (chicken)

[0071] [ka]

[0072] SEQ ID NO: 6 - Amino acid sequence of truncated collagen type 21 alpha 1 polypeptide from Gallus gallus (chicken) [ka]

[0073] SEQ ID NO:7 - Nucleotide sequence encoding truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0074] SEQ ID NO:8 - Amino acid sequence of truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0075] SEQ ID NO:9 - Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 1 [ka]

[0076] SEQ ID NO: 10 - Amino acid sequence of secretory signal sequence 1 [ka]

[0077] SEQ ID NO:11 - Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 2 [ka]

[0078] SEQ ID NO: 12 - Amino acid sequence of secretory signal sequence 2 [ka]

[0079] SEQ ID NO: 13—Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 3 [ka]

[0080] SEQ ID NO: 14 - Amino acid sequence of secretory signal sequence 3 [ka]

[0081] SEQ ID NO: 15—Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 4 [ka]

[0082] SEQ ID NO: 16 - Amino acid sequence of secretory signal sequence 4 [ka]

[0083] SEQ ID NO: 17—Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 5 [ka]

[0084] SEQ ID NO: 18 - Amino acid sequence of secretory signal sequence 5 [ka]

[0085] SEQ ID NO: 19—Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 6 [ka]

[0086] SEQ ID NO: 20 - Amino acid sequence of secretory signal sequence 6 [ka]

[0087] SEQ ID NO:21 - Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 7 [ka]

[0088] SEQ ID NO: 22 - Amino acid sequence of secretory signal sequence 7 [ka]

[0089] SEQ ID NO:23—Nucleotide sequence encoding secretory signal sequence designated as secretory signal sequence 8 [ka]

[0090] SEQ ID NO: 24 - Amino acid sequence of secretory signal sequence 8 [ka]

[0091] SEQ ID NO:25 - Codon-optimized nucleotide sequence encoding truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0092] SEQ ID NO:26 - Codon-optimized nucleotide sequence encoding truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0093] SEQ ID NO:27 - Codon-optimized nucleotide sequence encoding truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0094] SEQ ID NO:28 - Codon-optimized nucleotide sequence encoding truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0095] SEQ ID NO:29 - Codon-optimized nucleotide sequence encoding truncated collagen type 2 alpha 1 polypeptide from Acipenser schrenckii (Amur sturgeon) [ka]

[0096] SEQ ID NO:30 - Codon-optimized nucleotide sequence encoding truncated collagen type 21 polypeptide from Gallus gallus [ka]

[0097] In some embodiments, the non-naturally occurring polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 8. In some embodiments, the non-naturally occurring polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, etc., sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, and 8. Alternatively and / or additionally, the non-naturally occurring polypeptide is encoded by the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, and 25-30. In some embodiments, the non-naturally occurring polypeptide is encoded by a nucleic acid having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, etc., sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, and 25-30.

[0098] In some embodiments, the non-naturally occurring polypeptides provided herein may or may not contain one or more domains derived from naturally occurring collagen. Figure 6 shows an alignment of exemplary non-naturally occurring polypeptides of the present disclosure (e.g., truncated collagens) with the corresponding naturally occurring collagens. The top panel shows an alignment of the non-naturally occurring polypeptides of SEQ ID NO:2 and SEQ ID NO:6 with Gallus gallus type 21 alpha 1 collagen (e.g., SEQ ID NO:31). The bottom panel shows an alignment of the non-naturally occurring polypeptide of SEQ ID NO:8 with Acipenser schrenckii type 2 alpha 1 collagen. Figure 6 shows that the non-naturally occurring polypeptides can have one or more domains found in naturally occurring collagens (e.g., collagen triple helix repeat domains). Figure 6 further shows that the non-naturally occurring polypeptides can lack one or more domains found in naturally occurring collagens (e.g., von Willebrand factor type A (vWA) domain, laminin G domain, fibrillar collagen C-terminal domain). In some embodiments, the non-naturally occurring polypeptides provided herein can contain one or more collagen triple helix repeat domains. In some embodiments, the non-naturally occurring polypeptides provided herein may lack one or more of the von Willebrand factor type A (vWA) domain, the laminin G domain, and the fibrillar collagen C-terminal domain.

[0099] In some embodiments, a non-naturally occurring polypeptide (e.g., a recombinant polypeptide) comprises a secretory signal sequence. Any suitable secretory signal sequence (e.g., a hydrophobic signaling peptide, a Sec signal peptide, a Tat signal peptide, etc.) that can direct the secretion of a non-naturally occurring polypeptide (e.g., a recombinant polypeptide) into the periplasmic space and / or the extracellular space (e.g., when produced in a recombinant host cell) can be used. Exemplary secretory signal sequences include peptides having the amino acid sequence of any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, and 24. Alternatively and / or additionally, the secretory signal sequence comprises a peptide encoded by the nucleic acid sequence of any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, and 23. The secretory signal sequence is preferably located at the N-terminus of the non-naturally occurring polypeptide (e.g., a recombinant polypeptide). However, it is contemplated that the secretory signal sequence can be located at a position other than the N-terminus, provided that the secretory signal sequence remains functional.

[0100] A non-naturally occurring polypeptide (e.g., recombinant polypeptide) as described herein can be expressed or produced via a nucleic acid sequence encoding the non-naturally occurring polypeptide (e.g., recombinant polypeptide). Accordingly, another aspect of the present disclosure includes an expression vector comprising a nucleic acid sequence encoding a non-naturally occurring polypeptide (e.g., recombinant polypeptide). In some embodiments, the expression vector is a bacterial expression vector. In some embodiments, the expression vector is a yeast expression vector. In some embodiments, the expression vector is an insect expression vector. Any suitable expression vector can induce protein expression from an inserted nucleic acid encoding a non-naturally occurring polypeptide (e.g., recombinant polypeptide). Exemplary bacterial expression vectors can include pGEX vectors in which glutathione S-transferase is used as a fusion partner and gene expression is under the control of the tac promoter, or pET vectors using the T7 promoter (e.g., pET28 vectors, etc.). Exemplary yeast expression vectors can include pPIC vectors using the methanol-inducible AOX1 promoter. In some embodiments, the expression vector is in the form of a plasmid (e.g., including the form of a bacterial artificial chromosome, etc.) that exists independently in a host cell (e.g., a cell expressing a recombinant polypeptide). In some embodiments, the expression vector is stably integrated into the host cell chromosome by random or targeted integration.

[0101] In some embodiments, a nucleic acid sequence encoding a non-naturally occurring polypeptide (e.g., a recombinant polypeptide) is codon-optimized for expression in a non-animal cell, preferably a bacterial cell. As used herein, "codon-optimized" means that the codon composition has been improved for expression in a heterologous cell (e.g., a microbial cell, a bacterial cell, etc.) without altering the encoded amino acid sequence. Non-limiting examples of codon-optimized nucleic acid sequences (e.g., nucleic acid sequences encoding a non-naturally occurring polypeptide as described herein) include SEQ ID NOs: 25-30.

[0102] In some embodiments, an expression vector can contain one or more selection agents. The selection agents include certain sugars, including galactose-containing sugars, or antibiotics, including ampicillin, hygromycin, G418, and the like. Enzymes used to confer resistance to selection agents include β-galactosidase or β-lactamase. Alternatively and / or additionally, the expression vector can contain an inducible or constitutive promoter (e.g., a CMV promoter), and the nucleic acid encoding the recombinant protein can be operably linked to the inducible or constitutive promoter. For example, the expression vector can contain the tetracycline-inducible promoter pTET, the araC-ParaBAD-inducible promoter, or the IPTG-inducible lac promoter. As used herein, "operably linked" promoter and nucleic acid means that expression (e.g., transcription, translation, etc.) of the nucleic acid is under at least partial control of the promoter.

[0103] In some embodiments, a nucleic acid (e.g., a nucleic acid of any one of SEQ ID NOS: 1, 3, 5, 7, and 25-30) encoding a non-naturally occurring polypeptide (e.g., a recombinant polypeptide) and an expression vector can have overlaps of 20-50 bp, 20-40 bp, 20-30 bp, or 30-40 bp in length. Such overlaps can be added using PCR with a DNA polymerase (e.g., PRIMESTAR® GXL polymerase (www.takarabio.com / products / pcr / gc-rich-pcr / primestar-gxl-dna-polymerase)). Any suitable cloning system (e.g., IN-FUSION® Cloning (www.takarabio.com / products / cloning / in-fusion-cloning) or SGI Gibson assembly (us.vwr.com / store / product / 17613857 / gibson-assembly-hifi-1-step-kit-synthetic-genomics-inc)) can be used to assemble the opened expression vector and the insert nucleic acid encoding the non-naturally occurring polypeptide (e.g., recombinant polypeptide) into the final plasmid.

[0104] Such prepared expression vectors (or plasmids) can be used to generate genetically engineered or modified organisms or recombinant cells that produce the non-naturally occurring polypeptides (e.g., collagen, truncated collagen, or collagen fragments) described herein. Preferably, the recombinant cell contains at least one copy of a plasmid or stably integrated heterologous nucleic acid sequence encoding the non-naturally occurring polypeptide (e.g., collagen, truncated collagen, or collagen fragment, preferably a collagen, truncated collagen, or collagen fragment of or derived from Gallus gallus collagen and / or Acipenser schrenckii collagen). In some embodiments, the recombinant cell is a microbial cell. For example, if the expression vector is a bacterial expression vector, the expression vector can be inserted (e.g., via any suitable transformation method) into bacterial cells (e.g., E. coli, such as BL-21 cells) for protein expression, so that it exists independently in the bacterial cytoplasm (e.g., in the form of a plasmid) or is at least transiently and / or stably integrated into the bacterial chromosome.

[0105] The transformed cells can then be cultured in a suitable medium. Preferably, the suitable medium includes minimal medium, and the cells are frozen in 1.5 aliquots using vegetable glycerin at a 50:50 cell to glycerin ratio. For protein expression, one vial of frozen cultured cells can be cultured in a suitable amount of bacterial culture medium (e.g., minimal medium, 50 ml, 100 ml, etc.) for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or at least overnight at at least 36°C, preferably about 37°C, by continuously shaking the culture (e.g., at least 100 rpm, at least 200 rpm, at least 250 rpm, etc.). Tables 2 and 3 show exemplary formulations of minimal medium that can be used for cell cultivation and culture.

[0106] [Table 2]

[0107] [Table 3]

[0108] In some embodiments, the transformed cells may then be transferred to a larger volume of growth medium (e.g., minimal medium) and grown for at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, 5-10 hours, 5-9 hours, 6-9 hours, and / or until the cell density in the medium reaches an optical density (OD) of 600.

[0109] Furthermore, the fermentation process can be carried out at various temperatures ranging from 22°C to 33°C, 29°C to 33°C, 30°C to 32°C, 23°C to 29°C, or 25°C to 28°C. In some embodiments, the fermentation temperature can be maintained at a constant temperature, and the non-naturally occurring polypeptide can be purified once fermentation is complete. Alternatively, the fermentation temperature can be maintained for a desired period of time, and once the cell density reaches an OD600 of 10 to 20, the temperature can be reduced to induce protein production. In such embodiments, the temperature is typically reduced from 28°C to 25°C. Protein expression in the bacteria can be induced during fermentation by adding an inducing agent. For example, if an expression vector contains a lac promoter and a nucleic acid encoding a non-naturally occurring polypeptide (e.g., a truncated collagen, a collagen fragment, or a collagen) is under the control of the lac promoter, expression of the nucleic acid can be induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) at a concentration ranging from 0.1 to 1.5 mM, 0.1 to 1.0 mM, or 0.1 to 0.5 mM. Fermentation can be continued for 20 to 24 hours, or in some embodiments, 40 to 60 hours.

[0110] It is contemplated that such engineered recombinant cells (e.g., recombinant bacteria transformed with an expression vector) will intracellularly express a non-naturally occurring polypeptide (e.g., a truncated collagen, collagen fragment, or collagen) encoded by a nucleic acid in the expression vector. The polypeptide (e.g., a truncated collagen, collagen fragment, or collagen) expressed in such cells can then be secreted (via a secretory signal sequence) into the extracellular space (e.g., culture medium). Thus, in some embodiments, the culture medium can contain the secreted recombinant protein (e.g., a truncated collagen, collagen fragment, or collagen) encoded by the nucleic acid.

[0111] Accordingly, another aspect of the present disclosure includes a composition comprising a non-naturally occurring polypeptide (e.g., a recombinant collagen, truncated collagen, collagen fragment, or collagen) encoded by the nucleic acid. In some embodiments, the composition may include a recombinant cell comprising an integrated heterologous nucleic acid sequence encoding a non-naturally occurring polypeptide (e.g., a collagen, truncated collagen, or fragment thereof), and / or a culture medium (e.g., growth medium, culture medium, etc.) for the recombinant cell.

[0112] Alternatively and / or additionally, the composition may include recombinant protein purified from recombinant cells and / or culture medium. In some embodiments, recombinant protein is purified from culture medium in which recombinant cells have grown and secreted the recombinant protein. In some embodiments, the recombinant protein is conjugated with a tag (e.g., a histidine tag) to enable purification using an affinity purification technique known as immobilized metal affinity chromatography (IMAC). Alternatively, the recombinant protein may be purified via column chromatography. For example, recombinant protein may be purified by acid treatment of homogenized growth medium. In such an example, the pH of the growth medium (e.g., fermentation broth) may be lowered to 3-3.5 using 5-50% sulfuric acid. The recombinant cells are then separated using centrifugation. The supernatant of the acidified broth may be tested on a polyacrylamide gel to determine whether it contains recombinant protein in relatively higher abundance compared to the starting pellet. The resulting recombinant protein slurry is typically high in salt. Filtration steps may be used to reduce volume and salt, followed by concentration and diafiltration steps. For example, the filtration process is performed using 0.1 m 2 This can be done using an EMD Millipore Tangential Flow Filtration system with an ultrafiltration cassette of 0.2 m. When two cassettes are used in parallel, the total area of ​​filtration in this example is 0.2 m. 2 In this TFF step, a 5x volume reduction and a 19x salt reduction can be achieved. The final collagen slurry can be run on an SDS-PAGE gel to confirm the presence of recombinant protein. The purified recombinant proteins can then be analyzed on an SDS-PAGE gel to identify distinct, dark bands corresponding to the expected size of each protein. Further titer and purity determination can be performed using reverse-phase and size-exclusion HPLC chromatography. The purity of the purified recombinant protein is preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0113] In some embodiments, compositions comprising the non-naturally occurring polypeptides (e.g., recombinant proteins and / or purified recombinant proteins) provided herein can be formulated for consumption by an individual (e.g., a human, a patient, a human being, an animal, etc.). In some embodiments, the non-naturally occurring polypeptides (e.g., recombinant proteins and / or purified recombinant proteins) can be formulated for oral consumption as a dietary supplement. In some embodiments, the non-naturally occurring polypeptides (e.g., recombinant proteins and / or purified recombinant proteins) can be formulated for oral consumption as a food or food ingredient. In some embodiments, the non-naturally occurring polypeptides can be formulated as a protein supplement. Optionally, in such embodiments, the non-naturally occurring polypeptides (e.g., recombinant proteins and / or purified recombinant proteins) can be mixed with at least one of a carrier molecule, a preservative, and / or an additional edible ingredient. Thus, for example, the compositions can include vitamins (e.g., vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, etc.), minerals (e.g., calcium, zinc, copper, manganese, chromium, molybdenum (molundenum), boron, etc.), sugars (e.g., cellulose, dextrose, maltose, etc.), and / or natural extracts (e.g., herbs, ginseng, echinacea, green tea, glucosamine, omega-3, lutein, folic acid, cod liver oil, fish oil, coffee extract, etc.). Formulations suitable for consumption by an individual (e.g., human) include, but are not limited to, ready-to-mix powders, ready-to-drink beverages, functional shots, supplement tablets and capsules, coffee creamers, bars, bites or baked good, "non-bone" broths, non-dairy frozen novelties, gummies (e.g., candy), chocolate, and meat snacks. Non-limiting examples of formulations containing non-naturally occurring polypeptides are provided in Examples 4-6.

[0114] A composition, formulation, or product is "nutritional" or "nutritional" if it provides a significant amount of nutrients to the intended consumer, meaning that the consumer will assimilate all or part of the composition or formulation into cells, organs, and / or tissues. Generally, such assimilation into cells, organs, and / or tissues provides a benefit or utility to the consumer, for example, by maintaining or improving the health and / or natural function of the cells, organs, and / or tissues. A nutritional composition or formulation that is assimilated as described herein is referred to as "nutritional." As a non-limiting example, a polypeptide is nutritional if it provides a significant amount of polypeptide nutrients to the intended consumer, meaning that the consumer will assimilate all or part of the protein into cells, organs, and / or tissues, usually in the form of single amino acids or small peptides. "Nutritional" also refers to the process of providing a nutritional composition, formulation, product, or other material to a subject (e.g., a human or other mammal). A nutritional product need not be "nutritional complete," which means that when consumed in sufficient amounts, the product provides all the carbohydrates, lipids, essential fatty acids, essential and conditionally essential amino acids, vitamins, and minerals necessary for the consumer's overall health. Furthermore, a "nutritional complete protein" contains all the protein nutrients required (meaning the amount needed by an organism for physiological normality), but does not necessarily include vitamins and minerals, carbohydrates, or micronutrients such as lipids.

[0115] In preferred embodiments, the compositions or formulations are nutritional in that they provide polypeptides that can be broken down (e.g., by breaking peptide bonds, often referred to as protein digestion) into single amino acids and / or small peptides (e.g., 2, 3, or 4 amino acids, possibly up to 10 amino acids) in amounts sufficient to provide a "nutritional benefit." Furthermore, in certain embodiments, nutritional polypeptides are provided that cross the gastrointestinal wall and are absorbed into the bloodstream as small peptides (e.g., greater than a single amino acid but less than about 10 amino acids) or larger peptides, oligopeptides, or polypeptides (e.g., >11 amino acids). The nutritional benefit of polypeptide-containing compositions can be demonstrated, and optionally quantified, by several metrics. For example, a nutritional benefit is a benefit to a consuming organism that is equal to or exceeds at least about 0.5% of a reference daily intake of protein (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more than about 100% of a reference daily intake). Alternatively, a nutritional benefit is evidenced by a feeling and / or perception of fullness by the consumer. In other embodiments, the nutritional benefit is demonstrated by substantial uptake of the polypeptide component of the composition or formulation into the cells, organs, and / or tissues of the consumer, where such uptake typically refers to the use of a single amino acid or short peptide to produce the polypeptide de novo within the cell. "Consumer" or "consuming organism" refers to any animal capable of orally ingesting a product having a nutritional benefit. Typically, the consumer is a mammal, e.g., a healthy human, e.g., a healthy infant, child, adult, or elderly person. Alternatively, the consumer is a mammal, such as a human (e.g., an infant, child, adult, or elderly person), at risk of developing or suffering from (i) a lack of adequate nutrition and / or (ii) a disease, disorder, or condition characterized by its alleviation by a nutritional product of the present disclosure.An "infant" is usually a human under about 1 or 2 years of age, a "child" is usually a human under about 18 years of age, and an "elderly" or "senior adult" is a human about 65 years of age or older.

[0116] Nutritional polypeptides (e.g., the non-naturally occurring polypeptides described herein) are provided herein that can alter health status and treat, prevent, and reduce the severity of numerous diseases, disorders, and symptoms associated with the pathophysiology of amino acids. Because they are selected for their specific physiological benefits that improve health status and address many nutrition-related conditions, including gastrointestinal malabsorption, muscle wasting, diabetes or prediabetes, obesity, oncology, metabolic diseases, and other cellular and systemic diseases. Also provided are compositions and formulations that include nutritional polypeptides (e.g., the non-naturally occurring polypeptides described herein) as foods, beverages, medical foods, supplements, and medicines.

[0117] Nutritional polypeptides (e.g., the non-naturally occurring polypeptides described herein) can be evaluated for their physicochemical and functional properties (see, e.g., Example 7). Such properties can include digestibility, allergenicity, thermal stability, solubility, aggregation, toxicity, taste, and mouthfeel / texture properties.

[0118] In some embodiments, the formulation is incorporated into a food product that provides an advantage over a similar food product lacking the nutritional polypeptide (e.g., a non-naturally occurring polypeptide described herein), or the formulation is incorporated into other products, such as beverages or animal feed products. For example, the food product has a lower fat content, lower sugar content, and / or lower calorie content than a food product without the nutritional polypeptide (e.g., a non-naturally occurring polypeptide described herein). Preferably, the nutritional polypeptide (e.g., a non-naturally occurring polypeptide described herein) is present in the food product such that consumption of a nutritional amount of the food results in satiety. In some embodiments, gelatin, an animal-derived material, is replaced by a non-animal-derived product containing one or more nutritional polypeptides (e.g., a non-naturally occurring polypeptide described herein). Typically, the nutritional polypeptide (e.g., a non-naturally occurring polypeptide described herein) is present in an amount effective to replace gelatin in the product. The gelatin substitute is incorporated into a food, beverage, or animal feed product, and the formulation is substantially free of non-edible products.

[0119] Also provided herein are formulations comprising functional and / or nutritional amounts of nutritional polypeptides (e.g., non-naturally occurring polypeptides described herein) that increase the viscosity of food or beverage products, e.g., formulations comprising thickening nutritional polypeptides (e.g., non-naturally occurring polypeptides described herein) incorporated into foods that have advantages over similar foods lacking the nutritional polypeptides (e.g., non-naturally occurring polypeptides described herein). For example, the foods have a lower fat content, lower sugar content, and / or lower calorie content than foods without the nutritional polypeptides (e.g., non-naturally occurring polypeptides described herein). The thickening nutritional polypeptides (e.g., non-naturally occurring polypeptides described herein) can also be used as nutritionally preferable, low-calorie fat substitutes. Additionally, it may be desirable to add one or more polysaccharides or emulsifiers to the compositions and products, thereby further improving the smooth and soft mouthfeel.

[0120] In certain embodiments, the non-naturally occurring polypeptides of the present disclosure may be combined with other ingredients to provide formulated products, which may include carbohydrates, lipids, supplemental minerals, supplemental vitamins, excipients or buffers, flavorings, sweeteners, or coloring agents.

[0121] "Carbohydrate" refers to a sugar or sugar polymer. The terms "saccharide," "polysaccharide," "carbohydrate," and "oligosaccharide" can be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one for each carbon atom of the molecule. Carbohydrates usually have the molecular formula CnH2nOn. Carbohydrates can be monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. Most basic carbohydrates are monosaccharides, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two linked monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Oligosaccharides typically contain three to six monosaccharide units (e.g., raffinose, stachyose), while polysaccharides contain six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates can include modified saccharide units such as 2'-deoxyribose, in which the hydroxyl group has been removed, 2'-fluororibose, in which the hydroxyl group has been replaced with fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2'-fluororibose, deoxyribose, and hexose). Carbohydrates can exist in many different forms, including conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

[0122] As used herein, "lipid" includes fats, oils, triglycerides, cholesterol, phospholipids, and fatty acids in any form, including free fatty acids. Fats, oils, and fatty acids can be saturated, unsaturated (cis or trans), or partially unsaturated (cis or trans). In some embodiments, lipids include lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid, and the like. (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In some embodiments, the composition comprises at least one modified lipid, e.g., a lipid altered by cooking.

[0123] Additional components also include supplementary minerals or mineral sources.Examples of minerals include, but are not limited to, chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium and selenium.Suitable forms of any of the aforementioned minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelate minerals, mineral complexes, non-reactive minerals, such as carbonyl minerals and reduced minerals, and combinations thereof.

[0124] Additional ingredients can also include one or more supplementary vitamins.The vitamins can be fat-soluble or water-soluble vitamins.Suitable vitamins include, but are not limited to, vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid and biotin.Suitable forms of any of the above are vitamin salts, vitamin derivatives, compounds with the same or similar activity as vitamins, and vitamin metabolites.

[0125] The formulation may also contain an excipient or buffer. Non-limiting examples of suitable excipients include tastants, flavorants, buffers, preservatives, stabilizers, binders, compaction agents, lubricants, dispersion enhancers, disintegrants, flavorings, sweeteners, and coloring agents. Non-limiting examples of suitable buffers include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

[0126] The formulations may also contain preservatives. Non-limiting examples of suitable preservatives include organic acids naturally obtained from fermentation (e.g., citric acid, ascorbic acid, propionic acid), antimicrobial peptides (nisin), or other suitable preservatives (e.g., salt, calcium sorbate, sodium sorbate).

[0127] Binders, lubricants, dispersion enhancers, disintegrants, etc. can also be used as excipients. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohol, polyethylene glycol, polyol, saccharide, oligosaccharide, and combinations thereof. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. Non-limiting examples of suitable dispersing agents include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose such as high HLB emulsifier surfactants. Non-limiting examples of suitable non-effervescent disintegrants include starches (e.g., corn starch, potato starch, their pregelatinized starches, and modified starches), sweeteners, clays (e.g., bentonite), microcrystalline cellulose, alginates, sodium starch glycolate, gums (e.g., agar, guar gum, locust bean gum, karaya gum, pectin, and tragacanth gum). In some embodiments, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0128] Additional ingredients may also include flavorings, sweeteners, or coloring agents.The flavoring agent incorporated into the outer layer may be selected from synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof.Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts (e.g., sodium salt); dipeptide sweeteners (e.g., aspartame); dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (stevioside); chloro derivatives of sucrose, such as sucralose; and sugar alcohols (e.g., sorbitol, mannitol, sylitol), etc. Non-limiting examples of suitable colorants include Food, Drug and Cosmetic Colors (FD&C), Drug and Cosmetic Colors (D&C), and External Drug and Cosmetic Colors (Ext. D&C).

[0129] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules. Capsules typically include a core material containing a protein or composition and a shell wall encapsulating the core material. In some embodiments, the core material includes at least one of a solid, a liquid, and an emulsion. In some embodiments, the shell wall material includes at least one of soft gelatin, hard gelatin, and a polymer.

[0130] Powders or granules embodying the polypeptides and compositions disclosed herein can be incorporated into food products. In some embodiments, the food product is a drink for oral administration. Non-limiting examples of suitable drinks include fruit juices, fruit drinks, artificially flavored drinks, artificially sweetened drinks, carbonated drinks, sports drinks, liquid dairy products, shakes, alcoholic drinks, caffeinated drinks, infant formula, etc. Other means suitable for oral administration include aqueous and non-aqueous solutions, creams, pastes, emulsions, suspensions, and slurries, each of which may optionally contain at least one suitable solvent, preservative, emulsifier, suspending agent, diluent, sweetener, colorant, taste substance, flavorant, and flavoring agent.

[0131] Suitable examples of solid food products include, but are not limited to, food bars, snack bars, cookies, brownies, muffins, crackers, biscuits, creams or pastes, ice cream bars, frozen yogurt bars, and the like.

[0132] A formulation can include a nutritional polypeptide (e.g., a non-naturally occurring polypeptide as described herein) in an amount that is based on the concentration of the nutritional polypeptide (e.g., a non-naturally occurring polypeptide as described herein) (e.g., on a weight-to-weight basis), and the nutritional polypeptide (e.g., a non-naturally occurring polypeptide as described herein) comprises up to 100% by weight of the formulation, meaning that all or essentially all of the material present in the formulation is in the form of a nutritional polypeptide (e.g., a non-naturally occurring polypeptide as described herein). More usually, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% by weight present in the formulation is in the form of a nutritional polypeptide (e.g., a non-naturally occurring polypeptide as described herein). In some embodiments, the formulation comprises 10 mg, 100 mg, 500 mg, 750 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g or more than 100 g of nutritional polypeptide.

[0133] In some embodiments, the polypeptide or composition is provided in a dosage form designed to administer at least one polypeptide disclosed herein, wherein the total amount of polypeptide administered is selected from 0.1 g to 1 g, 1 g to 5 g, 2 g to 10 g, 5 g to 15 g, 10 g to 20 g, 15 g to 25 g, 20 g to 40 g, 25 g to 50 g, and 30 g to 60 g. In some embodiments, the dosage form is designed to administer at least one protein disclosed herein, wherein the total amount of protein administered is selected from about 0.1 g, 0.1 g to 1 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, and 100 g.

[0134] In some embodiments, the protein or composition is consumed at a rate of 0.1g to 1g per day, 1g to 5g per day, 2g to 10g per day, 5g to 15g per day, 10g to 20g per day, 15g to 30g per day, 20g to 40g per day, 25g to 50g per day, 40g to 80g per day, 50g to 100g per day or more.

[0135] In another aspect, the present disclosure provides methods for maintaining or increasing at least one of muscle mass, muscle strength, and functional activity in a subject. In some embodiments, the method includes providing a sufficient amount of a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure to the subject. In some embodiments, the subject is at least one of a medically critical elderly individual and an elderly individual suffering from protein-energy malnutrition. In some embodiments, the sufficient amount of a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject in conjunction with performing exercise. In some embodiments, the polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject via oral, enteral, or parenteral routes. In some embodiments, the polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject via the oral route. In some embodiments, the polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject via the enteral route.

[0136] In another aspect, the present disclosure provides methods for maintaining or achieving a desired body mass index in a subject. In some embodiments, the method comprises providing a sufficient amount of a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure to the subject. In some embodiments, the subject is at least one of a medically critical elderly individual and an elderly individual suffering from protein-energy malnutrition. In some embodiments, the sufficient amount of a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject in conjunction with exercise. In some embodiments, the polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject by oral, enteral, or parenteral routes.

[0137] In another aspect, the present disclosure provides methods of providing a polypeptide (e.g., a polypeptide of the present disclosure) to a subject having protein-energy malnutrition. In some embodiments, the method includes providing a sufficient amount of a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure to the subject. In some embodiments, the polypeptide of the present disclosure, the composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject by oral, enteral, or parenteral routes.

[0138] Polypeptides of the present disclosure are useful for treating sarcopenia or frailty after they have developed in a subject, or for preventing the onset of sarcopenia or frailty in a subject who is a member of a risk group. In some embodiments, all of the polypeptides consumed by the subject are polypeptides of the present disclosure. In some embodiments, polypeptides of the present disclosure are combined with proteins and / or free amino acids from other sources to provide the subject's total protein intake. In some embodiments, the subject is at least one of a medically critical elderly person and an elderly person suffering from protein-energy malnutrition. In some embodiments, a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject in conjunction with exercise. In some embodiments, a polypeptide of the present disclosure, a composition of the present disclosure, or a composition made by a method of the present disclosure is consumed by the subject by oral, enteral, or parenteral routes.

[0139] In some embodiments, incorporating at least one polypeptide or composition of the present disclosure into a subject's diet has at least one effect selected from inducing postprandial satiety (including by suppressing hunger), inducing thermogenesis, reducing glycemic response, positively affecting energy expenditure with a positive effect on lean body mass, reducing weight gain due to overeating, and reducing energy intake. In some embodiments, incorporating at least one polypeptide or composition of the present disclosure into a subject's diet has at least one effect selected from increasing body fat loss, reducing lean tissue loss, improving lipid profiles, and improving glucose tolerance and insulin sensitivity in the subject.

[0140] In some embodiments, a composition comprising a non-naturally occurring polypeptide (e.g., a recombinant protein and / or a purified recombinant protein) may be formulated for topical application. The topical application may be for medical or cosmetic purposes. In such embodiments, the composition may further comprise at least one of a carrier molecule (e.g., a vehicle), a preservative, and / or an additional edible ingredient. Any suitable carrier molecule is contemplated, and exemplary carrier molecules may include water, oil, alcohol, propylene glycol, or an emulsifier. Additionally, any suitable preservative is contemplated, and exemplary preservatives include zinc oxide, parabens, formaldehyde-releasing agents, isothiazolinones, phenoxyethanol, or organic acids such as benzoic acid, sodium benzoate, or butylene glycol, hexanediol, or potassium sorbate.

[0141] In one aspect, the composition comprising the non-naturally occurring polypeptide can be a personal care product (e.g., a cosmetic). In some embodiments, the composition is formulated for topical administration. The composition can contain other cosmetic ingredients suitable for human use. The personal care product can be useful for preventing or treating ultraviolet radiation damage to human skin or hair. The personal care product can be useful for enhancing skin firmness, elasticity, brightness, hydration, tactile or visual texture and / or stimulating collagen production. The personal care product can be useful for reducing skin redness. The personal care product can be applied to skin or hair. The compositions include, for example, masks, skin cleansers such as soaps, cleansing creams, cleansing lotions, facial cleansers, cleansing emulsions, cleansing pads, face washes, face and body creams and moisturizers, facial serums, face and body masks, facial toners and mists, eye creams and eye treatments, exfoliator formulas, lip balms and lipsticks, hair shampoos, hair conditioners and body shampoos, hair and scalp serums, hair mists and hair sprays, eye shadows, concealers, mascaras and other color cosmetics.

[0142] The composition comprising non-naturally occurring polypeptide can further comprise at least one additional component, including topical carrier or preservative.The topical carrier can comprise a topical carrier selected from the group consisting of liposome, biodegradable microcapsule, lotion, spray, aerosol, powder, biodegradable polymer, mineral oil, triglyceride oil, silicone oil, glycerin, glyceryl monostearate, alcohol, emulsifier, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane and water. Preservatives may include those selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1,3-diol, the cis isomer of 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidinium, 7-ethylbicyclooxazolidine, phenoxyethanol, butylene glycol, 1,2 hexanediol, methylparaben, sorbic acid, Germaben® II, rosemary extract, and EDTA.

[0143] Also provided herein in certain embodiments are methods for reducing skin damage, promoting repair of damaged skin, protecting skin from UV damage, and / or protecting skin cells from the effects of exposure to urban dust. In another embodiment, methods for increasing skin firmness, elasticity, brightness, hydration, tactile feel, or visual texture and / or stimulating collagen production are provided. These methods may include applying a composition comprising a non-naturally occurring polypeptide of the present disclosure to the skin of a subject. Without being bound by a particular theory or mechanism, the non-naturally occurring polypeptide in the composition may reduce skin damage by protecting against UV damage. In some cases, the non-naturally occurring polypeptide in the composition may promote repair of damaged skin by increasing cell viability. In some cases, the non-naturally occurring polypeptide in the composition may reduce skin damage and / or promote cell repair by increasing procollagen synthesis and / or promoting skin cell viability when applied to the skin. In some cases, the non-naturally occurring polypeptide reduces the formation of thymine-thymine (TT) dimer formation.

[0144] The methods provided herein encompass the use of compositions for treatments indicated for the methods, such as by the steps provided herein. In embodiments, the present disclosure provides the use of a composition provided herein (e.g., a non-naturally occurring polypeptide, or a formulation comprising a non-naturally occurring polypeptide) in a method for reducing skin damage, promoting repair of damaged skin, protecting skin from UV damage, and / or protecting skin cells from the effects of exposure to urban dust (e.g., by administering a composition provided herein to the skin of a subject). In embodiments, the present disclosure provides the use of a composition provided herein (e.g., a non-naturally occurring polypeptide, or a formulation comprising a non-naturally occurring polypeptide) in a method for enhancing the firmness, elasticity, brightness, hydration, tactile or visual texture of skin and / or stimulating collagen production.

[0145] Provided herein in certain embodiments are (e.g., topical) compositions or formulations comprising one or more non-naturally occurring polypeptides (e.g., for cosmetic use) provided herein. In some embodiments, the compositions provide any suitable amount of a polypeptide provided herein, such as any suitable amount (e.g., an amount suitable to provide a benefit when given or administered to an individual or cell). In some specific embodiments, the composition comprises an amount suitable to provide a beneficial effect to the skin of an individual when administered (e.g., topically) to the skin of the individual. In specific embodiments, the composition comprises 0.001% to 30% w / w of a polypeptide such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide). In more specific embodiments, the composition comprises 0.001% to 20% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide), 0.001% to 10% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide), 0.001% to 5% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide), 0.001% to 2% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide), 0.001% to 1% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide), 0.001% to 0.5% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide), and 0.001% to 0.2% w / w of a polypeptide, such as a polypeptide provided herein (or a non-naturally occurring collagen polypeptide).

[0146] In various embodiments, the concentration or amount of a non-naturally occurring polypeptide (e.g., recombinant protein) provided herein is present in any suitable amount in the compositions provided herein and can vary depending, for example, on the application or formulation (e.g., gel, capsule, liquid, powder, etc.). Exemplary concentrations of a non-naturally occurring polypeptide (e.g., recombinant protein) in a composition can be at least about 0.01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% (w / v or w / w) of the composition. Alternatively and / or additionally, exemplary concentrations of the non-naturally occurring polypeptide (e.g., recombinant protein) in the composition can be about 0.01%, about 0.05%, about 0.1%, about 0.2%, at least 0.5%, 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% (w / v or w / w) of the composition.Alternatively and / or additionally, exemplary concentrations of a non-naturally occurring polypeptide (e.g., recombinant protein) in a composition are from about 0.01% to about 99%, from about 0.05% to about 99%, from about 0.1% to about 99%, from about 0.1% to about 99%, from about 0.5% to about 99%, from about 0.1% to about 10%, from about 1% to about 99%, from about 5% to about 99%, from about 10% to about 99%, from about 15% to about 99%, from about 20% to about 99%, from about 25 ... Approximately 99%, approximately 25% to approximately 99%, approximately 30% to approximately 99%, approximately 35% to approximately 99%, approximately 40% to approximately 99%, approximately 45% to approximately 99%, approximately 50% to approximately 99%, approximately 55% to approximately 99%, approximately 60% to approximately 99%, approximately 65% ​​to approximately 99%, approximately 70% to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 99%, approximately 85% to approximately 99%, approximately 90% to approximately 99%, approximately 95% to approximately 99%, approximately 0.1% to approximately 90%, approximately 1 % to about 90%, about 5% to about 90%, about 10% to about 90%, about 15% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 35% to about 90%, about 40% to about 90%, about 45% to about 90%, about 50% to about 90%, about 55% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 8 The range may be 5% to about 90%, about 20% to about 80%, about 25% to about 80%, about 30% to about 80%, about 35% to about 80%, about 40% to about 80%, about 45% to about 80%, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, etc. Alternatively and / or additionally, exemplary concentrations of non-naturally occurring polypeptides (e.g., recombinant proteins) in the compositions can be about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, less than about 40%, etc.

[0147] Certain aspects of the present disclosure include methods for improving the appearance of a subject's skin, hair, and / or nails by administering a composition to the subject. Additionally and / or alternatively, the present disclosure includes methods for improving joint health and / or restoring bone density in a subject. In some embodiments, the subject has or is suspected of having osteoporosis and / or osteoarthritis. Alternatively and / or additionally, the present disclosure includes methods for improving gut health, altering or improving a subject's microbiome, or altering and / or reducing inflammation or tissue repair in a subject. In some embodiments, the composition is orally administered at a dose and schedule sufficient or effective to improve the appearance of a subject's skin, hair, and / or nails, improve joint health and / or restore bone density in a subject with osteoporosis and / or osteoarthritis, and / or improve gut health, altering or improving a subject's microbiome, or altering and / or reducing inflammation or tissue repair in a subject. Any suitable dose may be used as needed. In some embodiments, the dosage used is about 0.1 mg / kg to about 200 mg / kg, about 0.2 mg / kg to about 150 mg / kg, about 0.5 mg / kg to about 150 mg / kg, about 0.5 mg / kg to about 100 mg / kg, about 0.8 mg / kg to about 100 mg / kg, about 1.0 mg / kg to about 100 mg / kg, about 1.0 mg / kg to about 90 mg / kg, about 1.0 mg / kg to about 80 mg / kg, about 1.0 mg / kg to about 70 mg / kg, about 1.0 mg / kg to about 80 mg / kg, about 1.0 mg / kg to about 90 mg / kg, about 1.0 mg / kg to about 80 mg / kg, about 1.0 mg / kg to about 7 ...90 mg / kg, about 1.0 mg / kg to about 90 mg / kg, about 1.0 mg / kg to about 90 mg / kg, about 1.0 mg / kg to about 90 mg / kg, about 1. mg / kg, about 1.0 mg / kg to about 60 mg / kg, about 1.0 mg / kg to about 50 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, or about 5.0 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, or about 30 mg / kg. In some embodiments, the dose can be increased or decreased depending on the administration schedule.For example, the dose for administration to a subject (e.g., a human) can be increased or decreased to about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 1.0 mg / kg with each administration (e.g., for three consecutive administrations, the dose can be increased from 2.0 mg / kg to 2.2 mg / kg, 2.4 mg / kg, etc., respectively). In another example, the dose for administration to a subject (e.g., a human) can be increased and then decreased, or decreased and then increased, such that each dose is about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 1.0 mg / kg (e.g., for five consecutive administrations, the dose can be increased from 2.0 mg / kg to 2.2 mg / kg, 2.4 mg / kg, then 2.2 mg / kg and 2.0 mg / kg, etc., respectively).

[0148] In some embodiments, the administration schedule varies depending on the purpose, the sex, age or health condition of the subject.For example, in some embodiments, the composition is administered once a day, twice a day, three times a day, up to six times a day, every two days, every three days, every four days, every five days, every six days, etc. Alternatively and / or additionally, in some embodiments, the composition is administered multiple times at irregular intervals, or at extended or shortened intervals.

[0149] In certain embodiments, the composition is applied topically at a dose and / or schedule sufficient or effective to improve the appearance of the subject's skin, hair, and / or nails and / or reduce inflammation in the subject. In some cases, the dose varies depending on the target area of ​​topical application (e.g., hair, skin, wound, nails, etc.), and is about 0.1 g / inch of area for topical application. 2 ~approx. 10g / inch 2 , approximately 0.1g / inch 2 ~approximately 9g / inch 2 , approximately 0.1g / inch2 ~approx. 8g / inch 2 , approximately 0.1g / inch 2 ~Approx. 7g / inch 2 , approximately 0.1g / inch 2 ~approx. 6g / inch 2 , approximately 0.1g / inch 2 ~approximately 5g / inch 2 , approximately 0.1g / inch 2 ~approximately 4g / inch 2 , about 0.1g / inch 2 ~approximately 3g / inch 2 , approximately 0.5g / inch 2 ~approximately 5g / inch 2 , approximately 0.5g / inch 2 ~approximately 4g / inch 2 , approximately 1g / inch 2 ~approximately 4g / inch 2 or approximately 1g / inch 2 ~approximately 3g / inch 2 In certain embodiments, the dosage varies according to the purpose, sex, age, the severity of the damage to the area or health condition.For example, the composition can be applied to the target area of ​​the subject at least 3 times a day, at least 2 times a day, once a day, up to 6 times a day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, etc.

[0150] Skin Appearance and Quality: In some embodiments, provided herein are methods of improving the appearance and / or quality of skin, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide described herein) to an individual (e.g., an individual in need of improving the appearance and / or quality of their skin). In some embodiments, administration is orally or topically. In some cases, administration results in various changes or effects on the individual's skin. In some cases, the individual's skin exhibits increased proliferation and / or a reduced rate of cell death (e.g., when tested using a colorimetric assay to assess cellular metabolic activity (e.g., an MTT assay)). In some embodiments, the skin exhibits improved production of extracellular matrix (ECM) components (e.g., collagen, elastin, fibronectin, fibrillin) and / or reduced production of matrix-degrading proteins (e.g., matrix metalloproteinases (MMPs) and proteases). In certain cases, skin exhibits improved resistance or outcome when exposed to hazards such as photodamage (e.g., UV radiation), pollution (e.g., urban dust), and / or harsh skin care practices (e.g., retinoic acid, benzoyl peroxide, salicylic acid). In certain cases, such resistance or improved outcome is indicated by improved cell viability or proliferation (or reduced cell death), which can be assessed using an MTT viability assay, improved DNA repair, which can be assessed by thymidine dimer ELISA detection, reduced inflammation, which can be assessed by Luminex detection, or reduced reactive oxidative stress (ROS), which can be assessed by CM-H2DCFDA (a general oxidative stress indicator) detection.

[0151] In some cases, the skin exhibits reduced wrinkles and / or fine lines, reduced redness and / or hyperpigmentation of the skin, increased skin brightness, reduced pore size, reduced skin roughness, and / or reduced acne (e.g., as assessed using the CLARITY analysis). In certain cases, the skin exhibits improved skin elasticity, increased skin firmness, increased skin hydration, increased skin barrier function, increased skin collagen and elastin content, and / or increased dermal density.

[0152] Hair Quality: In some embodiments, provided herein are methods of improving the appearance and / or quality of hair, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide as described herein) to an individual (e.g., an individual in need of improved hair appearance and / or quality). In some embodiments, administration is orally or topically. In some cases, administration results in various changes or effects on the individual's hair. In certain cases, the hair exhibits improved hair fiber thickness and / or density, increased moisture, increased growth rate, reduced split ends, reduced frizz / increased static control, improved fiber alignment / shininess, increased combability, and / or increased resistance to breakage. In some cases, the hair exhibits improved hair growth, increased hair fiber diameter, increased combability, reduced hair loss, and / or increased hair tensile strength.

[0153] Nail Quality: In some embodiments, provided herein are methods of improving the appearance and / or quality of nails, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide as described herein) to an individual (e.g., an individual in need of improved nail appearance and / or quality). In some embodiments, administration is oral or topical. In some cases, administration results in various changes or effects on the individual's nails. In certain cases, nails exhibit improved (reduced) peeling, irregular nail edges and / or rough surfaces, frequent cracked / chipped nails, and / or increased nail growth rate.

[0154] Joint health: In some embodiments, provided herein are methods of improving joint health, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide as described herein) to an individual (e.g., an individual in need of improved joint health). In some embodiments, administration is orally. In some cases, administration results in various changes or effects on the individual's joint health. In certain cases, improved joint health is evidenced by a reduction in reported joint pain and / or an increase in range of joint mobility.

[0155] Inflammation: In some embodiments, provided herein are methods of ameliorating inflammatory effects, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide as described herein) to an individual (e.g., an individual in need of ameliorated inflammatory effects). In some embodiments, administration is orally or topically. In some cases, administration results in various changes or effects on inflammation in the individual. In certain cases, ameliorated inflammatory effects are demonstrated by reduced cytokine levels in the bloodstream (e.g., as assessed by Luminex detection) and / or restoration of healthy levels of immune cells (e.g., by blood differential counts).

[0156] Gut health: In some embodiments, provided herein are methods of improving gut health, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide as described herein) to an individual (e.g., an individual in need of improved gut health). In some embodiments, administration is orally. In some cases, administration results in various changes or effects on the individual's gut health. In certain cases, improved gut health is evidenced by improved intestinal motility and / or reduced gastrointestinal discomfort / pain.

[0157] Microbiome: Provided herein, in some embodiments, are methods of altering and / or improving the microbiome, such as by administering an effective amount of a product or composition provided herein (e.g., comprising a non-naturally occurring polypeptide as described herein) to an individual (e.g., an individual in need of microbiome improvement). In some embodiments, administration is orally. In some cases, administration results in various changes or effects on the individual's microbiome. In certain cases, the improved microbiome is demonstrated by increased microbial diversity and / or increased abundance of beneficial microorganisms (e.g., as assessed by 16S DNA sequencing of stool samples). [Example]

[0158] Example 1. Production of non-naturally occurring polypeptides of the present disclosure

[0159] This example demonstrates the production of recombinant polypeptides of the present disclosure by genetically engineered microorganisms and the purification process of the polypeptides so produced.

[0160] Polynucleotides represented by SEQ ID NOs: 1, 3, 5, and 7 were synthesized, and at least one of these polynucleotides was inserted into a pET vector. The overlap between the pET vector and SEQ ID NOs: 1, 3, 5, and 7 was designed to be 20–30 bp long and added using PCR with the PRIMESTAR® GXL polymerase enzyme (www.takarabio.com / products / pcr / gc-rich-pcr / primestar-gxl-dna-polymerase). The opened pET vector and insert DNA (e.g., the polynucleotide represented by SEQ ID NO: 1) were assembled into the final plasmid using IN-FUSION® Cloning (www.takarabio.com / products / cloning / in-fusion-cloning). In all cases, the nucleic acid sequence was preceded by a secretion signal sequence, as disclosed in SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, or 23. The sequences of the plasmids were verified by Sanger sequencing.

[0161] After transforming the cells with the final plasmid, they were grown in minimal medium and frozen in 1.5 aliquots using vegetable glycerol at a 50:50 cell-to-glycerol ratio. One vial of this frozen culture was revived in 50 ml of minimal medium at 37°C overnight at 200 rpm. The minimal medium formulation for this example is shown in Tables 2 and 3. The cells were then transferred to 300 ml of minimal medium and grown for 6-9 hours to reach an optical density (OD) of 5-10.

[0162] Fermentations were performed at various temperatures ranging from 25°C to 28°C. For some fermentations, the fermentation temperature was maintained constant, and the polypeptide was purified immediately upon completion of the fermentation. For other fermentations, the fermentation temperature was maintained for the desired time, and once the cell density reached an OD600 of 10-20, the temperature was lowered to induce protein production. Typically, the temperature was lowered from 28°C to 25°C. Induction was performed by adding IPTG to the medium at concentrations ranging from 0.1 to 0.5 mM. Fermentations were continued for 40-60 hours.

[0163] The recombinant polypeptide was purified as follows: the pH of the fermentation broth was lowered to 3-3.5 using 5-50% sulfuric acid. The cells were then separated using centrifugation or by centrifugation followed by microfiltration. The acidified broth supernatant was tested on a polyacrylamide gel and found to contain the recombinant protein in relatively higher abundance compared to the starting pellet. Concentration and diafiltration steps were performed using ultrafiltration to reduce volume and salts. The presence of the recombinant protein was confirmed by running the final polypeptide slurry on an SDS-PAGE gel.

[0164] To verify that the desired proteins were produced, supernatants from cultures of microorganisms containing SEQ ID NOs: 1, 3, 5, or 7 were collected and purified by lowering the pH as described above. When the acidified broths were analyzed by SDS-PAGE, bands corresponding to proteins of the expected sizes and relative purity were detected. As shown in Figure 1, intense, distinct bands were observed at the expected sizes for each respective protein. Samples were subsequently analyzed to quantify the titer and purity of the recombinant polypeptides by reverse-phase and size-exclusion HPLC chromatography and mass spectrometry, confirming the precise identity of each target protein.

[0165] Figures 2A-2C show SDS-PAGE gels of a non-naturally occurring polypeptide of the present disclosure before and after treatment at pH 3.0. Figure 2A shows an SDS-PAGE gel of a fermentation supernatant containing a non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO:2 before (lane 1) and after (lane 2) treatment at pH 3.0. The predicted molecular weight of such a polypeptide was approximately 17.9 kDa. The identity of the polypeptide was confirmed by mass spectrometry (data not shown). Figure 2B shows an SDS-PAGE gel of a fermentation supernatant containing a non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO:8 before (lane 3) and after (lane 4) treatment at pH 3.0. The predicted molecular weight of such a polypeptide was approximately 17.6 kDa. The identity of the polypeptide was confirmed by mass spectrometry (data not shown). Figure 2C shows an SDS-PAGE gel of fermentation supernatants containing the non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO:8 produced in various bacterial host strains before (lanes 3-5) and after (lanes 6-8) treatment at pH 3.0.

[0166] Example 2. Human clinical studies of non-naturally occurring polypeptides of the present disclosure

[0167] Skin appearance and quality: To evaluate the benefits of the recombinant polypeptides provided herein, patients are recruited and / or cultured human skin cells or patient-derived skin samples are provided. A non-naturally occurring polypeptide as described herein (or a product comprising a non-naturally occurring polypeptide as described herein) and a control product are administered (orally or topically) to separate cohorts (in vitro or in vivo) to evaluate various effects on the skin. The effects on the skin are evaluated quantitatively and / or qualitatively. For example, when a composition comprising a non-naturally occurring polypeptide is applied or administered to cultured human skin cells (primary culture or cell line) in vitro or to human skin tissue ex vivo, the cultured human skin cells or cells in the human skin tissue show increased proliferation or a reduced rate of cell death (e.g., when assessed using a colorimetric assay (e.g., MTT assay) to evaluate the metabolic activity of cells). In some cases, cells in such cultured human skin cells or human skin tissue contacted with or treated with a composition comprising a non-naturally occurring polypeptide described herein may show increased production of extracellular matrix (ECM) components (e.g., collagen, elastin, fibronectin, fibrillin) and decreased production of matrix-degrading proteins (e.g., matrix metalloproteinases (MMPs) and proteases) via RNA-seq transcriptome or proteome analysis. Evaluation of cells in such treated cultured human skin cells or human skin tissue demonstrates improved resistance or outcomes when exposed to hazards such as photodamage (e.g., UV radiation), pollution (e.g., urban dust), and harsh skin care practices (e.g., retinoic acid, benzoyl peroxide, salicylic acid).Such resistance or improved outcome is indicated by improved cell viability or proliferation (or reduced cell death) as assessed using an MTT viability assay, improved DNA repair as assessed by thymidine dimer ELISA detection, reduced inflammation as assessed by Luminex detection, and / or reduced reactive oxidative stress (ROS) as assessed by CM-H2DCFDA (a common oxidative stress indicator) detection.

[0168] In another example, when a composition comprising a non-naturally occurring polypeptide (e.g., as described herein) is applied orally or topically to the skin of a subject, the subject's skin is evaluated for a reduction in wrinkles and fine lines, a reduction in skin redness and hyperpigmentation, an increase in skin brightness, a reduction in pore size, a reduction in skin roughness, and a reduction in acne (e.g., when evaluated using the CLARITY analysis). The skin is further evaluated (before and after administration) to show changes in skin elasticity, changes in skin firmness, changes in skin hydration, changes in skin barrier function, changes in skin collagen and elastin content, and / or changes in dermal density.

[0169] Hair quality: The effect of a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) on hair (e.g., compared to a control product) is also evaluated. For example, when a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) is applied to hair or orally administered to a subject, hair quality is measured by measuring changes in hair fiber thickness and density, moisture, growth rate, split end occurrence, frizz / static control, fiber alignment / luster, combability, and / or resistance to breakage (e.g., measured by in vitro hair tress testing). In some cases, clinical tests measure changes in hair growth, hair fiber diameter, combability, hair loss, and / or hair tensile strength.

[0170] Nail quality: The effect of a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) on nails (e.g., the effect compared to a control product) is also evaluated. For example, nail quality is measured by measuring changes in nail hardness, nail peeling, irregular nail edges and rough nails, frequency of cracked / chipped nails, and / or nail growth rate when a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) is applied to the nails or orally administered to a subject.

[0171] Joint health: The effect of a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) on joints (e.g., the effect compared to a control product) is also evaluated. For example, joint quality is measured, such as by measuring changes in reported joint pain and / or range of joint mobility, when a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) is orally administered to a subject.

[0172] Inflammation: The effect of a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) on inflammation (e.g., the effect compared to a control product) is also evaluated. For example, when a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) is orally administered to a subject, changes in inflammation are measured, cytokine levels in the bloodstream are measured (e.g., assessed by Luminex detection), and / or immune cell levels are measured (e.g., by differential blood count).

[0173] Gut health: The effect of a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) on gut health (e.g., the effect compared to a control product) is also evaluated. For example, when a product provided herein (e.g., a product comprising a non-naturally occurring polypeptide as described herein) is orally administered to a subject, changes in gut motility and / or gastrointestinal discomfort / pain are measured.

[0174] The effects of the products provided herein (e.g., products containing a non-naturally occurring polypeptide as described herein) on the microbiome (e.g., effects compared to a control product) are also evaluated. For example, when a product provided herein (e.g., containing a non-naturally occurring polypeptide as described herein) is orally administered to a subject, changes in microbial diversity or the abundance of beneficial microorganisms are measured (which can be assessed by 16S DNA sequencing of stool samples). Such effects can also be demonstrated in vitro when the composition supports the growth of beneficial microorganisms in a co-culture broth.

[0175] Example 3. In vitro studies of non-naturally occurring polypeptides of the present disclosure

[0176] This example demonstrates the functional effects on cells in vitro after treatment with a non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO:2. The non-naturally occurring polypeptide of SEQ ID NO: 2 enhances the viability of human dermal fibroblasts.

[0177] Human primary fibroblasts were cultured for 24 hours in medium alone ( FIG. 3 ; "A") or medium containing 0.025% w / w ( FIG. 3 ; "B"), 0.05% w / w ( FIG. 3 ; "C"), or 0.1% w / w ( FIG. 3 ; "D") of a non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO: 2. Cell viability was assessed using an MTT colorimetric assay. As shown in FIG. 3 , fibroblasts treated with the polypeptide of SEQ ID NO: 2 exhibited increased cell viability compared to the medium-only control. The non-naturally occurring polypeptide of SEQ ID NO:2 increases the production of type I collagen in human dermal fibroblasts.

[0178] Human primary fibroblasts were cultured for 24 hours in medium alone (FIG. 4; "A") or medium containing 0.025% w / w (FIG. 4; "B"), 0.05% w / w (FIG. 4; "C"), or 0.1% w / w (FIG. 4; "D") of a non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO:2. Fibroblast type I collagen production was measured by analyzing supernatants by enzyme-linked immunosorbent assay (ELISA) for type I procollagen C-peptide, a readout for total secreted type I collagen. As shown in FIG. 4, fibroblasts treated with the polypeptide of SEQ ID NO:2 secreted higher levels of type I collagen than fibroblasts treated with the medium control. The non-naturally occurring polypeptide of SEQ ID NO: 2 increases the production of type I collagen in human tenocytes.

[0179] Human primary tenocytes were cultured for 24 hours in medium alone ( FIG. 5 ; "A") or medium containing 0.025% w / w ( FIG. 5 ; "B") or 0.05% w / w ( FIG. 5 ; "C") of a non-naturally occurring polypeptide having the amino acid sequence of SEQ ID NO: 2. Tenocyte type I collagen production was measured by analyzing supernatants by enzyme-linked immunosorbent assay (ELISA) for type I procollagen C-peptide, a readout for total secreted type I collagen. As shown in FIG. 5 , tenocytes treated with the polypeptide of SEQ ID NO: 2 secreted higher levels of type I collagen than cells treated with the medium control.

[0180] Example 4. Sports drinks containing non-naturally occurring polypeptides of the present disclosure

[0181] In this example, a non-naturally occurring polypeptide of the present disclosure was formulated in the form of a sports drink.

[0182] Sports Drink Formula:

[0183] 10g of SEQ ID NO:2 polypeptide / 12 oz

[0184] List of ingredients:

[0185] Water, Collagen Peptides, Sugar, Tangerine Juice Concentrate, Salt, Citric Acid, Monopotassium Phosphate, Sodium Citrate, Fruit and Vegetable Juices [for Color], Natural Flavors, Stevia

[0186] Variables tested:

[0187] 1.10g vs. 12g of SEQ ID NO:2 polypeptide / 12 oz serving

[0188] 2.0%~15% concentrated fruit juice

[0189] 3.7g-20g sugars per 12 oz serving

[0190] 4. Sweeteners: sucrose, monk fruit, stevia

[0191] 5. 0.05% to 0.30% citric acid

[0192] Example 5. Gummies containing non-naturally occurring polypeptides of the present disclosure

[0193] In this example, a non-naturally occurring polypeptide of the disclosure was formulated in the form of a gummy candy.

[0194] 2.5 g of polypeptide of SEQ ID NO: 2 and 100 mg of hyaluronic acid per 25 g

[0195] List of ingredients:

[0196] Tapioca syrup, sucrose, water, collagen peptides, citric acid, pectin, sodium citrate, natural flavors, sodium hyaluronate, fruit and vegetable juices [for color]

[0197] Variables tested:

[0198] 1. Order of addition - The polypeptide of SEQ ID NO: 2 must be in solution and added after the syrup cooking step.

[0199] 2. Various levels of SEQ ID NO:2 polypeptide (2%, 6%, 8%, 10%)

[0200] 3. The polypeptide of SEQ ID NO: 2 has a buffering effect; various citrate / citric acid levels tested

[0201] Example 6. Brownies containing non-naturally occurring polypeptides of the present disclosure

[0202] In this example, a non-naturally occurring polypeptide of the disclosure was formulated in the form of a brownie.

[0203] 3g of polypeptide of SEQ ID NO:2 / 40g

[0204] List of ingredients:

[0205] (For bread) Wheat flour, sucrose, cocoa powder, water, coconut oil, polypeptide of sequence number 2, olive oil, glycerin, vanilla extract, baking powder, salt, xanthan, lecithin.

[0206] Variables tested:

[0207] 1. Plain flour vs. bread flour

[0208] 2.3 g, 3.75 g, 5 g, or 9 g of polypeptide of SEQ ID NO: 2 per 40 g portion

[0209] 3. Reducing sugars 20% and 30%

[0210] Example 7. Properties of non-naturally occurring polypeptides of the present disclosure for nutritional uses

[0211] In this example, the non-naturally occurring polypeptide of SEQ ID NO: 2 was evaluated for various properties relevant to nutritional use.

[0212] viscosity

[0213] The non-naturally occurring polypeptides provided herein can be evaluated for viscosity in solution. In this example, the polypeptide of SEQ ID NO: 2 was shown to be soluble at up to 43% w / w at pH 4.5 and 50% w / w at pH 6.5 using a flow sweep on a DHR-II rheometer with 40 mm parallel plates at 25°C. The polypeptide of SEQ ID NO: 2 as a spray-dried powder was found to go into solution more slowly in water at or above 50°C than in water at ambient temperature. The results are shown in Figures 7A and 7B.

[0214] The interaction of the polypeptide of SEQ ID NO: 2 with hydrocolloids and oils was also evaluated. Blends of the polypeptide of SEQ ID NO: 2 with gum arabic were prepared in DI water and evaluated for viscosity at the ratios listed in Table 4. [Table 4]

[0215] Blends of the polypeptide of SEQ ID NO:2 and xanthan were prepared in DI water and evaluated for viscosity at various ratios. Figure 8 shows the results of this study. SDA represents the polypeptide of SEQ ID NO:2 spray dried at pH 6.5. SDB represents the polypeptide of SEQ ID NO:2 spray dried at 20% solids at pH 4.5.

[0216] Gel Hardness

[0217] The non-naturally occurring polypeptides of the present disclosure can be evaluated for gel hardness in solution. Briefly, a 5% protein solution (containing the polypeptide of SEQ ID NO: 2) was cross-linked with 100 μl of transglutaminase enzyme. The protein and enzyme mixture was deposited into a 12-well cell plate and incubated at 50°C for 2 hours. The gel was heated to 100°C for 10 minutes to inactivate the transglutaminase enzyme. The gel was cooled to ambient temperature and stored at 4°C overnight. Gel hardness was evaluated by casting 4 mL of the gel mixture into a 23 mm diameter well. Gels were allowed to return to ambient temperature before measurement, and gel firmness was recorded using a TA.XT Plus Texture Analyzer instrument as the force required to press a ½” stainless steel ball probe (TA-18) 2 mm down into the gel at 1 mm / sec. Protein solutions were prepared in DI water or 10 mM sodium phosphate buffer, pH 7.2. Solutions were adjusted to the target pH using 1 M HCl or 2 N NaOH before adding the enzyme. The results of this study are shown in Figure 9.

[0218] Protein solutions were prepared in DI water or 10 mM sodium phosphate buffer, pH 7.2. Before adding the enzyme, the solutions were adjusted to the target pH using 1 M HCl or 2 N NaOH. The results are shown in Figures 10A and 10B. At pH 5.5, the hardness of the cross-linked GL21 gel is 27-35 g. At pH 6.3-6.4, the hardness of the cross-linked polypeptide gel is 20-31 g.

[0219] Emulsion Properties

[0220] The non-naturally occurring polypeptides described herein can be evaluated for emulsion properties in solution. The protein solution at pH 4.5 was mixed with canola oil in a 5:1 ratio and homogenized using an IKA Ultra Turrax at 10,000 rpm for 10 minutes. After 24 hours, the emulsion stability was evaluated in 12 mL conical tubes at ambient temperature.

[0221] Foaming and foam stability

[0222] The non-naturally occurring polypeptides described herein can be evaluated for foaming properties in solution. 10 mL of 5% w / w polypeptide solutions of various lots of SEQ ID NO: 2 were shaken in 50 mL conical tubes for 2 minutes. The bubble volume and bubble collapse time were recorded, and the results are shown in Table 5. [Table 5]

[0223] Sensory Considerations

[0224] The non-naturally occurring polypeptides of the present disclosure can be evaluated for sensory properties, including odor and flavor. Various lots of spray-dried polypeptides of SEQ ID NO: 2 were evaluated in dry or solution form, and the results are shown in Table 6. [Table 6]

[0225] solubility

[0226] The non-naturally occurring polypeptides of the present disclosure can be evaluated for solubility. The effect of agglomeration using lecithin solution on a polypeptide of SEQ ID NO: 2 in the form of a compressed powder was evaluated to increase particle size and improve solubility. 2.5 g of protein powder was added to 50 mL of water. Wettability was evaluated by observing sedimentation within 20 seconds. Dissolution was evaluated by slow stirring for 40 seconds and then stopping for 60 seconds. The results are shown in Table 7. [Table 7-1] [Table 7-2]

[0227] The viscosity of a solution of the polypeptide of SEQ ID NO: 2 in a 20% w / w solution was also measured at 25° C., and the results are shown in FIG.

[0228] An 8% w / w protein solution (containing the polypeptide of SEQ ID NO:2) at pH 5.5 and 100 μl of transglutaminase enzyme were homogenized with 50% w / w oil on ice using a Polytron at 26,000 rpm for 1 minute. 4 g of the mixture was deposited into each well of a 12-well cell plate and incubated at 50°C for 2 hours. The gel was heated to 100°C for 10 minutes to inactivate the enzyme. The gel was cooled to ambient temperature and stored at 4°C overnight. The polypeptide of SEQ ID NO:2 stabilized the high-oil emulsion during the cross-linking reaction to form a protein-oil gel. The results are shown in the figure.

[0229] Example 8. Confirmation of the polypeptide sequence of the product and absence of hydroxyproline residues

[0230] Mass spectrometry was used to confirm the sequence of the polypeptide of SEQ ID NO: 2 produced by the methods of the present disclosure. Tables 8 and 9 provide the results of peptide mapping of this polypeptide. [Table 8] [Table 9]

[0231] Analyses were also performed to evaluate any amino acid or peptide modifications present in the constructed polypeptide of SEQ ID NO: 2 (Table 10). In a few cases, further confirmatory analyses were performed to differentiate between the presence of hydroxyproline residues and methionine oxidation. For example, based on fragmentation results from MS / MS scans, tryptic peptide T1 (sequence DTGFPGMPGR) was shown to contain methionine oxidation rather than proline hydroxylation. Based on these results, tryptic peptide 1 (T1) was conclusively determined to have oxidation at methionine position 7, with no evidence of hydroxyproline at positions 5 or 8. Similarly, despite the presence of an additional methionine at position 83 in tryptic peptide 9 (T9), there were no detectable levels of methionine oxidation, hydroxyproline at positions 77, 85, 92, 95, and 97, or hydroxylysine at position 98 of the polypeptide. Thus, the truncated collagen polypeptides of the present disclosure also differ from naturally occurring collagen polypeptides by lacking hydroxyproline residues. [Table 10]

[0232] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the embodiments of the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. In certain embodiments, for example, the following items are provided: (Item 1) A non-naturally occurring polypeptide comprising an amino acid sequence having (i) at least 80% sequence identity to SEQ ID NO: 31, together with an N-terminal truncation, a C-terminal truncation, or both; or (ii) at least 80% sequence identity to SEQ ID NO: 32, together with an N-terminal truncation, a C-terminal truncation, or both. (Item 2) 2. The non-naturally occurring polypeptide of claim 1, comprising an amino acid sequence having (i) at least 85% sequence identity to SEQ ID NO: 31, together with an N-terminal truncation, a C-terminal truncation, or both; or (ii) at least 85% sequence identity to SEQ ID NO: 32, together with an N-terminal truncation, a C-terminal truncation, or both. (Item 3) 3. The non-naturally occurring polypeptide of item 1 or 2, comprising an amino acid sequence having (i) at least 90% sequence identity to SEQ ID NO: 31, together with an N-terminal truncation, a C-terminal truncation, or both; or (ii) at least 90% sequence identity to SEQ ID NO: 32, together with an N-terminal truncation, a C-terminal truncation, or both. (Item 4) 4. The non-naturally occurring polypeptide of any one of items 1 to 3, comprising an amino acid sequence having (i) at least 95% sequence identity to SEQ ID NO: 31, together with an N-terminal truncation, a C-terminal truncation, or both; or (ii) at least 95% sequence identity to SEQ ID NO: 32, together with an N-terminal truncation, a C-terminal truncation, or both. (Item 5) 5. The non-naturally occurring polypeptide of any one of items 1 to 4, comprising an amino acid sequence having (i) at least 98% sequence identity to SEQ ID NO: 31, together with an N-terminal truncation, a C-terminal truncation, or both; or (ii) at least 98% sequence identity to SEQ ID NO: 32, together with an N-terminal truncation, a C-terminal truncation, or both. (Item 6) 6. The non-naturally occurring polypeptide of any one of items 1 to 5, comprising: (i) the amino acid sequence of SEQ ID NO: 31 with an N-terminal truncation, a C-terminal truncation, or both; or (ii) the amino acid sequence of SEQ ID NO: 32 with an N-terminal truncation, a C-terminal truncation, or both. (Item 7) 7. The non-naturally occurring polypeptide according to any one of items 1 to 6, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31 together with an N-terminal truncated portion. (Item 8) 8. The non-naturally occurring polypeptide according to item 7, wherein the N-terminal truncated portion is an N-terminal truncated portion of 50 to 600 amino acids. (Item 9) 7. The non-naturally occurring polypeptide according to any one of items 1 to 6, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31 together with a C-terminal truncated portion. (Item 10) 10. The non-naturally occurring polypeptide according to item 9, wherein the C-terminal truncated portion is a C-terminal truncated portion of 50 to 250 amino acids. (Item 11) 7. The non-naturally occurring polypeptide of any one of items 1 to 6, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31, together with both an N-terminal truncation and a C-terminal truncation. (Item 12) Item 12. The non-naturally occurring polypeptide according to Item 11, wherein the N-terminal truncated portion is an N-terminal truncated portion of 50 to 600 amino acids and the C-terminal truncated portion is a C-terminal truncated portion of 50 to 250 amino acids. (Item 13) 13. The non-naturally occurring polypeptide according to any one of items 1 to 12, comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. (Item 14) 13. The non-naturally occurring polypeptide according to any one of items 1 to 12, consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. (Item 15) 7. The non-naturally occurring polypeptide according to any one of items 1 to 6, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32 together with an N-terminal truncated portion. (Item 16) 16. The non-naturally occurring polypeptide according to item 15, wherein the N-terminal truncated portion is an N-terminal truncated portion of 50 to 750 amino acids. (Item 17) 7. The non-naturally occurring polypeptide according to any one of items 1 to 6, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32 together with a C-terminal truncated portion. (Item 18) 18. The non-naturally occurring polypeptide according to item 17, wherein the C-terminal truncated portion is a C-terminal truncated portion of 50 to 250 amino acids. (Item 19) 7. The non-naturally occurring polypeptide of any one of items 1 to 6, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 32, together with both an N-terminal truncation and a C-terminal truncation. (Item 20) 20. The non-naturally occurring polypeptide according to Item 19, wherein the N-terminal truncated portion is an N-terminal truncated portion of 50 to 750 amino acids and the C-terminal truncated portion is a C-terminal truncated portion of 50 to 250 amino acids. (Item 21) 21. The non-naturally occurring polypeptide according to any one of items 1 to 6 or 15 to 20, comprising the amino acid sequence of SEQ ID NO:8. (Item 22) 21. The non-naturally occurring polypeptide according to any one of items 1 to 6 or 15 to 20, consisting of the amino acid sequence of SEQ ID NO: 8. (Item 23) 23. The non-naturally occurring polypeptide of any one of items 1 to 22, wherein the non-naturally occurring polypeptide has a total truncation of between 50 amino acids and 900 amino acids. (Item 24) 24. The non-naturally occurring polypeptide according to any one of items 1 to 23, wherein the non-naturally occurring polypeptide is 50 to 250 amino acids in length. (Item 25) 25. The non-naturally occurring polypeptide of any one of items 1 to 24, wherein the non-naturally occurring polypeptide does not include one or more of a laminin G domain, a von Willebrand factor type A (vWA) domain, and a fibrillar collagen C-terminal domain. (Item 26) 26. The non-naturally occurring polypeptide of any one of items 1 to 25, wherein the non-naturally occurring polypeptide comprises one or more collagen triple helix repeats. (Item 27) 27. The non-naturally occurring polypeptide according to any one of items 1 to 26, wherein the non-naturally occurring polypeptide is a monomer. (Item 28) 28. The non-naturally occurring polypeptide according to any one of items 1 to 27, wherein the non-naturally occurring polypeptide does not form a stable triple helix structure of naturally occurring collagen. (Item 29) 29. The non-naturally occurring polypeptide of any one of items 1 to 28, wherein the non-naturally occurring polypeptide is substantially free of other collagen chains. (Item 30) 30. The non-naturally occurring polypeptide of any one of items 1 to 29, wherein the non-naturally occurring polypeptide has a non-naturally occurring level of hydroxylation compared to naturally occurring collagen. (Item 31) 31. The non-naturally occurring polypeptide of any one of items 1 to 30, wherein less than 10% of the prolines present in the non-naturally occurring polypeptide are hydroxylated. (Item 32) 32. The non-naturally occurring polypeptide of any one of items 1 to 31, wherein the non-naturally occurring polypeptide is not hydroxylated. (Item 33) 33. The non-naturally occurring polypeptide of any one of items 1 to 32, wherein the non-naturally occurring polypeptide has a non-naturally occurring level of glycosylation compared to naturally occurring collagen. (Item 34) 34. The non-naturally occurring polypeptide of any one of items 1 to 33, wherein the non-naturally occurring polypeptide protein contains less than 5 wt.% glycosylation. (Item 35) 35. A composition comprising 0.001% to 30% w / w of the non-naturally occurring polypeptide according to any one of items 1 to 34. (Item 36) 36. The composition of claim 35, wherein the composition is formulated for consumption by an individual. (Item 37) 37. The composition of claim 36, wherein the composition is a dietary supplement. (Item 38) 38. The composition of item 36 or 37, wherein the individual is a human. (Item 39) 39. A method for improving the appearance of a subject's skin, hair and / or nails, and / or improving the health of the bones, muscles and / or joints of said subject, said method comprising administering to said subject the composition of any one of items 35 to 38. (Item 40) 40. The method of claim 39, wherein the administering step comprises orally administering to the subject. (Item 41) 35. A recombinant cell comprising at least one copy of a heterologous nucleic acid sequence encoding the non-naturally occurring polypeptide of any one of items 1 to 34. (Item 42) 42. The recombinant cell according to item 41, wherein the recombinant cell is a microbial cell. (Item 43) 43. The recombinant cell of item 42, wherein the microbial cell is a bacterial cell. (Item 44) 44. The recombinant cell of item 43, wherein the bacterial cell is a cell of the species Escherichia coli. (Item 45) 45. The recombinant cell of any one of items 41 to 44, wherein the recombinant cell lacks an enzyme that hydroxylates one or more amino acids of the non-naturally occurring polypeptide. (Item 46) 46. ​​The recombinant cell according to any one of items 41 to 45, wherein the recombinant cell lacks prolyl 4-hydroxylase and / or prolyl 3-hydroxylase.

Claims

1. A polypeptide comprising an amino acid sequence having at least 90% sequence identity to a truncated version of SEQ ID NO:31, wherein the truncated version of SEQ ID NO:31 is truncated by 50 to 600 amino acids at the N-terminus and by 50 to 250 amino acids at the C-terminus compared to full-length SEQ ID NO:31, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2, and wherein the polypeptide increases type I collagen production.

2. 2. The polypeptide of claim 1, comprising an amino acid sequence having at least 95% sequence identity to a truncation of SEQ ID NO:

31.

3. 3. The polypeptide of claim 1 or 2, comprising an amino acid sequence having at least 98% sequence identity to a truncation of SEQ ID NO:

31.

4. The polypeptide according to any one of claims 1 to 3, comprising the amino acid sequence of a truncation of SEQ ID NO:

31.

5. The polypeptide according to any one of claims 1 to 4, comprising the amino acid sequence of SEQ ID NO:

6.

6. The polypeptide according to any one of claims 1 to 5, consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

6.

7. The polypeptide of any one of claims 1 to 6, wherein the polypeptide has a total truncation of between 100 and 850 amino acids.

8. The polypeptide of any one of claims 1 to 7, wherein the polypeptide does not contain one or more of a laminin G domain, a von Willebrand factor type A (vWA) domain, and a fibrillar collagen C-terminal domain.

9. The polypeptide of any one of claims 1 to 8, wherein the polypeptide comprises one or more collagen triple helix repeats.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide is a monomer.

11. The polypeptide of any one of claims 1 to 10, wherein the polypeptide does not form the stable triple helix structure of naturally occurring collagen.

12. The polypeptide according to any one of claims 1 to 11, wherein the polypeptide does not contain other collagen chains.

13. 13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide has a reduced level of hydroxylation compared to naturally occurring collagen.

14. 14. The polypeptide of any one of claims 1 to 13, wherein less than 10% of the prolines present in the polypeptide are hydroxylated.

15. The polypeptide of any one of claims 1 to 14, wherein the polypeptide is not hydroxylated.

16. 16. The polypeptide of any one of claims 1 to 15, wherein the polypeptide has a reduced level of glycosylation compared to naturally occurring collagen.

17. 17. The polypeptide of any one of claims 1 to 16, wherein the polypeptide protein contains less than 5 wt.% glycosylation.

18. A composition comprising 0.001% to 30% w / w of a polypeptide according to any one of claims 1 to 17.

19. 20. The composition of claim 18, wherein the composition is a product formulated for oral consumption by an individual.

20. 20. The composition of claim 19, wherein the product is a dietary supplement, a food, a beverage, a medical food, a supplement, or a medicine.

21. 21. The composition of claim 19 or 20, wherein the individual is a human.

22. 20. The composition of claim 18, wherein the composition is a personal care product.

23. 23. The composition of claim 22, wherein the personal care product is formulated for topical application to the skin, hair and / or nails of an individual.

24. 24. The composition of claim 22 or 23, wherein the personal care product is a cosmetic.

25. 25. The composition of any one of claims 22 to 24, wherein the personal care product is selected from the group consisting of skin cleansers, cleansing creams, cleansing lotions, facial cleansers, cleansing emulsions, cleansing pads, facial cleansers, face creams, face moisturizers, body creams, body moisturizers, facial serums, face masks, body masks, facial toners, facial mists, eye creams, eye treatments, exfoliating formulas, lip balms, lipsticks, hair shampoos, hair conditioners, body shampoos, hair serums, scalp serums, hair mists, hair sprays, eye shadows, concealers, mascaras, and color cosmetics.

26. 26. The composition of any one of claims 18 to 25, for improving the appearance of the skin, hair and / or nails of a subject, and / or improving the bone, muscle and / or joint health of said subject, and / or improving the gut health of a subject, and / or altering and / or reducing inflammation or tissue repair in a subject, and / or altering or improving the microbiome of a subject.

27. A recombinant cell comprising at least one copy of a heterologous nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 17.

28. 28. The recombinant cell of claim 27, wherein the recombinant cell is a microbial cell.

29. 29. The recombinant cell of claim 28, wherein the microbial cell is a bacterial cell.

30. 30. The recombinant cell of claim 29, wherein the bacterial cell is an Escherichia coli (Escherichia coli) cell.

31. 31. The recombinant cell of any one of claims 27 to 30, wherein the recombinant cell lacks an enzyme that hydroxylates one or more amino acids of the polypeptide.

32. 32. The recombinant cell of any one of claims 27 to 31, wherein the recombinant cell is deficient in prolyl 4-hydroxylase and / or prolyl 3-hydroxylase.

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