Recombinant silk polypeptides and compositions made thereof

Abstract

Disclosed herein are recombinant silk / silk-like polypeptides and compositions made thereof.

Description

RECOMBINANT SILK POLYPEPTIDES AND COMPOSITIONS MADE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 607,785, filed December 8, 2023, the entire disclosure of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 5, 2024, is named 59844_SeqListing.xml and is 9,104 bytes in size.FIELD

[0003] The present invention relates to recombinant silk / silk-like polypeptides and compositions made thereof such as, for example, cosmetic and personal care compositions.BACKGROUND

[0004] Silk is a structural protein that has many qualities that make it desirable for use in applications such as skincare and cosmetics. Recent technology has resulted in the scalable production of various recombinant spider silk polypeptides and polypeptides that are derived from recombinant spider silk polypeptides using various host organisms.

[0005] Accordingly, there is a need for new recombinant silk / silk-like polypeptides providing desired properties.SUMMARY

[0006] In some embodiments, provided herein are recombinant polypeptides comprising a repeat domain comprising an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1 . In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 1 by conservative amino acid substitutions. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 1 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

[0007] In some embodiments, provided herein is a recombinant polypeptide comprising a repeat domain comprising an amino acid sequence 100% identical to SEQ ID NO: 1.

[0008] In some embodiments, provided herein are recombinant polypeptides comprising a repeat domain comprising an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 2 by conservative amino acid substitutions. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 2 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

[0009] In some embodiments, provided herein is a recombinant polypeptide comprising a repeat domain comprising an amino acid sequence 100% identical to SEQ ID NO: 2.

[0010] In some embodiments, provided herein are recombinant polypeptides comprising a repeat domain comprising an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 3 by conservative amino acid substitutions. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 3 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

[0011] In some embodiments, provided herein is a recombinant polypeptide comprising a repeat domain comprising an amino acid sequence 100% identical to SEQ ID NO: 3.

[0012] In some embodiments, provided herein are recombinant polypeptides comprising a repeat domain comprising an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 4 by conservative amino acid substitutions. In some embodiments, the amino acid sequence of the repeat domain only differs from SEQ ID NO: 4 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

[0013] In some embodiments, provided herein is a recombinant polypeptide comprising a repeat domain comprising an amino acid sequence 100% identical to SEQ ID NO: 4.

[0014] In some embodiments, the recombinant polypeptide further comprises an N- terminal non-repetitive domain and / or a C-terminal non-repetitive domain.

[0015] Also provided herein are expression constructs encoding a recombinant polypeptide described herein and host cells comprising the expression constructs. In some embodiments, the host cell is a yeast cell such as Pichia pastoris.

[0016] Further provided herein are cosmetic, skin, or hair care compositions, comprising: one or more active ingredients for cosmetic, skin, or hair care, and a recombinant polypeptide described herein.

[0017] In some embodiments, provided herein are cosmetic, skin, or hair care compositions having a silicone replacement, comprising: one or more active ingredients for cosmetic, skin, or hair care, and a silicone replacement component comprising a recombinant polypeptide described herein, wherein the composition is substantially free of silicone.DETAILED DESCRIPTION

[0018] The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the aspects.Definitions

[0019] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0020] The term “stability”, as used herein with respect to silk proteins, refers to the ability of the product not to form a gelation, discoloration or turbidity that is due to the self-aggregation of silk proteins. For example, U.S. Patent Publication No. 2015 / 0079012 (Wray et al.) is directed to the use of humectant, including glycerol to increase the shelf-stability of skincare products comprising full-length silk fibroin. U.S. Patent No. 9,187,538 is directed to a skincare formulation comprising full-length silk fibroin that is shelf stable for up to 10 days. Both of these publications are incorporated herein by reference in their entirety.

[0021] The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.

[0022] An “isolated” RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.

[0023] An “isolated” organic molecule e.g., a silk protein) is one which is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured. The term does not require that the biomolecule has been separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.

[0024] The term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1 ) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0025] An endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it.

[0026] A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0027] The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

[0028] The term “polypeptide” encompasses both naturally occurring and non-naturally occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

[0029] The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.

[0030] The term “polypeptide fragment” refers to a polypeptide that has a deletion, e.g., an amino-terminal and / or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

[0031] A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.) As used herein,homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0032] When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties {e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365- 89 (herein incorporated by reference).

[0033] The twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nded. 1991), which is incorporated herein by reference. Stereoisomers e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, E-N,N,N- trimethyllysine, c-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxyterminal end, in accordance with standard usage and convention.

[0034] The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0035] Sequence homology for polypeptides, which is sometimes also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned tovarious substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1 .

[0036] A useful algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul etal., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0037] Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

[0038] Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62. The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art.

[0039] Polypeptide sequences can also be compared using FASTA, a program in GCG Version 6.1 . FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63- 98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1 , herein incorporated by reference.

[0040] Throughout this specification and aspects, the word “comprise” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0041] The term “glass transition” as used herein refers to the transition of a substance or composition from a hard, rigid or “glassy” state into a more pliable, “rubbery” or “viscous” state.

[0042] The term “glass transition temperature” as used herein refers to the temperature at which a substance or composition undergoes a glass transition.

[0043] The term “melt transition” as used herein refers to the transition of a substance or composition from a rubbery state to a less-ordered liquid phase.

[0044] The term “melting temperature” as used herein refers to the temperature range over which a substance undergoes a melt transition.

[0045] The term “plasticizer” as used herein refers to any molecule that interacts with a polypeptide sequence to prevent the polypeptide sequence from forming tertiary structures and bonds and / or increases the mobility of the polypeptide sequence.

[0046] The term “powder” as used herein refers to a composition that is present in granular form, which may or may not be complexed or agglomerated with a solvent such as water or serum. The term “dry powder” may be used interchangeably with the term “powder;” however, “dry powder” as used herein simply refers to the gross appearance of the granulated material and is not intended to mean that the material is completely free of complexed or agglomerated solvent unless otherwise indicated. Dry powder may be produced by spray-drying, lyophilization, and / or according to methods known in the art.

[0047] The term “carrier” refers to a recombinant protein used for surface hydration, surface cleansing, surface defense, surface detoxification, surface exfoliation, surface improvement, coloring, and / or delivery of various additives or solvents, including, but not limited to, water, glycerin, alcohols, siloxane, oils, humectants, emollients, occlusive agents, active agents, and / or cosmetic adjuvants to a surface like skin, hair, or nails. The carrier as used herein comprises an outer shell and hollow core.

[0048] The term “cosmetics” as used herein includes make-up, foundation, skin care, hair care, and nail care products.

[0049] The term “make-up” as used herein refers to products that leave color on the face, including foundation, blacks and browns, i.e., mascara, concealers, eye liners, brow colors, eye shadows, blushers, lip colors, powders, solid emulsion compact, and so forth.

[0050] The term “foundation” as used herein refers to liquid, cream, mousse, pancake, compact, concealer or like product created or reintroduced by cosmetic companies to even out the overall coloring of the skin.

[0051] The term “skin care products” as used herein refer to those used to treat or care for, or somehow moisturize, improve, or clean the skin. Products contemplated by the phrase “skin care products” include, but are not limited to, creams, mists, serums, cleansing gels, ampules, adhesives, patches, bandages, toothpaste, anhydrous occlusive moisturizers, antiperspirants, deodorants, personal cleansing products, powder laundry detergent, fabric softener towels, occlusive drug delivery patches, nail polish, powders, tissues, wipes, hair conditioners-anhydrous, shaving creams, and the like.

[0052] The term “sagging” as used herein means the laxity, slackness, or the like condition of skin that occurs as a result of loss of, damage to, alterations to, and / or abnormalities in dermal elastin, muscle and / or subcutaneous fat.

[0053] The terms “treating” or “treatment” as used herein refer to the treatment (e.g., alleviation or elimination of symptoms and / or cure) and / or prevention or inhibition of the condition e.g., a skin condition) or relief of symptoms.

[0054] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.Recombinant Silk Polypeptides

[0055] The invention provides recombinant silk or silk-like polypeptides.

[0056] Silk polypeptides are characteristically composed of a repeat domain (REP) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In some embodiments, both the C-terminal and N-terminal domains may be between 75-350 amino acids in length. The repeat domain exhibits a hierarchical architecture. The repeat domain comprises a series of blocks (also called repeat units). The blocks are repeated, sometimes perfectly and sometimes imperfectly (making up a quasi-repeat domain), throughout the silk repeat domain. The length and composition of blocks varies among different silk types and across different species. In some cases, blocks may be arranged in a regular pattern, forming larger macro-repeats that appear multiple times (usually 2-8) in the repeat domain of the silk sequence. Repeated blocks inside a repeat domain or macro-repeat, and repeated macro-repeats within the repeat domain, may be separated by spacing elements. In some embodiments, block sequences comprise a glycine rich region followed by a polyA region. In some embodiments, short (-1-10) amino acid motifs appear multiple times inside of blocks.

[0057] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or 100% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1 .

[0058] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1 , where the amino acid sequence only differs from SEQ ID NO: 1 by conservative amino acid substitutions.

[0059] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1 , where the amino acid sequence only differs from SEQ ID NO: 1 by substitutions of amino acid residues in SEQ ID NO: 1 with serine (S), glycine (G), glutamine (Q), and / or alanine (A).

[0060] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or 100% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.

[0061] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2, where the amino acid sequence only differs from SEQ ID NO: 2 by conservative amino acid substitutions.

[0062] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2, where the amino acid sequence only differs from SEQ ID NO: 2 by substitutions of amino acid residues in SEQ ID NO: 2 with serine (S), glycine (G), glutamine (Q), and / or alanine (A).

[0063] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or 100% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.

[0064] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3, where the amino acid sequence only differs from SEQ ID NO: 3 by conservative amino acid substitutions.

[0065] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3, where the amino acid sequence only differs from SEQ ID NO: 3 by substitutions of amino acid residues in SEQ ID NO: 3 with serine (S), glycine (G), glutamine (Q), and / or alanine (A).

[0066] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or 100% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.

[0067] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4, where the amino acid sequence only differs from SEQ ID NO: 4 by conservative amino acid substitutions.

[0068] In some embodiments, the recombinant polypeptide comprises or consists of a repeat domain having an amino acid sequence at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4, where the amino acid sequence only differs from SEQ ID NO: 4 by substitutions of amino acid residues in SEQ ID NO: 4 with serine (S), glycine (G), glutamine (Q), and / or alanine (A).

[0069] The amino acid sequences of SEQ ID NOS: 1-4 are shown in the table below.

[0070] In some embodiments, the recombinant polypeptide comprises an alanine composition of 12-40% of the amino acid sequence of the recombinant polypeptide, a glycine composition of 25-50% of the amino acid sequence of the co-polymer, a proline composition of 9-20%> of the amino acid sequence of the co-polymer, a p-turn composition of 15-37% of the amino acid sequence of the copolymer, a GPG amino acid motif content of 18-55% of the amino acid sequence of the copolymer, and a polyalanine amino acid motif content of 9-35% of all amino acids of the recombinant polypeptide.

[0071] In some embodiments, the recombinant polypeptide also includes an N-terminal non- repetitive domain between 75-350 amino acids in length, and a C-terminal non-repetitive domain between 75-350 amino acids in length. In some embodiments, the quasi-repeat domain is 500-5000, 119-1575, or 900-950 amino acids in length. In other embodiments, the mass of the recombinant polypeptide is 40-400, 12.2-132, or 70-100 kDa. In some embodiments, the alanine composition is 16-31% or 15-20% of the amino acid sequence of the recombinant polypeptide . In other embodiments, the glycine composition is 29-43%) or 38-43%) of the amino acid sequence of the recombinant polypeptide. In some embodiments, the proline composition is 11-16% or 13-15% of the amino acid sequence of the recombinant polypeptide. In other embodiments, the p-turn composition is 18- 33% or 25-30% of the amino acid sequence of the recombinant polypeptide . In some embodiments, the GPG amino acid motif content is 22-47% or 30-45% of the amino acid sequence of the copolymer. In other embodiments, the poly alanine amino acid motif content is 12-29% of the amino acid sequence of the recombinant polypeptide .

[0072] In some embodiments, the recombinant polypeptide comprises 0 or more N-terminal domains (NTD), 1 or more repeat domains (REP), and 0 or more C-terminal domains (CTD). In some aspects of the embodiment, the recombinant polypeptide is >100 amino acids of a single polypeptide chain.

[0073] In some embodiments, non-repetitive N- and C-terminal domains can be selected for synthesis. In some embodiments, N-terminal domains can be by removal of the leading signal sequence, e.g., as identified by SignalP (Peterson, T.N., et. Al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785- 786 (2011).

[0074] In some embodiments, the N-terminal domain and / or C-terminal domain sequences can be derived from Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope bruennichi, Argiope trifasciata, Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis Spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis, Latrodectus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0075] In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to a 3X FLAG nucleotide coding sequence (e.g., coding for 3 copies of FLAG tag: GDYKDDDDKDYKDDDDKDYKDDDDK (SEQ ID NO: 5)). In some embodiments, the silk polypeptide nucleotide coding sequence is operatively linked to other affinity tags such as 6- 8 His residues.

[0076] In some embodiments, the molecular weight of the recombinant polypeptide may range from 20 kDa to 2000 kDa, or greater than 20 kDa, or greater than 10 kDa, or greater than 5 kDa, or from 5 to 400 kDa, or from 5 to 300 kDa, or from 5 to 200 kDa, or from 5 to 100 kDa, or from 5 to 50 kDa, or from 5 to 500 kDa, or from 5 to 1000 kDa, or from 5 to 2000 kDa, or from 10 to 400 kDa, or from 10 to 300 kDa, or from 10 to 200 kDa, or from 10 to 100 kDa, or from 10 to 50 kDa, or from 10 to 500 kDa, or from 10 to 1000 kDa, or from 10 to 2000 kDa, or from 20 to 400 kDa, or from 20 to 300 kDa, or from 20 to 200 kDa, or from 40 to 300 kDa, or from 40 to 500 kDa, or from 20 to 100 kDa, or from 20 to 50 kDa, or from 20 to 500 kDa, or from 20 to 1000 kDa, or from 20 to 2000 kDa.Characterization of Recombinant Spider Silk Polypeptide Powder Impurities and Degradation

[0077] Different recombinant spider silk polypeptides have different physiochemical properties such as melting temperature and glass transition temperature based on the strength and stability of the secondary and tertiary structures formed by the proteins. Silk polypeptides form beta sheet structures in a monomeric form. In the presence of other monomers, the silk polypeptides form a three-dimensional crystalline lattice of beta sheet structures. The beta sheet structures are separated from, and interspersed with, amorphous regions of polypeptide sequences.

[0078] Beta sheet structures are extremely stable at high temperatures - the melting temperature of beta-sheets is approximately 257°C as measured by fast scanning calorimetry. See Cebe et al., Beating the Heat - Fast Scanning Melts Silk Beta Sheet Crystals, Nature Scientific Reports 3:1130 (2013). As beta sheet structures are thought to stay intact above the glass transition temperature of silk polypeptides, it has been postulated that the structural transitions seen at the glass transition temperature of recombinant silk polypeptides are due to increased mobility of the amorphous regions between the beta sheets.

[0079] Plasticizers lower the glass transition temperature and the melting temperature of silk proteins by increasing the mobility of the amorphous regions and potentially disrupting beta sheet formation. Suitable plasticizers used for this purpose include, but are not limited to, water and polyalcohols (polyols) such as glycerol, triglycerol, hexaglycerol, and decaglycerol. Other suitable plasticizers include, but are not limited to, Dimethyl Isosorbite; adiptic acid; amide of dimethylaminopropyl amine and caprylic / capric acid; acetamide; and any combination thereof.

[0080] As hydrophilic portions of silk polypeptides can bind ambient water present in the air as humidity, water will almost always be present, the bound ambient water may plasticize silk polypeptides. In some embodiments, a suitable plasticizer may be glycerol, present either alone or in combination with water or other plasticizers. Other suitable plasticizers are discussed above.

[0081] In addition, in instances where recombinant silk polypeptides are produced by fermentation and recovered as recombinant silk polypeptide powder from the same, there may be impurities present in the recombinant silk polypeptide powder that act as plasticizers or otherwise inhibit the formation of tertiary structures. For example, residual lipids and sugars may act as plasticizers and thus influence the glass transition temperature of the protein by interfering with the formation of tertiary structures.

[0082] Various well-established methods may be used to assess the purity and relative composition of recombinant silk polypeptide powder or composition. Size Exclusion Chromatography separates molecules based on their relative size and can be used to analyze the relative amounts of recombinant silk polypeptide in its full-length polymeric and monomeric forms as well as the amount of high, low and intermediate molecular weight impurities in the recombinant silk polypeptide powder. Similarly, Rapid High Performance Liquid Chromatography may be used to measure various compounds present in a solution such as monomeric forms of the recombinant silk polypeptide. Ion Exchange Liquid Chromatography may be used to assess the concentrations of various trace molecules in solution, including impurities such as lipids and sugars. Other methods of chromatography and quantification of various molecules such as mass spectrometry are well established in the art.

[0083] Depending on the embodiment, the recombinant silk polypeptide may have a purity calculated based on the amount of the recombinant silk polypeptide in its monomeric form by weight relative to the other components of the recombinant silk polypeptide powder. In various instances, the purity can range from 50% by weight to 90% by weight, depending onthe type of recombinant silk polypeptide and the techniques used to recover, separate and post-process the recombinant silk polypeptide powder.

[0084] Both Size Exclusion Chromatography and Reverse Phase High Performance Liquid Chromatography are useful in measuring full-length recombinant silk polypeptide, which makes them useful techniques for determining whether processing steps have degraded the recombinant silk polypeptide by comparing the amount of full-length silk polypeptide in a composition before and after processing. In various embodiments of the present invention, the amount of full-length recombinant silk polypeptide present in a composition before and after processing may be subject to minimal degradation. The amount of degradation may be in the range 0.001 % by weight to 10% by weight, or 0.01 % by weight to 6% by weight, e.g. less than 10% or 8% or 6% by weight, or less than 5% by weight, less than 3% by weight or less than 1% by weight.Expression Vectors and Host Cells

[0085] The expression vectors of the present invention can be produced following the teachings of the present specification in view of techniques known in the art.

[0086] Expression constructs and host cells described in U.S. Patent Nos. 11 ,306,127, 11 ,634,729, 10,906,947, and 11 ,214,785 (all of which are hereby incorporated by reference herein in their entireties) may be used in combination with nucleic acids encoding the recombinant polypeptides described herein in order to produce the polypeptides.

[0087] In some embodiments of the present invention, host cells transformed with nucleic acid molecules encoding the recombinant polypeptides are provided. In some embodiments of the present invention, these cells carry the nucleic acid sequences of the present invention on vectors, which may but need not be freely replicating vectors. In other embodiments of the present invention, the nucleic acids have been integrated into the genome of the host cells.

[0088] A variety of host organisms can be engineered / transformed to comprise a recombinant polypeptide expression system. Preferred organisms for expression of a recombinant silk polypeptide include yeast, fungi, and gram-positive bacteria. In certain embodiments, the host organism is Arxula adeninivorans, Aspergillus aculeatus, Aspergillus awamori, Aspergillus ficuum, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus tubigensis, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus methanolicus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Candida boidinii, Chrysosporium lucknowense, Fusarium graminearum , Fusarium venenatum,Kluyveromyces lactis, Kluyveromyces marxianus, Myceliopthora thermophila , Neurospora crassa, Ogataea polymorpha, Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purpurogenum, Penicillium roqueforti, Phanerochaete chrysosporium, Pichia angusta, Pichia methanolica, Pichia (Komagataella) pastoris, Pichia polymorpha, Pichia stipitis, Rhizomucor miehei, Rhizomucor pusillus, Rhizopus arrhizus, Streptomyces lividans, Saccharomyces cerevisiae, Schwanniomyces occidentalis, Trichoderma harzianum, Trichoderma reesei, or Yarrowia lipolytica.

[0089] In preferred aspects, the methods provide culturing host cells for direct product secretion for easy recovery without the need to extract biomass. In some embodiments, the recombinant polypeptides are secreted directly into the medium for collection and processing.Polypeptide purification

[0090] The recombinant polypeptides produced by gene expression in a recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. For example, purification / isolation methods described in U.S. Patent Application Publication No. 2022 / 0017580, 2022 / 0289790, and 2022 / 0372086 (all of which are hereby incorporated by reference herein in their entireties) may be used in combination with the recombinant polypeptides described herein.

[0091] In an embodiment, a commercially available expression / secretion system can be used, whereby the recombinant polypeptide is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression / secretion vectors are not used, an alternative approach involves purifying the recombinant block copolymer polypeptide from cell lysates (remains of cells following disruption of cellular integrity) derived from prokaryotic or eukaryotic cells in which a polypeptide was expressed. Methods for generation of such cell lysates are known to those of skill in the art. In some embodiments, recombinant block copolymer polypeptides are isolated from cell culture supernatant.

[0092] The recombinant polypeptides may be purified by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant polypeptide or nickel columns for isolation of recombinant polypeptides tagged with 6-8 histidine residues at their N-terminus or C-terminus. Alternative tags may comprise the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.

[0093] Additionally, the method of the present invention may preferably include a purification method, comprising exposing the cell culture supernatant containing expressed block copolymer polypeptides to ammonium sulphate of 5-60% saturation, preferably 10- 40% saturation.Silicone replacement Component

[0094] In various embodiments, the recombinant silk polypeptide may be used as a silicone replacement within a personal care and / or cosmetic composition for providing benefits to cosmetics, skin and hair that would normally be associated with linear silicones and silicone elastomers.

[0095] The silicone replacement component includes the recombinant silk polypeptide. The silicone replacement component can consist of the recombinant silk polypeptide. The silicone replacement component can include the recombinant silk polypeptide with a solvent and / or one or more additives, such as preservatives and chelating agents. The silicone replacement component can include the recombinant silk polypeptide in an amount, based on the total weight of the silicone replacement component, of about 1 wt% to about 40 wt% or in any other suitable amount needed to achieve a final desired loading the recombinant silk polypeptide in the cosmetic, skin or hair care composition.

[0096] Without intending to be limited by theory, in various embodiments of the present invention, inducing the silicone replacement Component may be used in applications where it is desirable to prevent the aggregation of the monomeric recombinant silk polypeptide into its crystalline polymeric form or to control the transition of the recombinant silk polypeptide into its crystalline polymeric form at a later stage in processing. In other embodiments, such inducing is not required.

[0097] In one specific embodiment, the silicon elastomer replacement component may be used to prevent aggregation of the recombinant silk polypeptide prior to blending the recombinant silk polypeptide with a second polymer. In another specific embodiment, the silicon elastomer replacement component may be used to create a base for a cosmetic or skincare product where the recombinant silk polypeptide is present in the base in its monomeric form. In this embodiment, having the recombinant silk polypeptide in its monomeric form in a base allows for the controlled aggregation of the monomer into its crystalline polymeric form upon contact with skin or through various other chemical reactions.

[0098] In various embodiments, the temperature to which the silicon elastomer replacement component having the recombinant silk polypeptide is heated will be minimized in order to minimize or entirely prevent degradation of the recombinant silk polypeptide. In specificembodiments, the recombinant silk melt will be heated to a temperature of less than 120°C, less than 100°C, less than 80°C, less than 60°C, less than 40°C, or less than 20°C. Often the melt will be at a temperature in the range 10°C to 120°C, 10°C to 100°C, 15°C to 80°C, 15°C to 60°C, 18°C to 40°C or 18°C to 22°C during processing. In other embodiments, the silicon elastomer replacement component is not heated. In such embodiments, the presence of heat is not required to form a silicon elastomer replacement component.

[0099] The amount of degradation of the recombinant silk polypeptide may be measured using various techniques. As discussed above, the amount of degradation of the recombinant silk polypeptide may be measured using Size Exclusion Chromatography to measure the amount of full-length recombinant silk polypeptide present. In various embodiments, the recombinant silk polypeptide is degraded in an amount of less than 6.0 weight % after it is formed into a molded body. In another embodiment, the recombinant silk polypeptide is degraded in an amount of less than 4.0 weight % after molding, less than 3.0 weight %, less than 2.0 weight %, or less than 1 .0 weight %, such that the amount of degradation may be in the range 0.001% by weight to 10%, 8%, 6%, 4%, 3%, 2% or 1% by weight, or 0.01% by weight to 6%, 4%, 3%, 2% or 1% by weight. In another embodiment, the recombinant silk protein in the composition is substantially non-degraded. In a similar embodiment, the recombinant silk protein in the composition is substantially non-degraded over a period of time, at least 1 day, 1 month, 1 year, or 5 years.

[0100] In some embodiments, the silicone replacement component is physically stable. In various embodiments, the component remains in its material form, e.g., a powder, for a prolonged period of time, with a prolonged shelf life. On prolonged use, the silicone replacement component remains substantially stable. In some embodiments, the silicone replacement component has stability substantially the same as the stability of a silicone and / or silicone elastomer.

[0101] In some embodiments, the silicone replacement component has material properties substantially similar to the material properties of a silicone and / or silicone elastomer. In various embodiments, the silicone replacement component has substantially similar rheology as a silicone and / or silicone elastomer and / or imparts to a composition of the disclosure similar rheology as inclusion of a silicone and / or silicone elastomer.

[0100] In most embodiments of the present invention, the silicone replacement component is in a powder form. The silicone replacement component can include the recombinant silk polypeptide in a powder. In some embodiments, the silicone replacement component is spray-dried. In other embodiments, the silicone replacement component is freeze-dried or vacuum-dried. The terms "spray-drying" and "spray-dried" are used herein for simplicity butthe skilled person will appreciate that freeze-drying or lyophilization and vacuum drying can be substituted for spray-drying as appropriate. These silicone replacement components may be stored dry.

[0101] In some embodiments, the recombinant silk protein is more stable in a dried form than in an aqueous slurry. In some embodiments, spray-dried recombinant silk is obtained as follows: a slurry composition comprising extracted recombinant silk is kept chilled during the drying step. It is pumped to a tall form spray dryer where the moisture content of the resulting powder is tightly controlled. As the protein powder is hydroscopic, the final powder collection and packout is performed to minimize reintroduction of moisture. The design of the packaging material should minimize moisture and light exposure.

[0102] In some embodiments, recovery and separation of the recombinant silk polypeptide from a cell culture is performed as follows: i) extraction and separation, ii) urea removal by ultrafiltration, ill) washing by precipitation, iv) salt removal and protein concentration, and v) spray drying.

[0103] In some embodiments, to freeze-dry a composition it is cooled until it solidifies and placed under reduced pressure to cause the most volatile ingredients in the composition to sublime. The solid residue may form a single mass which requires milling to form a fine powder. A typical freeze-dried powder comprises porous irregular shaped particles and readily hydrates. As freeze-drying does not require strong heat it is used to produce powders which comprise volatile ingredients. In some embodiments, the silicone replacement component is deep freeze-dried at a temperature below about -100°C.

[0104] After formation of the silicone replacement component, the crystallinity of the silicone replacement component can increase, thereby strengthening the composition. In some embodiments, the silicone replacement component stays the same or decreases. In some embodiments, the crystallinity index of the silicone replacement component as measured by X-ray crystallography is from 2% to 90%. In some other embodiments, the crystallinity index of the silicone replacement component as measured by X-ray crystallography is at least 3%, at least 4%, at least 5%, at least 6%, or at least 7%.

[0105] In some embodiments of the present invention, the silicone replacement component is a solid or film. In some embodiments, the silicone replacement component is a powder. In some embodiments, the solid or film will be substantially homogeneous meaning that the material, as inspected by light microscopy, has a low amount or does not have any inclusions or precipitates. In some embodiments, light microscopy may be used to measure birefringence which can be used as a proxy for alignment of the recombinant silk into a three-dimensional lattice. Birefringence is the optical property of a material having arefractive index that depends on the polarization and propagation of light. Specifically, a high degree of axial order as measured by birefringence can be linked to high tensile strength. In some embodiments, recombinant silk solids and films will have minimal birefringence. In various embodiments, the solid is a bead. In some other embodiments, the solid functions as an exfoliant. The recombinant silk solid may be in the form of a gentle skin scrub for the skin. In some embodiments, the material form is a roll, pellet, sheet, or flake.

[0106] In some embodiments, the recombinant silk protein comprises a hollow core and / or a shell. In some embodiments, the recombinant silk protein ranges from about 1 pm to about 30 pm in diameter, about 5 pm to about 20 pm, or about 10 pm to about 50 pm in diameter, while recombinant silk protein in water ranges from about 20 to about 80 pm in diameter, about 30 pm to about 70 pm, or about 40 pm to about 100 pm in diameter. Prior to incorporating into the compositions of the disclosure, the recombinant silk protein hollow powder can be milled and incorporated as a milled powder.Solvents

[0107] In some embodiments, the silicone replacement component can include one or more solvents. For example, the recombinant silk polypeptide can be suspended in a solvent. The solvent can be an aqueous solvent, an alcohol, or an oil-based solvent. For example, the solvent can be one or more of water, glycerin, deionized water, olive oil, and pentylene glycol. For example, the recombinant silk polypeptide can be treated with a solvent such that the hollow core contains the solvent such as liquid water or glycerin, either in form of liquid water itself, or as a liquid aqueous solution, as an emulsion containing liquid water, or as an aqueous dispersion. In some embodiments, the silicone replacement component comprises about a 25 wt% solution in glycerin.

[0108] In some embodiments, the solvent is water. Without intending to be limited by theory, subjecting the recombinant silk polypeptide to a solvent such as water results in a recombinant silk polypeptide that has expanded or swelled, wherein the protein functions as a carrier containing the solvent (e.g., water). These compositions can be stored dry and partially rehydratable after immersion in water to directly form a liquid or semi-liquid aqueous suspension of expanded particles.

[0109] In some embodiments, the recombinant silk protein may expand a portion of the hollow core. In some other embodiments, the recombinant silk protein may expand a portion of the shell. In such embodiments where the solvent is water, the recombinant silk protein transforms into a hydrogel. In other embodiments where the solvent is water, the recombinant silk protein transforms into a paste. In various embodiments, heat and / or pressure may be added to further process the recombinant silk protein compositions.

[0110] In some embodiments, a solvent is generally present in a proportion ranging from 55 to 90% by weight relative to the total weight of the recombinant silk polypeptide. This range includes all specific values and subranges there between, including 60%, 65%, 70%, 75%, 80%, and 85% by weight. In some embodiments, the recombinant silk protein is insoluble in various solvents, including water at various different pH levels, glycerin, alcohols, siloxane, and oils.

[0111] In some embodiments, the solvent is an aqueous type. In such embodiments, the solvent is water. The solvent may have a pH ranging from 6 to 12. In some embodiments, the solvent has a pH of 6. In some other embodiments, the solvent has a pH ranging from 0 to 5, from 2 to 7, from 4 to 9, from 6 to 11 , from 8 to 13, or from 10 to 14.

[0112] In other embodiments, the solvent includes a mixture of various volatile organic solvents, in order to obtain relatively short drying times. In some embodiments, the solvent is an alcohol.

[0113] Solvents may include water, ethyl alcohol, toluene, methylene chloride, isopropanol, n-butyl alcohol, castor oil, organopolysiloxane oils, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulphoxide, dimethyl formamide and tetrahydrofuran.

[0114] In some embodiments, the organopolysiloxane oil may be volatile, non-volatile, or a mixture of volatile and non-volatile silicones. The term “non-volatile” as used in this context refers to those silicones that are liquid under ambient conditions and have a flash point (under one atmospheric of pressure) of or greater than about 100°C. The term “volatile” as used in this context refers to all other silicone oils. Suitable organopolysiloxanes can be selected from a wide variety of silicones spanning a broad range of volatilities and viscosities. Suitable silicones are disclosed in U.S. Pat. No. 5,069,897, issued Dec. 3, 1991 , which is incorporated by reference herein in its entirety. Examples of suitable organopolysiloxanes include, but are not limited to, polyalkylsiloxanes, alkyl substituted dimethicones, dimethiconols, polyalkylaryl siloxanes, and mixtures thereof. For instance, polyalkylsiloxanes, dimethicones and cyclomethicones may be used.

[0115] In some embodiments, the solvent is a vegetable oil and hydrogenated vegetable oil. In some embodiments, the solvent is a free fatty acid. Examples of vegetable oils and hydrogenated vegetable oils include safflower oil, castor oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, sesame oil, sunflower seed oil, partially and fully hydrogenated oils from the foregoing sources, and mixtures thereof. Animal fats and oils, e.g., cod liver oil, lanolin and derivatives thereof such as acetylated lanolin and isopropyl lanolate, may be used. Alsouseful are C4-C20 alkyl ethers of polypropylene glycols, C1-C20 carboxylic acid esters of polypropylene glycols, and di-Cs-Cso alkyl ethers, examples of which include PPG-14 butyl ether, PPG-15 stearyl ether, dioctyl ether, dodecyl octyl ether, and mixtures thereof.

[0116] The compositions of the present invention may be substantially free of semi-solid hydrocarbons such as petrolatum, lanolin and lanolin derivatives, sterols (e.g., ethoxylated soya sterols), high molecular weight polybutenes and cocoa butter. By “substantially free,” as used herein, means that the concentration of the semi-solid hydrocarbons is less than 10%, or less than 5% or less than 2% or 0%.Recombinant Silk Proteins as a Cosmetics Formulation

[0117] In various embodiments, the recombinant silk protein is compounded into a silk cosmetic or skincare product e.g., solutions applied to the skin or hair). Specifically, the recombinant silk protein can be incorporated into a silicone replacement component to be used as a base for a cosmetic or skincare product where the recombinant silk polypeptide is present in the base in its monomeric or less-crystalline form. In some embodiments, the silicone replacement component may be used as a base for a cosmetic or skincare product where the recombinant silk polypeptide is present in the base in a semi-crystalline form. In such embodiments, the recombinant silk polypeptide is not present in the base in its monomeric form.

[0118] In other embodiments, the recombinant silk protein is compounded into a silk cosmetic or skincare product that contains silicone.

[0119] In most embodiments, the cosmetic formulations are physically stable. In such embodiments, the recombinant silk protein and any other ingredients remain in its formulation for a prolonged period of time, with a prolonged shelf life. On prolonged use, the silicone replacement component remains substantially stable and the ingredients do not precipitate out of the formulation.

[0120] The composition of the invention may be used to apply the silk protein to the skin, nails, hair or mucous membranes, by contacting the composition with the skin, nails, hair or mucous membranes of a subject. Preferably, the inventive composition is used with human subjects.

[0121] In most embodiments, the cosmetic formulations are non-toxic or non-allergenic to subject hosts to which the cosmetic is applied. It is also desirable in the art to produce cosmetic compositions for hair and epidermal contact which will not permanently stain tissue and which can be removed by ordinary washing with aqueous detergents.

[0122] The solids, films, emulsions, hydrogels, and other material forms discussed in various embodiments may contain various humectants, emollients, occlusive agents, active agents, and cosmetic adjuvants, depending on the embodiment and the desired efficacy of the formulation. In some embodiments, the recombinant silk protein functions as a carrier. In some embodiments, the recombinant silk protein is a carrier, delivering one or more agents to a surface such as skin, hair, or nails.

[0123] In some embodiments, the cosmetic formulation comprises a plasticizer. Suitable concentrations of plasticizer by weight in the composition ranges from, e.g., 1 to 60% by weight, 10 to 60% by weight, 10 to 50% by weight, 10 to 40% by weight, 15 to 40% by weight, 10 to 30% by weight, or 15 to 30% by weight. In some embodiments, the plasticizer is glycerol. In some embodiments, the plasticizer is triethanolamine, trimethylene glycol, polyethylene glycol, propylene glycol, sorbitol, sucrose, a saturated fatty acid, or an unsaturated fatty acid.

[0124] In the instance where water is used as a plasticizer, a suitable concentration of water by weight in the composition ranges from, e.g., 5 to 80% by weight, 15 to 70% by weight, 20 to 60% by weight, 25 to 50% by weight, 19 to 43% by weight, or 19 to 27% by weight. Where water is used in combination with another plasticizer, it may be present in a range of, e.g., 5 to 50% by weight, 15 to 43% by weight or 19 to 27% by weight.

[0125] In some embodiments, suitable plasticizers may include polyols e.g., glycerol), water, lactic acid, ascorbic acid, phosphoric acid, ethylene glycol, propylene glycol, triethanolamine, acid acetate, propane-1 ,3-diol or any combination thereof. In various embodiments, the amount of plasticizer can vary according to the purity and relative composition of the recombinant silk protein. For example, a higher purity powder may have less impurities such as a low molecular weight compound that may act as a plasticizer and therefore require the addition of a higher percentage by weight of plasticizer.

[0126] In some embodiments, the composition comprises a humectant or emollient. The term “humectant” as used herein refers to a hygroscopic substance that forms a bond with water molecules. Suitable humectants include, but are not limited to glycerol, propylene glycol, polyethylene glycol, pentalyene glycol, tremella extract, sorbitol, dicyanamide, sodium lactate, hyaluronic acid, aloe vera extract, alpha-hydroxy acid and pyrrolidonecarboxylate (NaPCA).

[0127] The term “emollient” as used herein refers to a compound that provide skin a soft or supple appearance by filling in cracks in the skin surface. Suitable emollients include, but are not limited to shea butter, cocao butter, squalene, squalane, octyl octanoate, sesame oil, grape seed oil, natural oils containing oleic acid e.g., sweet almond oil, argan oil, olive oil,avocado oil), natural oils containing gamma linoleic acid e.g., evening primrose oil, borage oil), natural oils containing linoleic acid e.g., safflower oil, sunflower oil), or any combination thereof.

[0128] In some instances, an emollient or humectant may be an occlusive agent, and the disclosure contemplates inclusion of an occlusive agent into the composition in various embodiments. The term “occlusive agent” refers to a compound that forms a barrier on the skin surface to retain moisture. Other suitable occlusive agents may include, but are not limited to beeswax, canuba wax, ceramides, vegetable waxes, lecithin, allantoin. Without intending to be limited by theory, the film-forming capabilities of the silicone replacement component presented herein make an occlusive agent that forms a moisture retaining barrier because the recombinant silk polypeptides act attract water molecules and also act as humectants.

[0129] Optionally, the cosmetic formulation comprises an active agent. The term “active agent” refers to any compound that has a known beneficial effect in a hair care, skincare, or cosmetic formulation, including pigment in cosmetic formulations. Various active agents include, but are not limited to, acetic acid ( / .e., vitamin C), alpha hydroxyl acids, beta hydroxyl acids, zinc oxide, titanium dioxide, retinol, niacinamide, other recombinant proteins (either as full length sequences or hydrolyzed into subsequences or “peptides”), copper peptides, curcuminoids, glycolic acid, hydroquinone, kojic acid, l-ascorbic acid, alpha lipoic acid, azelaic acid, lactic acid, ferulic acid, mandelic acid, dimethylaminoethanol (DMAE), resveratrol, natural extracts containing antioxidants (e.g. green tea extract, pine tree extract), caffeine, alpha arbutin, coenzyme Q-10, and salicylic acid.

[0130] The term “cosmetic adjuvant” refers to various other agents used to create a cosmetic product with commercially desirable properties, including, without limitation, surfactants, emulsifiers, preserving agents and thickeners.

[0131] As described herein, in various embodiments, the recombinant silk protein may form a semi-solid or gel-like structure that is dispersible. In various embodiments where the recombinant silk protein is compounded into a skin care formulation, the recombinant silk protein may form a non-reversible three-dimensional structure such as a gel or film that transforms into a dispersible liquid upon the surface of the skin.

[0132] In various embodiments, the recombinant silk protein may be suspended in water (“aqueous suspended protein”) to form a silicone replacement component in the form of a film, gel, or base that can be incorporated ( / .e., compounded) in a cosmetic or skincare formulation. Depending on the embodiment, the amount of recombinant silk protein to water in the aqueous suspended protein can vary, as can the relative ratio of recombinant silkpolypeptide powder to additive in the recombinant silk protein. In some embodiments, the silicone replacement component will comprise 10-33% recombinant silk polypeptide powder by weight. In some embodiments, a different solvent than water will be used. In some embodiments, the recombinant silk protein is suspended in water to create an aqueous suspended protein that is 1 -40% recombinant silk protein and 60-99% water. In a specific embodiment, the silicone replacement component is suspended in water to create an aqueous suspended protein that is 10% recombinant silk polypeptide powder by weight, 30% additive by weight and 60% water by weight based on the total weight of the silicone replacement component. In a specific embodiment, the protein is suspended in water to create an aqueous suspended protein that is 6% recombinant silk polypeptide powder by weight, 18% additive by weight and 76% water by weight based on the total weight of the silicone replacement component. In a specific embodiment, the protein is suspended in water to create an aqueous suspended protein that is 10% recombinant silk polypeptide powder by weight and 90% water by weight based on the total weight of the silicone replacement component.

[0133] Depending on the embodiment, the aqueous suspended protein may be optionally heated and agitated when it is re-suspended in water. In some embodiments, heating and agitating the aqueous suspended protein may result in a phase transformation of the recombinant silk polypeptides in the aqueous suspended protein. Specifically, heating and agitating the aqueous suspended protein results in three distinct phases that are assessed by centrifugation: 1) a gel phase that is distinct from the supernatant after centrifugation; 2) a colloidal phase that can be filtered from the supernatant after centrifugation; and 3) a solution phase that remains after filtering the colloidal phase from the supernatant. Various combinations of heat, agitation and centrifugation may be used, provided that the aqueous suspended protein must not be subject to prolonged heat in order to prevent degradation of the recombinant silk polypeptides. In a specific embodiment, the protein is subjected to gentle agitation at 90°C for 5 minutes and centrifuged at 16,000 RCF for 30 minutes.

[0134] In various embodiments, either the various phases of the aqueous suspended protein ( / .e., colloidal phase, gel phase and solution) or the aqueous suspended protein may be incorporated in a cosmetic or skincare formulation to provide a source of recombinant silk protein. Depending on the embodiment, the aqueous suspended protein may be subject to agitation with or without heat before incorporating into a skincare formulation. Optionally, the aqueous suspended protein may be separated in the above-discussed phases by centrifugation and / or filtering. Depending on the embodiment, the skincare formulation may be an emulsion (e.g., a cream or serum) or a primarily aqueous solution e.g., a gel). In certain embodiments, the recombinant silk protein may be incorporated into any of thecosmetic, skin care, or hair care formulations described herein without aqueous resuspension. In these compositions, a homogenizer or similar equipment may be used to ensure that the recombinant silk protein is uniformly distributed in the composition.

[0135] In some embodiments, the aqueous suspended protein may be subject to heat and agitation, then cast onto a flat surface and dried into a film. In some embodiments, the aqueous suspended protein may be cast onto a flat surface and dried into a film without being subjected to heat and / or agitation. In such embodiments, the aqueous suspended protein may be cast onto a flat surface and dried into a film without being subjected to additional processing. In some embodiments, the aqueous suspended protein may be incorporated into an emulsion, then cast onto a flat surface and dried into a film. Depending on the embodiment, various different drying conditions may be used. Suitable drying conditions include drying at 60°C or at 80°C with and without a vacuum. In embodiments that use a vacuum, 15 Hg is a suitable amount of vacuum. Other methods of drying are well established in the art.

[0136] In various embodiments, the films comprising the aqueous suspended protein alone have a low melting temperature. In various embodiments, the films comprising the aqueous suspended protein alone have melting temperature that is less than body temperature (around 34-36°C) and melts upon contact with skin. Without intending to be limited by theory, the recombinant silk polypeptide forms enough intermolecular interactions to make a semisolid structure ( / .e., film); however, this structure is reversible upon skin contact and can be re-formed after dispersion on the skin surface. In various embodiments, the film will have reduced crystallinity compared to the recombinant silk protein or the recombinant silk powder, as measured by Fourier-transform infrared spectroscopy (FTIR). In various embodiments, the films comprising the aqueous suspended protein do not melt upon contact with skin. In such embodiments, the film functions as a barrier. In various embodiments, the film is a hydrophobic film of low density. The film or barrier may range from about 1 pm to about 50 pm in thickness, from about 10 pm to about 30 pm, or from about 20 pm to about 40 pm in thickness. Upon contact with skin, the barrier may be formed on the surface of the epidermal layer, materializing a robust, non-specific adherence is made to the skin surface. In some embodiments, the thickness of the film changes depending on the concentration of recombinant silk protein and surface area of application.

[0137] In some embodiments, the barrier is long-lasting and prevents against one or more environmental stressors, including wind, humidity, harsh additives, pollution, abrasion, dirt, and grease. The barrier may withstand abrasion equivalent to at least 100 rubs by hand, at least 200 rubs, at least 400 rubs, at least 600 rubs, or at least 800 rubs.

[0138] In one specific embodiment, the aqueous suspended protein or the protein may be incorporated (e.g., homogenized) into an emulsion, then cast on a flat surface and lyophilized to create a porous film. Depending on the embodiment, various techniques may be used for lyophilization, including freezing the film at -80°C for 30 minutes. Other lyophilization techniques will be well known to those skilled in the art.

[0139] In various embodiments, the above-described films can be used as a topical skincare agent. This film may be applied directly to the skin and can be re-hydrated to form a dispersible viscous substance that is incorporated into the skin. As discussed herein, various emollients, humectants, active agents, and other cosmetic adjuvants may be incorporated into the film. This film may be applied directly to the skin and adsorb to the skin due to contact with the skin, or after gently rubbing the film into the skin. In some embodiments, the film may be applied directly the skin and adsorb to the skin without additional rubbing or contact. In some embodiments, the protein resuspended in an aqueous solution may be applied to the face and then exposed to a coagulant such as propylene glycol via mist to form a gellable mask.

[0140] Depending on the embodiment, the film that is cast may be a flat film ( / .e., with no surface variability) or may be cast on a mold that incorporates microstructures. In a specific embodiment, the film that is cast on a mold that incorporates microneedle structures to prick the surface of the skin and assist in delivery of active agents.

[0141] In an alternate embodiment, the aqueous suspended protein may be added to an emulsion that is used as a cosmetic product. The emulsion may be applied to skin or hair and then allowed to form a film on the surface of the skin upon drying. As discussed herein, various emollients, humectants, active agents, and other cosmetic adjuvants may be incorporated into the emulsion.

[0142] In some embodiments, the compositions of the disclosure may be liquid or semisolid, such as creams, lotions, and gels. The compositions useful in the subject invention may be made into a wide variety of product forms that are known in the art. These include, but are not limited to, powders, lotions, creams, gels, patches, serums, ampules, powders, sticks, sprays, ointments, pastes, mousses, ointments, liquids, emulsions, foams, or aerosols. These product forms may comprise several types of additives, as further discussed herein, including, but not limited to, solutions, aerosols, emulsions, gels, solids, and liposomes. The compounds which are active in the compositions and methods of this invention may be delivered topically by any means known to those of skill in the art.

[0143] In some other embodiments, the compositions may be basic cosmetic compositions such as facial cleansers, such as toilet water, cream, essence, cleansing foam and cleansingwater; pack and body oil; color cosmetic compositions such as foundation, lipstick, mascara, and make-up base; hair product compositions such as shampoo, rinse, hair conditioner and hair gel; soap; and the like. The cosmetic formulation can be prepared in any method known in the art, using the silicone replacement component described herein, optionally together with at least one carrier and / or additive, which are commonly used in the field of preparing cosmetic compositions.

[0144] In some embodiments, the compositions comprise at least one cosmetic agent. Examples of cosmetic agents include emollients, humectants, colorants, pigments, fragrances, moisturizers, viscosity modifiers and any other cosmetic forming agent. One or more cosmetic agents can be included in the cosmetic composition. In another embodiment, additional active ingredients as known in the art and described herein may also be used, including, but not limited to, a skin softener, a skin permeation enhancer, a colorant, an aromatic, an emulsifier, and a thickener. Also, the cosmetic composition may further comprise a perfumery, a pigment, a bactericidal agent, an antioxidant, a preservative, and / or a moisturizer, as well as inorganic salts and synthetic polymer substances, for, e.g., the purpose of improving physical properties.

[0145] The composition may also be delivered topically via a lotion. Single emulsion skin care preparations, such as lotions and creams, of the oil-in-water type and water-in-oil type are well-known in the cosmetic art and are useful in the subject invention. Multiphase emulsion compositions, such as the water-in-oil-in-water type, are also useful in the subject invention. In general, such single or multiphase emulsions contain water, emollients, and emulsifiers as essential ingredients.

[0146] The compositions of the present invention can also be formulated into a solid formulation (e.g., a wax-based stick, soap bar composition, powder, bead, exfoliant, or a wipe containing liquid or powder).

[0147] The compositions of this invention can be formulated as a gel (e.g., an aqueous gel using a suitable gelling agent(s)). Suitable gelling agents for aqueous gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymer and hydrogenated ethylene / propylene / styrene copolymer. Such gels typically comprise between about 0.1% and 5%, by weight, of such gelling agents. In some embodiments, such compositions include a combination of recombinant silk protein, water (Aqua), sodium C14-16 olefin sulfonate, glycerin, cocoa betaine, sodium benzoate, sodium hydroxide, calcium gluconate, sodium hyaluronate,propanediol, xanthan gum, gluconolactone, and tetrasodium glutamate diacetate. In some embodiments, compositions comprise a cleansing detergent, soap, serum, or toner. In a specific embodiment, the serum is aqueous-based. In another specific embodiment, the toner is alcohol-based.

[0148] The compositions useful in the present invention may be formulated as emulsions. If the composition is an emulsion, in some embodiments, from about 1% to about 10% or from about 2% to about 5% of the composition comprises an emulsifier. Emulsifiers may be nonionic, anionic or cationic. Suitable emulsifiers are disclosed in, for example, INCI Handbook, pp. 1673-1686. Lotions and creams can be formulated as emulsions. In some embodiments, the composition is an emulsion and the recombinant silk protein is an emulsifier. In some embodiments, the composition is an emulsion, the recombinant silk protein is an emulsifier, and the composition is free of other emulsifiers.

[0149] Yet another type of composition may be an ointment. An ointment may comprise a simple base of animal or vegetable oils or semi-solid hydrocarbons. An ointment may comprise from about 2% to about 10% of an emollient in addition to from about 0.1% to about 2% of a thickening agent. Examples of thickening agents include, e.g., cellulose derivatives (methyl cellulose and hydroxyl propylmethylcellulose), synthetic high molecular weight polymers e.g., carboxyvinyl polymer and polyvinyl alcohol), plant hydrocolloids e.g., karaya gum and tragacanth gum), clay thickeners {e.g., colloidal magnesium aluminum silicate and bentonite), carboxyvinyl polymers, carboxylic acid polymers, crosslinked polyacrylates, polyacrylamides, xanthan gum, and mixtures thereof.

[0150] The compositions useful in the subject invention may contain, in addition to the aforementioned components, a wide variety of additional oil-soluble materials and / or water- soluble materials conventionally used in compositions for use on skin, hair, and nails at their art-established levels.

[0151] The compositions of the present invention may be directly applied to the skin or may be applied onto other delivery implements such as wipes, sponges, brushes, and the like. The compositions may be used in products designed to be left on the skin, wiped from the skin, or rinsed off of the skin.

[0152] In some embodiments, the composition improves the appearance of skin, such as increasing skin firmness / plumpness, increasing elasticity, improving overall skin health, increasing hydration, accelerating and / or improving wound healing, improving pollution defense, reducing dermatological aging, decreasing skin fragility, preventing and reversing loss of collagen and / or elastin, preventing skin atrophy, promoting / accelerating cell turnover, increasing genetic expression, improving skin texture, preventing and decreasing fine linesand wrinkles, improving skin tone, enhancing skin thickness, decreasing pore size, minimizing skin discoloration, restoring skin luster, minimizing signs of fatigue, improving skin barrier function, minimizing skin dryness, preventing, reducing, or treating hyperpigmentation, improving the mitochondrial function of the skin, improves exfoliation, reduces toxicity, mattifying skin, reducing oxidative stress levels, attenuating pollution induced oxidative stress, attenuating UVA or UVB induced oxidative stress, or any combination thereof.

[0153] The compositions of various embodiments defend against pollutants and other irritants. As a result, many skin conditions, such as acne, the redness associated with rosacea (adult acne), and other inflammatory conditions can be actively managed by application of the cosmetic formulations.Coagulants

[0154] In some embodiments, a recombinant silk polypeptide containing composition and / or a silicone replacement component as described herein is exposed to a coagulant. This can change the properties of the composition / component to facilitate controlled aggregation of silk in the silk-based composition. In some embodiments, composition / component is submerged in a coagulant. In some embodiments, the composition / component is exposed to a coagulant mist or vapor. In one embodiment, an aqueous protein composition comprises or is submerged with or mixed with a coagulant. In some embodiments, a silk-based solid or semi-solid, such as a film, is submerged in or exposed to a vapor comprising coagulant. In some embodiments, methanol is used as an effective coagulant.

[0155] In some embodiments, alcohol (e.g., isopropanol, ethanol, or methanol) can be used as a coagulant or solvent. In some embodiments, 60%, 70%, 80%, 90% or 100% alcohol is used as a coagulant. In some embodiments, a salt can be used as a coagulant. Examples of salts include, but are not limited to, ammonium sulfate, sodium chloride, sodium sulfate, and other protein precipitating salts effective at a temperature from 20 to 60°C.

[0156] In some embodiments, a combination of one or more of water, acids, solvents, and salts, including, but not limited to, the following classes of chemicals of Brbnsted-Lowry acids, Lewis acids, binary hydride acids, organic acids, metal cation acids, organic solvents, inorganic solvents, alkali metal salts, and alkaline earth metal salts can be used as a coagulant. In some embodiments, the acids comprise dilute hydrochloric acid, dilute sulfuric acid, formic acid, or acetic acid. In some embodiments, the solvents comprise ethanol, methanol, isopropanol, t-butyl alcohol, ethyl acetate, propylene glycol, or ethylene glycol. In some embodiments, the salts comprise LiCI, KC1 , BeCl2, MgCl2, CaCl2, NaCI, ZnCh,FeCh, ammonium sulfate, sodium sulfate, sodium acetate, or other salts of nitrates, sulfates or phosphates. In some embodiments, the coagulant is at a pH from 2.5 to 7.5.Other additives

[0157] In some embodiments, a composition in accordance with the disclosure and / or the silicone replacement component thereof can include one or more additives. This can change the properties of the composition as it interacts with the skin. In some embodiments, the silkbased composition is submerged in the additive. In some embodiments, the composition / component is exposed to the additive mist or vapor. In one embodiment, an aqueous protein composition comprises or is submerged with or mixed with the additive. In some embodiments, a silk-based solid or semi-solid, such as a film, is submerged in or exposed to a vapor comprising the additive. In some embodiments, the silk-based gel is exposed to the additive prior to hallow powder formation (e.g., the silk-based gel and additive are co-spray dried together).

[0158] The additive can itself be inert or it can possess dermatological benefits of its own. The additive should also be physically and chemically compatible with the essential components described herein, and should not unduly impair stability, efficacy or other use benefits associated with the compositions of the present invention. The type of additive utilized in the present invention depends on the type of product form desired for the composition. In some embodiments, the additive is an acid textile dye.

[0159] Pigments are frequently added to cosmetic formulations to achieve a desired color for application to the skin. Such pigments are known and the concentrations required to achieve a desired coloring are readily determinable. Pigments may be inorganic or organic. Inorganic pigments include iron oxides (red, black, brown colors), manganese violet, ultramarines (green, blue, pink, red, or violet aluminum sulfosilicates), aquamarines, copper powder, mica, clays, silica, and titanium dioxide. Organic dyes that have been certified by the US FDA for cosmetic use generally have the prefix “D&C” and a suffix of a color and a number (for example, D&C Green #3).

[0160] Certain embodiments of the present invention contain from about 0% to about 30%, from about 1% to about 20%, from about 2% to about 15%, or from about 5% to about 15% of a colorant, on an anhydrous pigment weight basis. These are usually aluminum, barium or calcium salts or lakes. Dyes may be present at a concentration of from about 0% to about 3% and pearlizing agents and the like from 0% to about 10%. Such dyes in combination with recombinant silk proteins are stable and have a long shelf-life. The shelf-life of such compositions may be about 6 months, about 1 year, or about 2 years. In some embodiments, the shelf-life of such compositions may be at least 5 years.

[0161] There are no specific limitations as to the pigment, colorant, or filler powders used in the composition. Each may be a body pigment, inorganic white pigment, inorganic colored pigment, pearling agent, and the like. Specific examples are talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, aluminum magnesium silicate, silica, titanium dioxide, zinc oxide, red iron oxide, yellow iron oxide, black iron oxide, ultramarine, polyethylene powder, methacrylate powder, polystyrene powder, silk powder, crystalline cellulose, starch, titanated mica, iron oxide titanated mica, bismuth oxychloride, and the like.

[0162] Additional pigment / powder fillers include, but are not limited to, inorganic powders such as gums, chalk, Fuller's earth, kaolin, sericite, muscovite, phlogopite, synthetic mica, lepidolite, biotite, lithia mica, vermiculite, aluminum silicate, starch, smectite clays, alkyl and / or trialkyl aryl ammonium smectites, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed aluminum starch octenyl succinate barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal tungstate, magnesium, silica alumina, zeolite, barium sulfate, calcined calcium sulfate (calcined gypsum), calcium phosphate, fluorine apatite, hydroxyapatite, ceramic powder, metallic soap (zinc stearate, magnesium stearate, zinc myristate, calcium palmitate, and aluminum stearate), colloidal silicone dioxide, and boron nitride; organic powder such as polyamide resin powder (nylon powder), cyclodextrin, methyl polymethacrylate powder, copolymer powder of styrene and acrylic acid, benzoguanamine resin powder, polyethylene tetrafluoride) powder, and carboxyvinyl polymer, cellulose powder such as hydroxyethyl cellulose and sodium carboxymethyl cellulose, ethylene glycol monostearate; and inorganic white pigments such as magnesium oxide. Other useful powders are disclosed in U.S. Pat. No. 5,688,831 , to El-Nokaly et al., issued Nov. 18, 1997, herein incorporated by reference in its entirety. These pigments and powders can be used independently or in combination.

[0163] Besides the silk protein, the composition according to the invention can further comprise a film-forming substance. Examples of film-forming substances include, e.g., cellulose derivatives, nitrocellulose, acrylic polymers or copolymers, acrylic, styrene, acrylate-styrene and vinyl resins, vinyl copolymers, polyester polymers, arylsulphonamide resins, and alkyde resins.

[0164] In some embodiments, the composition may include an amphoteric surfactant, a phospholipid, or a wax.

[0165] Examples of other additives include, but are not limited to, cannabidiol, foaming surfactants, depigmentation agents, reflectants, detangling / wet combing agents, amino acids and their derivatives, antimicrobial agents, allergy inhibitors, anti-acne agents, anti-aging agents, anti-wrinkling agents antiseptics, analgesics, antitussives, antipruritics, localanesthetics, anti-hair loss agents, hair growth promoting agents, hair growth inhibitor agents, antihistamines, antiinfectives, inflammation inhibitors, anti-emetics, anticholinergics, vasoconstrictors, vasodilators, wound healing promoters, peptides, polypeptides and proteins, deodorants and antiperspirants, medicament agents, skin emollients and skin moisturizers, skin firming agents, hair conditioners, hair softeners, hair moisturizers, vitamins, tanning agents, skin lightening agents, antifungals, depilating agents, shaving preparations, external analgesics, perfumes, counterirritants, hemorrhoidals, insecticides, poison ivy products, poison oak products, burn products, anti-diaper rash agents, prickly heat agents, make-up preparations, vitamins, herbal extracts, retinoids, flavenoids, sensates, anti-oxidants, skin conditioners, hair lighteners, chelating agents, cell turnover enhancers, sunscreens, anti-edema agents, collagen enhancers, and mixtures thereof.

[0166] Examples of suitable vitamins nonexclusively include vitamin B complex, including thiamine, nicotinic acid, biotin, pantothenic acid, choline, riboflavin, vitamin B6, vitamin B12, pyridoxine, inositol, carnitine; vitamins A, C, D, E, K and their derivatives such as vitamin A palmitate and pro-vitamins, (e.g., panthenol (pro vitamin B5) and panthenol triacetate) and mixtures thereof.

[0167] Examples of sunscreen agents include, but are not limited to, avobenzone, benzophenones, bornelone, butyl paba, cinnamidopropyl trimethyl ammonium chloride, disodium distyrylbiphenyl disulfonate, paba, potassium methoxycinnamate, butyl methoxydibenzoylmethane, octyl methoxycinnamate, oxybenzone, octocrylene, octyl salicylate, phenylbenzimidazole sulfonic acid, ethyl hydroxypropyl aminobenzoate, menthyl anthranilate, aminobenzoic acid, cinoxate, diethanolamine methoxycinnamate, glyceryl aminobenzoate, titanium dioxide, zinc oxide, oxybenzone, Padimate O, red petrolatum, and mixtures thereof.

[0168] The amount of additive to be combined with the composition may vary depending upon, for example, the ability of the additive to penetrate through the skin, hair, or nail; the specific additive chosen; the particular benefit desired; the sensitivity of the user to the additive; the health condition, age, and skin, hair, and / or nail condition of the user; and the like. In sum, the additive is used in a “safe and effective amount,” which is an amount that is high enough to deliver a desired skin, hair, or nail benefit or to modify a certain condition to be treated, but is low enough to avoid serious side effects, at a reasonable risk to benefit ratio within the scope of sound medical judgment.

[0169] The invention illustratively disclosed herein suitably may be practiced in the absence of any component, ingredient, or step which is not specifically disclosed herein. Several examples are set forth below to further illustrate the nature of the invention and the mannerof carrying it out. However, the invention should not be considered as being limited to the details thereof.

[0170] The compositions and methods of the present invention provide for skin equal or better performance for softness, quick absorption, easy spreadability (or “playtime”), lightweight film formation, and non-greasy afterfell as compared to compositions containing silicone elastomers. Additionally, if the skin is being treated with an SPF composition, then the invention provides equal or better performance for low white cast. The compositions and methods of the present invention provide for hair equal or better performance for long-lasting wear, shine, non-greasiness, frizz control, adding thickness to the hair, styling retention, electrostatic properties, resistance to heat, and UV-radiation and pollution defense.Equivalents and Scope

[0171] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended aspects.

[0172] In the aspects, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Aspects or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0173] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” and “consisting essential of” is thus also encompassed and disclosed.

[0174] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0175] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0176] Section and table headings are not intended to be limiting.

Claims

WHAT IS CLAIMED IS:1 . A recombinant polypeptide comprising a repeat domain comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1 .

2. The recombinant polypeptide of claim 1 , wherein the repeat domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1.

3. The recombinant polypeptide of claim 1 , wherein the repeat domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1.

4. The recombinant polypeptide of claim 1 , wherein the repeat domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1.

5. The recombinant polypeptide of claim 1 , wherein the repeat domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1.

6. The recombinant polypeptide of claim 1 , wherein the repeat domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 1 , over the full length of SEQ ID NO: 1.

7. The recombinant polypeptide of any of claims 1-6, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 1 by conservative amino acid substitutions.

8. The recombinant polypeptide of any of claims 1-6, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 1 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

9. The recombinant polypeptide of claim 1 , wherein the repeat domain comprises an amino acid sequence 100% identical to SEQ ID NO: 1 .

10. A recombinant polypeptide comprising a repeat domain comprising an amino acid sequence at least 95% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.11 . The recombinant polypeptide of claim 10, wherein the repeat domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.

12. The recombinant polypeptide of claim 10, wherein the repeat domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.

13. The recombinant polypeptide of claim 10, wherein the repeat domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.

14. The recombinant polypeptide of claim 10, wherein the repeat domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.

15. The recombinant polypeptide of claim 10, wherein the repeat domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 2, over the full length of SEQ ID NO: 2.

16. The recombinant polypeptide of any of claims 10-15, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 2 by conservative amino acid substitutions.

17. The recombinant polypeptide of any of claims 10-15, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 2 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

18. The recombinant polypeptide of claim 10, wherein the repeat domain comprises an amino acid sequence 100% identical to SEQ ID NO: 2.

19. A recombinant polypeptide comprising a repeat domain comprising an amino acid sequence at least 95% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.

20. The recombinant polypeptide of claim 19, wherein the repeat domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.21 . The recombinant polypeptide of claim 19, wherein the repeat domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.

22. The recombinant polypeptide of claim 19, wherein the repeat domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.

23. The recombinant polypeptide of claim 19, wherein the repeat domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.

24. The recombinant polypeptide of claim 19, wherein the repeat domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 3, over the full length of SEQ ID NO: 3.

25. The recombinant polypeptide of any of claims 19-24, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 3 by conservative amino acid substitutions.

26. The recombinant polypeptide of any of claims 19-24, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 3 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

27. The recombinant polypeptide of claim 19, wherein the repeat domain comprises an amino acid sequence 100% identical to SEQ ID NO: 3.

28. A recombinant polypeptide comprising a repeat domain comprising an amino acid sequence at least 95% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.

29. The recombinant polypeptide of claim 28, wherein the repeat domain comprises an amino acid sequence at least 96% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.

30. The recombinant polypeptide of claim 28, wherein the repeat domain comprises an amino acid sequence at least 97% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.31 . The recombinant polypeptide of claim 28, wherein the repeat domain comprises an amino acid sequence at least 98% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.

32. The recombinant polypeptide of claim 28, wherein the repeat domain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.

33. The recombinant polypeptide of claim 28, wherein the repeat domain comprises an amino acid sequence at least 99.5% identical to SEQ ID NO: 4, over the full length of SEQ ID NO: 4.

34. The recombinant polypeptide of any of claims 28-33, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 4 by conservative amino acid substitutions.

35. The recombinant polypeptide of any of claims 23-33, wherein the amino acid sequence of the repeat domain only differs from SEQ ID NO: 4 by substitutions with serine (S), glycine (G), glutamine (Q), or alanine (A).

36. The recombinant polypeptide of claim 28, wherein the repeat domain comprises an amino acid sequence 100% identical to SEQ ID NO: 4.

37. The recombinant polypeptide of any of claims 1-36, further comprising an N- terminal non-repetitive domain and / or a C-terminal non-repetitive domain.

38. An expression construct encoding the recombinant polypeptide of any of claims 1 - 37.

39. A host cell comprising the expression construct of claim 38.

40. The host cell of claim 39, wherein the host cell is a yeast cell.41 . The host cell of claim 40, wherein the host cell is Pichia pastoris.

42. A cosmetic, skin, or hair care composition, comprising: one or more active ingredients for cosmetic, skin, or hair care, and the recombinant polypeptide of any of claims 1 - 37.

43. A cosmetic, skin, or hair care composition having a silicone replacement, comprising: one or more active ingredients for cosmetic, skin, or hair care, and a siliconereplacement component comprising the recombinant polypeptide of any of claims 1 - 37, wherein the composition is substantially free of silicone.

44. The composition of claim 43, wherein the silicone replacement component is present in a loading level less than conventional loading levels of a silicone while providing at least the same performance.

45. The composition of claim 44, wherein the performance is one or more of imparting a matte finish; detangling of hair; imparting a soft feel to skin; imparting a silky, smooth, and powdery feel; decrease in glossiness on the skin; enhanced shine on the hair; vibrant and efficient pigment delivery; easy spreadability; quick absorption time; wrinkle blurring effect; hair style retention; heat resistance for hair; UV and pollution defense; and increased viscosity.

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