WO2023150696A2 - Compositions and methods for delivering hydrophobic bioactive agents - Google Patents

Compositions and methods for delivering hydrophobic bioactive agents Download PDF

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Publication number
WO2023150696A2
WO2023150696A2 PCT/US2023/061962 US2023061962W WO2023150696A2 WO 2023150696 A2 WO2023150696 A2 WO 2023150696A2 US 2023061962 W US2023061962 W US 2023061962W WO 2023150696 A2 WO2023150696 A2 WO 2023150696A2
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Prior art keywords
recombinant
protein
lactoglobulin
lactoglobulin protein
native
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PCT/US2023/061962
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French (fr)
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WO2023150696A9 (en
WO2023150696A3 (en
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Timothy GEISTLINGER
Janine Lin
Vaibhav Bhatt
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Perfect Day, Inc.
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Publication of WO2023150696A2 publication Critical patent/WO2023150696A2/en
Publication of WO2023150696A3 publication Critical patent/WO2023150696A3/en
Publication of WO2023150696A9 publication Critical patent/WO2023150696A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the use of recombinant milk proteins for delivery of hydrophobic bioactive agents.
  • the present invention relates to recombinant ⁇ -lactoglobulin proteins having improved binding of such hydrophobic bioactive agents, to compositions comprising such recombinant ⁇ -lactoglobulin proteins, and to methods for producing and using such compositions.
  • milk proteins evolved to bind valuable nutrients, such as, for example, minerals and hydrophobic compounds, and thereby to help solubilize, stabilize, and/or protect from degradation or inactivation of such nutrients, and to carry them from mother to neonate.
  • strategies may be developed for using milk proteins as high-quality, natural food components that deliver bioactive agents to promote health and treat disorders (see, for example, Chen et al. 2006. Trends Food Sci Technol. 17:272).
  • a major shortcoming of some milk proteins as vehicles for delivery of bioactive agents is that their binding of certain bioactive agents in not stringent enough to prevent premature release of the bioactive agents (e.g., release prior to having reached the site of action of the bioactive agents, due to, for example, early temporal release or release under certain environmental conditions).
  • a recombinant ⁇ -lactoglobulin protein comprising a modification compared to a native ⁇ -lactoglobulin protein that improves binding of a hydrophobic bioactive agent to the recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is native Bos taurus ⁇ - lactoglobulin protein (SEQ ID NO: 1), native Capra hircus ⁇ -lactoglobulin protein (SEQ ID NO: 3), or native Ovis aries ⁇ -lactoglobulin protein (SEQ ID NO: 5), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I A37M, A37V, A37Q, A37D, A37G, A37S.
  • the native ⁇ -lactoglobulin protein is native Bos taurus ⁇ - lactoglobulin protein (SEQ ID NO: 1), native Capra hircus ⁇ -lactoglobulin protein (SEQ ID NO: 3), or native
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is native Homo sapiens P ⁇ lactoglobulin protein (SEQ ID NO: 2), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: A37L, A37L A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41 L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69N, K69N, K
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is Equus asinus ⁇ - lactoglobulin protein (SEQ ID NO: 4), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43L V43L, L46I, L46F.
  • L46M, L46V, L46Y, L46T I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71F, I71Y, I71W, I71 V, F105I, F105V, L 123I, L123V, L123Y, L123F, L144I, L144F, L144M, L144R, L144V, H147R, H147K, and H147D.
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is native Equus caballus ⁇ -lactoglobulin protein (SEQ ID NO: 6), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W.
  • SEQ ID NO: 6 native Equus caballus ⁇ -lactoglobulin protein
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is native Equus caballus ⁇ -lactoglobulin protein (SEQ ID NO: 7), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T.
  • SEQ ID NO: 7 native Equus caballus ⁇ -lactoglobulin protein
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is native Equus asinus ⁇ - lactoglobulin protein (SEQ ID NO: 8), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L391, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69N, K69N, K
  • the present invention comprises the recombinant ⁇ -lactoglobulin protein of paragraph [0006], wherein the native ⁇ -lactoglobulin protein is native Ovis aries ⁇ - lactoglobulin protein (SEQ ID NO: 9), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43L V43L, L46I, L46F, L46M, L46V, L46Y, L46T.
  • SEQ ID NO: 9 native Ovis aries ⁇ - lactoglobulin protein
  • the present invention comprises the composition of paragraph [0014], wherein the composition further comprises a bioactive agent bound to the recombinant ⁇ - lactoglobulin protein .
  • the present invention comprises the composition of paragraph [0014] or [0015], wherein the bioactive agent is bound to the recombinant ⁇ -lactoglobulin protein via covalent bonding.
  • the present invention comprises the composition of paragraph [0014] or [0015], wherein the bioactive agent is bound to the recombinant ⁇ -lactoglobulin protein via non-covalent bonding.
  • the present invention comprises the composition of paragraph [0014] or [0015], wherein the bioactive agent is bound to the recombinant ⁇ -lactoglobulin protein via a linker peptide.
  • the present invention comprises the composition of paragraphs [0014] through [0018], wherein the bioactive agent is bound to a solvent-exposed region of the recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the composition of paragraphs [0014] through [0018], wherein the bioactive agent is bound to a not-solvent-exposed region of the recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the composition of paragraphs [0014] through [0020], wherein the composition comprises between 0.1% and 100% by mass of a recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the composition of paragraphs [0014] through [0021], wherein the recombinant ⁇ -lactoglobulin protein is a monomer.
  • the present invention comprises the composition of paragraphs [0014] through [0021], wherein the recombinant ⁇ -lactoglobulin protein is a polymer or polymer network comprising linked repeated protein monomers, wherein the repeated protein monomers comprise or consist of the recombinant ⁇ -lactoglobulin protein of paragraphs [0006] through [0013], or of dimers, trimers, or tetramers of the recombinant ⁇ -lactoglobulin protein of paragraphs [0006] through [0013],
  • a recombinant expression construct consisting of a polynucleotide comprising: a promoter sequence, an optional secretion signal sequence, a protein coding sequence encoding the recombinant ⁇ -lactoglobulin protein of paragraphs [0006] through [0013], and a termination sequence; wherein: the promoter sequence is operably linked in sense orientation to the optional secretion signal sequence and the protein coding sequence, the optional secretion signal sequence is operably linked in sense orientation to the protein coding sequence, and the one or more terminator sequences are operably linked to the protein coding sequence.
  • a recombinant host cell comprising the recombinant expression construct of paragraph [0024].
  • the recombinant host cell of paragraph [0026] wherein the recombinant host cell is derived from a bacterium.
  • a method for obtaining the recombinant host cell of paragraphs [0026] through [0030] comprises: obtaining a polynucleotide encoding the recombinant ⁇ -lactoglobulin protein of paragraphs [0003] through [0013], or the recombinant expression construct of paragraph [0024], or the recombinant vector of paragraph [0024]; and introducing the polynucleotide, recombinant expression construct, or recombinant vector, respectively, into a host cell.
  • the present invention comprises the method of paragraph [0032], wherein the method further comprises purifying the recombinant ⁇ -lactoglobulin protein from the fermentation broth to obtain a preparation comprising the recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the method of paragraph [0032] or [0033], wherein the method further comprises post-processing the recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the method of paragraph [0035], wherein the method further comprises polymerizing the recombinant ⁇ -lactoglobulin protein to obtain a polymer or polymer network comprising or consisting of the recombinant ⁇ -lactoglobulin protein.
  • the present invention comprises the method of paragraph [0035] or [0036], wherein the method further comprises binding the bioactive agent to the recombinant ⁇ - lactoglobulin protein.
  • Amino acids can be referred to herein by their single-letter codes, amino acid names, or three-letter codes.
  • the single-letter codes, amino acid names, and three-letter codes are as follows: G Glycine (Gly), P - Proline (Pro), A - Alanine (Ala), V Valine (Vai), L - Leucine
  • Tyrosine (Tyr), W -Tryptophan (Trp), H - Histidine (His), K - Lysine (Lys), R - Arginine (Arg),
  • Amino acid residues are denoted by a first letter for the amino acid, followed by a number that specifies the position of the amino acid in a reference sequence (e.g., any of SEQ ID NOs: 1-9).
  • Amino acid substitutions are denoted by a first letter for the amino acid that is to be replaced, followed by a number that specifies the position of the amino acid to be replaced in a reference sequence (e.g., any of SEQ ID NOs: 1-9), and a second leter that is to be substituted at the position in place of the amino acid that is to be replaced,
  • x, y, and/or z may refer to “x” alone, “y” alone, “z” alone, “x, y, and z", “(x and y) or z", “(x and z) or y”, “(y and z) or x", "x and y” alone, “x and z” alone, “y and z” alone, or “x or y or z”.
  • the term “at least” or “one or more” as used herein refers to one, two, three, four, five, six, seven, eight, nine, ten, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more, or all of the elements subsequently listed.
  • corresponding native ⁇ -lactoglobulin protein refers to a native ⁇ -lactoglobulin protein that is identical to a recombinant ⁇ -lactoglobulin protein that is compared to the “corresponding native ⁇ -lactoglobulin protein” except that it does not comprise a modification as provided herein in the recombinant ⁇ -lactoglobulin protein.
  • encoding refers to a polynucleotide that comprises a coding sequence that when placed under the control of appropriate regulatory sequences is transcribed into mRNA that may be translated into a polypeptide.
  • a coding sequence generally starts at a start codon (e.g., ATG) and ends at a stop codon (e.g., UAA, UAG and UGA).
  • a coding sequence may contain a single open reading frame, or several open reading frames (e.g., separated by introns).
  • the term "essentially free of” as used herein refers to the indicated component being either not detectable in the indicated composition by common analytical methods, or to the indicated component being present in such trace amount as to not be functional.
  • the term "functional” as used in this context refers to not materially contributing to properties of the composition comprising the trace amount of the indicated component, or to not having material activity (e.g., chemical activity, enzymatic activity) in the indicated composition comprising the trace amount of the indicated component, or to not having health-adverse effects upon use or consumption of the composition comprising the trace amount of the indicated component,
  • material activity e.g., chemical activity, enzymatic activity
  • the term “materially contributing’' as used herein refers to the indicated component contributing to an attribute of a composition to such extent that in the absence of the component (e.g., in a reference composition that is identical to the composition except that it lacks the indicated component) the attribute is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less present/active/
  • filamentous fungus refers to an organism from the filamentous form of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge. UK).
  • a filamentous fungus is distinguished from a yeast by its hyphal elongation during vegetative growth.
  • fungus refers to organisms of the phyla Ascomycotas, Basidiomycota, Zygomycota, and Chythridiomycota, Oomycota, and Glomeromycota. It is understood, however, that fungal taxonomy is continually evolving, and therefore this specific definition of the fungal kingdom may be adjusted in the future.
  • fungal host cell refers to a host cell that is obtained from a fungus.
  • homolog refers to a protein that comprises an amino acid sequence that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%) identical to a sequence of amino acids of a similar length (i.e., a length that is within +/- 20% of the length of the query amino acid sequence) comprised in a reference protein, and that has a functional property that is similar (e.g., is within 50%, within 40%, within 30%, within 20%, or within 10% of) or identical to that of the reference protein.
  • the term includes polymorphic variants, interspecies homologs (e.g., ortholog
  • host cell refers not only to the particular subject cell but to the progeny of such cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the subject cell, but are still included within the scope of the term “host cell” as used herein.
  • identity or “identical” in the context of two or more polynucleotide or polypeptide sequences as used herein refer to the nucleotide or amino acid residues that are the same when the two or more polynucleotide or polypeptide sequences, respectively , are aligned for maximum correspondence.
  • the "identity” may exist over a region of the sequences being compared (e.g.. over the length of a functional domain) or over the full length of the sequences.
  • a “region” is considered to be a continuous stretch of at least 6, 9, 14, 19, 24, 29, 34, 39, or more nucleotides, or of at least 2, 6, 10, 14, 18, 22, 26, 30, or more amino acids.
  • typically one sequence acts as a reference sequence to which one or more test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman (1981. Adv Appl Math. 2:482), by the homology alignment algorithm of Needleman & Wunsch (1970. J Mol Biol. 48:443), by the search for similarity method of Pearson & Lipman (1988. Proc Natl Acad Sci USA.
  • mammal-produced milk refers to a milk produced by a mammal.
  • mammals include cow, human, sheep, wild sheep, goat, buffalo, camel, horse, donkey, alpaca, yak, llama, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, and woolly mammoth.
  • the term "native” as used herein refers to what is found in nature in its unmodified state (e.g.. a cell that is not genetically modified by a human, and that is maintained under conditions [e.g., level of oxygenation, pH, salt concentration, temperature, and nutrient (e.g., carbon, nitrogen, sulfur) availability] that are not defined by a human).
  • conditions e.g., level of oxygenation, pH, salt concentration, temperature, and nutrient (e.g., carbon, nitrogen, sulfur) availability
  • operably linked refers to an arrangement of elements that allows them to be functionally related.
  • a promoter sequence is operably linked to a protein coding sequence if it controls the transcription of the protein coding sequence
  • a secretion signal sequence is operably linked to a protein if the secretion signal sequence directs the protein through the secretion system of a cell.
  • An "operably linked” element may be in contiguous linkage with another element, or act in trans or at a distance to another element.
  • functions that may be operably linked include control of transcription, control of translation, protein folding, and protein secretion.
  • polymer refers to a molecule that is composed of repeated molecular units that are covalently linked, either directly with each other or via intermediary molecules.
  • polymer network refers to a network of polymers that are crosslinked with each other via covalent bonds.
  • suitable covalent crosslinks include amide bonds (e.g., lactam bridges, native chemical ligation bonds, Staudinger ligation bonds) and disulfide bonds.
  • polynucleotide refers to a polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1,000) nucleotides.
  • the term includes both sense and antisense strands of DNA molecules (e.g., cDNA, genomic DNA, synthetic DNA) and RNA molecules (e.g., mRNA, synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, and/or chemical modifications.
  • a polynucleotide may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels; methylation; substitution of one or more of the naturally occulting nucleotides with an analog; internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates
  • charged linkages e.g., phosphoroth
  • modified nucleotides are described in the art (see, for example, Malyshev et al. 2014. Nature 509:385; Li et al. 2014. J. Am. Chem. Soc. 136:826). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding or other chemical interaction. Such molecules are known in the art and include, for example, molecules in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications may include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" polynucleotides.
  • a polynucleotide may be in any topological conformation.
  • a polynucleotide may be single- stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular', or in a padlocked conformation.
  • polynucleotide sequence refers to a sequence of nucleotides that are comprised in a polynucleotide or of which a polynucleotide consists.
  • protease refers to a protein that can hydrolyze (i.e., cleave) a peptide bond (e.g., members of enzyme classification groups EC 3.4).
  • polypeptide and protein as used herein can be interchanged, and refer to a naturally -occurring or a naturally not occurring polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100) amino acids.
  • a “polypeptide” or “protein” may have an active structure or lack a functional structure, comprise coded and/or non-coded amino acids, comprise amino acids that occur in nature and/or amino acids that do not occur in nature, comprise chemically modified and/or biochemically modified and/or derivatized amino acids, comprise unmodified and/or modified peptide backbones, and/or be monomeric (i.e., having a single chain) or polymeric (i.e., having of two or more chains, which may be covalently or non-covalently associated).
  • amino acid sequence refers to a sequence of amino acids that is comprised in a "polypeptide” or “protein”, or of which a “polypeptide” or “protein” consists, Amino acids comprised in an amino acid sequence are sequentially numbered by counting from the N-terminus of the polypeptide, A “polypeptide” or “protein may be isolated from naturally occurring systems (e.g., plant or animal lifeforms), synthesized in solution or on solid support, or produced recombinantly.
  • promoter sequence refers to a polynucleotide that directs transcription of a downstream polynucleotide in a cell.
  • a promoter sequence may include necessary nucleotides near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter sequence may also optionally include distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
  • protease recognition or cleavage sequence or “recognition or cleavage sequence for a protease” as used herein refers to an amino acid sequence in a polypeptide that is preferably recognized by a protease and in which a peptide bond is cleaved by the protease.
  • the general nomenclature of positions in protease recognition or cleavage sequences are defined as described by Schechter & Berger (1967. Biochem Biophys Res Common. 27(2):157; 1968. Biochem Biophys Res Commun.
  • recombinant ⁇ -lactoglobulin refers to a recombinantly produced polypeptide (i.e., a polypeptide that is produced in a recombinant host cell, or to a polypeptide that is synthesized from a recombinant polynucleotide) that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a native ⁇ -lactoglobulin protein (e.g., a native Bos taurus
  • SEQ ID NO: 6 a native a native Equus caballus ⁇ -lactoglobulin protein [amino acids 19 to 181 of UniProt sequence P07380, SEQ ID NO: 7], a native Equus asinus ⁇ - lactoglobulin protein [UniProt sequence P13613, SEQ ID NO: 8], a native Ovis aries musimon ⁇ - lactoglobulin protein [e.g., UniProt sequence P67975, SEQ ID NO: 9] ).
  • recombinant host cell refers to a host cell that comprises a recombinant polynucleotide.
  • a recombinant host cell may produce a polynucleotide or polypeptide not found in the native (non -recombinant) form of the host cell, or a recombinant host cell may produce a polynucleotide or polypeptide at a level that is different from that in the native (non-recombinant) form of the host cell. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such a cell.
  • a recombinant host cell may be an isolated cell or cell line grown in culture, or may be a cell which resides in a living tissue or organism.
  • the term "recombinant polynucleotide” as used herein refers to a polynucleotide that is removed from its naturally occurring environment, or a polynucleotide that is not associated with all or a portion of a polynucleotide abutting or proximal to the polynucleotide when it is found in nature, or a polynucleotide that is operatively linked to a polynucleotide that it is not linked to in nature, or a polynucleotide that does not occur in nature, or a polynucleotide that contains a modification that is not found in that polynucleotide in nature (e.g., insertion, deletion, or point mutation introduced artificially, e.g., by human intervention), or a polynucleotide that is integrated into a chromosome at a heterologous site.
  • a modification that is not found in that polynucleotide in nature
  • a polynucleotide is also considered “recombinant” if it contains a genetic modification that does not naturally occur.
  • an endogenous polynucleotide is considered a "recombinant polynucleotide” if it contains an insertion, deletion, or substitution of one or more nucleotides that is introduced artificially (e.g., by human intervention).
  • Such modification may introduce into the polynucleotide a point mutation, substitution mutation, deletion mutation, insertion mutation, missense mutation, frameshift mutation, duplication mutation, amplification mutation, translocation mutation, or inversion mutation.
  • the term includes a polynucleotide in a host cell's chromosome, as well as a polynucleotide that is not in a host cell’s chromosome (e.g., a polynucleotide that is comprised in an episome).
  • a recombinant polynucleotide in a host cell or organism may replicate using the in vivo cellular machinery of the host cell; however, such recombinant polynucleotide, although subsequently replicated intracellularly, is still considered recombinant for purposes of this invention.
  • regulatory element refers a polynucleotide sequence that mediates, modulates, or regulates expression (e.g., transcription, post-transcriptional events, translation) of a polynucleotide to which the regulatory element is operably linked.
  • secretion signal refers to a peptide that is operably linked to the N -terminus of a protein, and that mediates the delivery of the protein via the intracellular secretory pathway of a host cell in which the protein is produced (i.e,, synthesized) to the exterior of the host cell.
  • operable linkage of a recombinant protein with a secretion signal requires removal of a start codon of the polynucleotide sequence encoding the recombinant protein.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • two or more refers to two, three, four, five, six, seven, eight, nine, ten, or more, or all of the elements subsequently li sted.
  • vector refers to a nucleic acid that can carry a polynucleotide sequence to be introduced into a host cell.
  • vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), vims particles (e.g., comprising heterologous polynucleotides), DNA constructs (e.g., produced by cloning or PCR amplification), and linear double- stranded molecules (e.g., PCR fragments).
  • BACs bacterial artificial chromosomes
  • YACs yeast artificial chromosomes
  • vims particles e.g., comprising heterologous polynucleotides
  • DNA constructs e.g., produced by cloning or PCR amplification
  • linear double- stranded molecules e.g., PCR fragments
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • Other vectors may be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • yeast refers to any organism of the order Saccharomycetales. Vegetative growth of yeast is by budding /b lebbing of a unicellular thallus, and carbon catabolism may be fermentative.
  • % by mass refers to a percentage value for a mass as determined in a hydrated composition, such that the composition includes the mass of powder as well as the mass of the hydrating agent, with 100% fixed as the percentage value for the entire hydrated composition.
  • the term refers to a percentage value for a mass as determined relative to the eventual entire hydrated composition (with 100% fixed as the percentage value for that entire eventual hydrated composition).
  • a recombinant ⁇ -lactoglobulin protein that comprises a modification compared to a corresponding native ⁇ -lactoglobulin protein, wherein the modification improves binding of a hydrophobic bioactive agent to the recombinant ⁇ - lactoglobulin protein.
  • a recombinant ⁇ -lactoglobulin protein according to the above may be loaded with one or more of a number of desirable hydrophobic bioactive agents, and may then be administered to a subject (e.g., a human or other animal) where it can provide the one or more hydrophobic bioactive agents such that they can exert their bioactivity.
  • the rate of binding of the one or more hydrophobic bioactive agents may be controlled by the type and/or number of modifications comprised in the recombinant ⁇ -lactoglobulin protein (e.g., the more modifications, the greater the binding affinity for the one or more hydrophobic bioactive compounds by the recombinant ⁇ -lactoglobulin protein).
  • the modification that improves binding of a hydrophobic bioactive compound to the recombinant ⁇ -lactoglobulin protein may be a modification that increases binding affinity of the hydrophobic bioactive agent to the recombinant ⁇ -lactoglobulin protein.
  • the modification may, for example, comprise or consist of a substitution of an amino acid in a region of the ⁇ -lactoglobulin protein that can bind the hydrophobic bioactive agent with an amino acid that can provide for a stronger, non-covalent interaction (e.g., electrostatic interaction, van der Waals bonding, hydrogen bonding) with the hydrophobic bioactive agent.
  • the modification typically has minimal impact on protein structure of the recombinant ⁇ -lactoglobulin protein compared to that of the corresponding native ⁇ -lactoglobulin protein. Such minimal impact may be achieved by creating the modification on a solvent-exposed loop, avoiding changing amino acids in beta-sheet or alpha-helixes, selecting conservative amino acid substitutions (i.e., substitutions of amino acids having similar biochemical properties), and/or selecting amino acid deletions, substitutions, and/or additions that do not create steric hindrances between side chains of amino acids in a three-dimensional conformation of the recombinant ⁇ - lactoglobulin protein (as determined, for example, by examination using PyMol [Schrodinger, New York, NY] and multi -sequence alignments [e.g., of orthologs of native ⁇ -lactoglobulin proteins; for example, using MUSCLE (Edgar, 2004, Nucleic Acids Res, 32: 1792-1797)]).
  • the modification that improves binding of a hydrophobic bioactive agent to a recombinant ⁇ -lactoglobulin protein may be comprised in or in the vicinity (e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) of a region of a corresponding native ⁇ -lactoglobulin protein that can bind hydrophobic compounds.
  • Non-limiting examples of regions of native ⁇ -lactoglobulin protein that can bind hydrophobic compounds include regions spanning from approximately amino acid 21 to approximately amino acid 124 (i.e., “central binding cavity” or “beta-barrel region”), and from approximately amino acid 135 to approximately amino acid 150 (“interface region”) of native Bos taurus ⁇ -lactoglobulin protein (SEQ ID NO: 1), and corresponding regions in orthologs (e.g., SEQ ID NOs: 2-9).
  • the modification that improves binding of a hydrophobic bioactive agent to a recombinant ⁇ -lactoglobulin protein may consist of a single modification (e.g., a single amino acid substitution, deletion, or addition).
  • Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L.
  • Such single modification may be an amino acid substitution selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I84L, I84M, I84F, I84V, I84Y, I84W, N90L N90K, N90R, N90Q, N90S, N90T, N90D, L122L L122V,
  • Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y. I56A, L58I, L58W.
  • Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41L V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58L L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69L K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84
  • Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39I, L39M, L39W, L39F, L39V, V411, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71 F, I71 Y, I71W, I71 V, F105I, F105V, L123L L123V, L123Y, L123F, L144I, L144F, L144M, L144R, and L144V of native Equus caballus ⁇ - lactoglobulin protein (SEQ ID NO: 7).
  • Such single modification may be an amino acid substitution selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y. L58M, E62A.
  • Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, K60
  • the modification that improves binding of a hydrophobic bioactive agent to a recombinant ⁇ -lactoglobulin protein may consist of two or more modifications (e.g., two or more amino acid substitutions, deletions, or additions, or combinations thereof).
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69N, K
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, L461.
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L.39W, L39F, L39V, V411, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71F, I71Y, I71W, I71V, F105I, F105V, L123I, L123V, L123Y, L123F, L144I, L144F, L144M, L144R, L144V, H147R, H147R, H
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M. L39W. L39F, L39V. V41I, V41 M.
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39I, L39M, L39W, L39F, L39V, V41L V41M, V41L. V43I, V43L, L46I, L46E L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W. L58Y, L58M, I71L, I71F, I71Y.
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: A37L, A371, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41 L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58L L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69L K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, V92I, V92L, V92M, V
  • Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M. L39W. L39F, L39V.
  • the recombinant ⁇ -lactoglobulin protein may comprise a further modification.
  • the recombinant ⁇ -lactoglobulin protein may comprise a modification that introduces a non-native protease recognition or cleavage sequence in or in the vicinity (e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) of a solvent- exposed region of the corresponding native ⁇ -lactoglobulin protein (i.e., a region that is on the surface of a three-dimensional structure of the corresponding native ⁇ -lactoglobulin protein), a lipid-binding region of the corresponding native ⁇ -lactoglobulin protein (i.e., a region in a three- dimensional structure of the corresponding native ⁇ -lactoglobulin protein that can bind a lipid); and/or an allergenic epitope comprised in the corresponding native ⁇ -lactoglobulin protein.
  • Such further modification may facilitate cleavage and/or denaturation of the recombinant ⁇ - lactoglobulin protein in the gastrointestinal tract of a subject (e.g., (e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig, human])), and thereby attenuate or essentially eliminate allergenicity of the recombinant ⁇ -lactoglobulin protein, and/or facilitate in vi vo release of the bound hydrophobic bioactive agent from the recombinant ⁇ -lactoglobulin protein.
  • a subject e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig, human]
  • suitable modifications that introduce a non-native protease recognition or cleavage sequence are disclosed, for example, in PCT publication WO2021168343, published August 26, 2021.
  • the hydrophobic bioactive agent of which binding to the recombinant ⁇ -lactoglobulin protein according to any of the above may be any hydrophobic bioactive agent.
  • hydrophobic bioactive agents include hydrophobic nutraceuticals (i.e., hydrophobic compounds that have physiological benefit or provide protection against disease), hydrophobic compounds that have micro-/biocidal activity, hydrophobic compounds that have anti-viral activity, lipids, and hydrophobic therapeutics (i.e., hydrophobic compounds that treat disease, disorder, or injury: e.g., hydrophobic compounds that have anti- cancer activity, treat pain [e.g., acute pain, chronic pain], heal or soothe skin defects [i.e., defects in skin that disrupt the continuity of epithelium, such as, for example rashes, sores, abrasions, bums, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers, diabetic ulcers, diabetic fool ulcers, and decubitus ulcers, itchy skin], or have anesthetic effects).
  • hydrophobic nutraceuticals i.e., hydrophobic compounds that have physiological benefit or provide protection against disease
  • Non-limiting examples of hydrophobic nutraceuticals include lipid- soluble pro- vitamins and vitamins and lipid-soluble antioxidants.
  • Non-limiting examples of lipid-soluble vitamins include vitamin A (retinoids), vitamin D (e.g., vitamin D2, vitamin D3), vitamin E (e.g., tocopherols [e.g., alpha- tocopherol, beta- tocopherol, gamma- tocopherol, delta-tocopherol], tocotrienols [e.g., alpha-tocotrienol, beta- tocotrienol, gamma-tocotrienol, delta-tocotrienol]), vitamin K (e.g., vitamin K1 (phylloquinone), vitamin K2 (menaquinone), vitamin K3 (menadione), vitamin K4 (menadiol diacetate), vitamin K5), and derivatives.
  • vitamin A retinoids
  • vitamin D e.g., vitamin D2, vitamin D3
  • vitamin E e.g., tocopherols [e.g., alpha- tocopherol, beta- tocopherol,
  • Non-limiting examples of lipid-soluble antioxidants include fatty acids (e.g., linolenic, linoleic, oleic, palmitic) and derivatives (e.g., lipoic acid), carotenoids (e.g., a-carotene, ⁇ - carotene, ⁇ -carotene, lutein, zeaxanthin, astaxanthin), fat-soluble polyphenols (e.g., astaxanthin, olive oil), coenzyme Q 10 , and derivatives and mixtures thereof.
  • fatty acids e.g., linolenic, linoleic, oleic, palmitic
  • derivatives e.g., lipoic acid
  • carotenoids e.g., a-carotene, ⁇ - carotene, ⁇ -carotene, lutein, zeaxanthin, astaxanthin
  • fat-soluble polyphenols e.g
  • Non-limiting examples of hydrophobic compounds that have micro-fbiocidal activity include 4-aminododecylpyridinium chloride, 4-acetylaminododecylpyridinium chloride, 4- benzoylaminododecylpyridinium bromide and 4-( 1 -naphthoyl) aminododecylpyridinium bromide (Zhao & Sun. 2008. J Appl Microbiol. 104(3):824), hydrophobic compounds extracted from garlic (e.g., allicin, vinyldithiin, ajoene, and diallyl polysulfides; see, for example, Nakamoto et al. 2020. Exp Ther Med. 19(2): 1550).
  • garlic e.g., allicin, vinyldithiin, ajoene, and diallyl polysulfides; see, for example, Nakamoto et al. 2020. Exp Ther Med. 19(2): 15
  • Non-limiting examples of hydrophobic compounds that have anti-viral activity are known in the art (see, for example, Hadfield et al. 1999. Proc Natl Acad Sci USA. 96(26): 14730: Anasir et al. 2021. J Biomed Sci. 28, 10; and Linnakoski et al. 2018. Front Microbiol. 9:2325).
  • Non-limiting examples of lipids include octanoic acid (caprylic acid), lionoleic acid, lauric acid, myristic acid, palmitic acid, succinic acid, hepta-decanoic acid, 12-bromododecanoic acid, and oleic acid.
  • Non-limiting examples of hydrophobic compounds that have anti-cancer activity include all-trans-retinol, all-trans retinoic acid [ATR.A], all-trans-retinyl acetate, curcumin, epigallocatechin gallate (EGCG), geinistein, 3,3-diindolylmethane (DII), indole-3-carbinol, and resveratrol.
  • the hydrophobic bioactive agent may be bound to the recombinant ⁇ -lactoglobulin protein according to any of the above (e.g,, a recombinant ⁇ -lactoglobulin protein according to any of the above) via non-covalent bonding (e.g., electrostatic interaction, van der Waals bonding, hydrogen bonding).
  • non-covalent bonding e.g., electrostatic interaction, van der Waals bonding, hydrogen bonding.
  • a recombinant expression construct consisting of a polynucleotide comprising: i) a promoter sequence (e.g., a polynucleotide sequence for any of the promoters disclosed herein), ii) an optional secretion signal sequence (i.e., a sequence that encodes a peptide that mediates the delivery of a nascent protein attached to the peptide to the exterior of the cell in which the nascent protein is synthesized: e.g., a polynucleotide sequence encoding any of the secretion signals disclosed herein), iii) a recombinant ⁇ -lactoglobulin protein coding sequence (i.e., a polynucleotide sequence encoding a recombinant ⁇ -lactoglobulin protein according to any of the above, optionally comprising a tag polypeptide (e.g., any of the tag
  • the recombinant expression construct may further comprise an operably linked sequence encoding for an affinity purification tag, such that the expressed recombinant ⁇ - lactoglobulin protein includes a peptide sequence for affinity purification.
  • affinity purification tag may be operably linked such that when expressed the affinity purification tag is present either at or toward the amino terminus, the carboxy terminus, or both.
  • Such affinity purification tag may comprise a maltose binding protein (MBP) tag, a glutathione-S-transferase (GST) tag, a poly(His) tag, a hexa(His) tag, a FLAG-tag, a V5-tag, a VSV-tag, an E-tag, an NE- tag, a hemagglutinin (Ha)-tag, Strep-tag, and a Myc-tag.
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • the recombinant expression construct may further comprise a sequence for integration by homologous (i.e., targeted integration) or nonhomologous recombination into the genome of a host cell.
  • the recombinant expression construct may comprise at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1,000, or at least 10,000 base pairs that have sufficient identity with a target sequence in the genome of the host cell to enhance the probability of homologous recombination of the recombinant expression construct.
  • Such homologous sequence may be non-coding or coding.
  • the optional secretion signal sequence and/or recombinant ⁇ -lactoglobulin protein coding sequence comprised in a recombinant expression construct according to any of the above may be codon-optimized for expression in a recombinant host cell according to any of the above.
  • a recombinant expression construct according to any of the above may be isolated.
  • a recombinant expression construct according to any of the above may be generated upon integration of a fragment of the recombinant expression construct into the genome of a host cell (e.g., the genome of a recombinant host cell according to any of the above).
  • a polynucleotide comprising a recombinant ⁇ -lactoglobulin protein coding sequence (optionally operably linked to a secretion signal sequence) may be stably integrated within the genome of a host cell such that one or more regulatory elements of an endogenous gene locus become operably linked to the recombinant ⁇ -lactoglobulin protein coding sequence, thereby generating a recombinant expression construct according to any of the above.
  • a recombinant expression construct according to any of the above may comprise any promoter sequence that is active in a recombinant host cell according to any of the below.
  • the promoter sequence may be a constitutive promoter sequence (i.e., a promoter sequence that is active under most environmental and developmental conditions), or an inducible or repressible promoter sequence (i.e., a promoter sequence that is active only under certain environmental or developmental conditions [e.g., in presence or absence of certain factors, such as, but not limited to, carbon (e.g., glucose, galactose, lactose, sucrose, cellulose, sophorose, gentiobiose, sorbose, disaccharides that induce the cellulase promoters, starch, tryptophan, thiamine, methanol), phosphate, nitrogen, or other nutrient; temperature; pH; osmolarity; heavy metals or heavy metal ions; inhibitors; stress; catabolites; and combinations thereof]).
  • carbon e.g., glucose, galactose, lactose, sucrose, cellulose, sophorose, gentiobiose,
  • the promoter sequence may consist of a single promoter sequence, or of two or more promoter sequences (e.g., combination of two or more promoters or functional parts thereof arranged in sequence, combination of an inducible and a constitutive promoter).
  • the two or more promoter sequences may be identical, or at least two of the two or more promoter sequences may not be identical.
  • the promoter sequence may comprise or consist of a bidirectional promoter sequence (i.e., a polynucleotide that initiates transcription in both orientations by recruiting transcription factors, for example generated by fusing two identical or different promoters in opposite directions).
  • a bidirectional promoter sequence i.e., a polynucleotide that initiates transcription in both orientations by recruiting transcription factors, for example generated by fusing two identical or different promoters in opposite directions.
  • Non-limiting examples of suitable promoter sequences include promoter sequences that are functional in a bacterial host cell, including T7 promoter, T5 promoter, Tac promoter, pL/pR promoter, phoA promoter, lacUV5 promoter, trc promoter, trp promoter, cstA promoter, xylA promoter, manP promoter, malA promoter, lacA promoter, aprE promoter, AaprE promoter, srfA promoter, p43 promoter, ylbA promoter, oB promoter, veg promoter, PG1 promoter, PG6 promoter, ⁇ PL promoter, ⁇ PR promoter, and spa promoter, and functional parts and combinations thereof,
  • Non-limiting examples of suitable promoter sequences include promoter sequences that are functional in a fungal host cell, including xlnA promoter, xynl promoter, xyn2 promoter, xyn3 promoter, xyn4 promoter, bxll promoter, cbhl promoter, cbh2 promoter, egl1 promoter, egl2 promoter, egl3 promoter, egl4 promoter, egl5 promoter, glaA promoter, agdA promoter, gpdA promoter, gpdl promoter, AOX 1 promoter, GAP1 promoter, MET3 promoter, ENO1 promoter, GPD1 promoter, PDC1 promoter, TEF1 promoter, AXE1 promoter.
  • CIP1 promoter CIP1 promoter, GH61 promoter, PKI1 promoter, RP2 promoter, ADH1 promoter, CUP1 promoter, GAL1 promoter, PGK1 promoter, YPT1 promoter, LAC4 promoter, LAC4-PB 1 promoter, FLD1 promoter, MOX promoter.
  • a recombinant expression construct according to any of the above may optionally comprise any secretion signal sequence that is active in a recombinant host cell according to any of the below.
  • the optional secretion signal sequence may encode a secretion signal that mediates translocation of the nascent recombinant ⁇ -lactoglobulin protein into the ER post-translationally (i.e., protein synthesis precedes translocation such that the nascent recombinant ⁇ -lactoglobulin protein is present in the cell cytosol prior to translocating into the ER) or co-translationally (i.e., protein synthesis and translocation into the ER occur simultaneously).
  • Non-limiting examples of suitable secretion signal sequences include secretion signal sequences that are functional in a bacterial host cell, including secretion signal sequences of genes encoding any of the following proteins: PelB, OmpA, Bla, PhoA, PhoS, MalE, LivK, LivJ, MglB, AraF, AmpC, RbsB, MerP, CpdB, Lpp, LamB, OmpC, PhoE, OmpF, TolC, BtuB, and LutA, and functional parts and combinations thereof.
  • Non-limiting examples of suitable secretion signal sequences include secretion signal sequences that are functional in a fungal host cell, including secretion signal sequences of genes encoding any of the following proteins: CBH1, CBH2, EGL1, EGL2, XYN1, XYN2, BXL1, HFB1, HFB2, GLAA, AMYA, AMYC, AAMA, alpha mating factor, SUC2, PHO5, INV, AMY, LIP, PIR, OST1, and ⁇ - glucosidase, and functional parts and combinations thereof.
  • a recombinant expression construct according to any of the above may comprise any termination sequence that is active in a recombinant host cell according to any of the below.
  • Non-limiting examples of suitable termination sequences include termination sequences include termination sequences of the adhl , amaA, amdS, amyA, aox1, cbh1, cbh2, cyc1 , egl1 , egl2, gal1, gap1, glaA, gpd1 , gpdA, pdc1, pgk1 tef1 , tps1 , trpC, xyn1, xyn2, xyn3, and xyn4 genes, and functional parts and combinations thereof.
  • the termination sequence may consist of a single termination sequence, or of two or more termination sequences, wherein the two or more termination sequences may be identical, or at least two of the two or more termination sequences may be not identical.
  • the termmation sequence may consist of a bidirectional termination sequence.
  • a recombinant expression construct according to any of the above may further comprise additional regulatory elements.
  • Non-limiting examples of regulatory elements include promoter sequences, termination sequences, transcriptional start sequences, translational start sequences, translation stop sequences, enhancer sequences, activator sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions, upstream activation sequences (UAS), introns, operators (i.e., sequences of nucleic acids adjacent to a promoter that comprise a protein- binding domain where a repressor protein can bind and reduce or eliminate activity of the promoter), efficient RNA processing signals (e.g., splicing signals, polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., ribosome binding sites [e.g., Shine-Dalgamo sequences]), sequences that enhance protein stability, sequences that enhance protein secretion, and combinations thereof.
  • UAS upstream activation sequences
  • operators i.e., sequences of nucleic acids adjacent to
  • a recombinant vector that comprises a recombinant expression construct according to any of the above or a fragment thereof (e.g., a polynucleotide that comprises a recombinant ⁇ -lactoglobulin protein coding sequence and optional secretion signal sequence, which upon integration into the genome of a host cell creates a recombinant expression construct according to any of the above),
  • the recombinant vector may comprise a single recombinant expression construct according to any of the above, or two or more recombinant expression constructs according to any of the above, which may be identical or at least two of which may be not identical (e.g., differ from each other in a promoter sequence, a secretion signal, a ⁇ -lactoglobulin protein coding sequence, a termination sequence, and/or an additional regulatory element).
  • the recombinant vector comprises two or more recombinant expression constructs
  • the two or more recombinant expression constructs may encode the same recombinant ⁇ -lactoglobulin protein.
  • the two or more recombinant expression constructs encoding the same recombinant ⁇ -lactoglobulin protein differ from each other in a promoter sequence, secretion signal sequence, termination sequence, and/or additional regulatory element.
  • the recombinant vector may further comprise one or more other elements suitable for propagation of the recombinant vector in a recombinant host cell.
  • Non-limiting examples of such other elements include origins of replication and selection markers. Origins of replication and selection markers are known in the art, and include bacterial and fungal origins of replication (e.g., AMA1, ANSI).
  • Selection markers may be resistance genes (i.e., polynucleotides that encode proteins that enable host cells to detoxify an exogenously added compound [e.g., an antibiotic compound]), auxotrophic markers (i.e., polynucleotides that encode proteins that permit a host cell to synthesize an essential component (usually an amino acid) while grown in media that lacks that essential component), or color markers (i.e., genes that encode proteins that can produce a color).
  • resistance genes i.e., polynucleotides that encode proteins that enable host cells to detoxify an exogenously added compound [e.g., an antibiotic compound]
  • auxotrophic markers i.e., polynucleotides that encode proteins that permit a host cell to synthesize an essential component (usually an amino acid) while grown in media that lacks that essential component
  • color markers i.e., genes that encode proteins that can produce a color).
  • Non-limiting examples of suitable selection markers include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine 5' -phosphate decarboxylase), sC
  • the selection marker may comprise an alteration that decreases production of the selective marker, thus increasing the number of copies needed to permit a recombinant host cell comprising the recombinant vector to survive under selection. Selection may also be accomplished by co-transformation, wherein the transformation is carried out with a mixture of two vectors and the selection is made for one vector only.
  • the recombinant vector may further comprise sequences for integration by homologous (i.e,, targeted integration) or nonhomologous recombination into the genome of a host cell.
  • the recombinant expression construct may comprise at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1,000, or at least 10,000 base pairs that have sufficient identity with a target sequence in the genome of the host cell to enhance the probability of homologous recombination of the recombinant expression construct.
  • Such homologous sequence may be non-coding or coding.
  • a recombinant vector according to any of the above may be isolated.
  • a recombinant host cell that is capable of producing a recombinant ⁇ -lactoglobulin protein according to any of the above, wherein the recombinant host cell comprises a recombinant expression construct according to any of the above.
  • the recombinant host cell may comprise a single recombinant expression construct according to any of the above, or comprise two or more recombinant expression constructs according to any of the above.
  • the two or more recombinant expression constructs may be identical, or at least two of the two or more recombinant expression constructs may differ from each other (e.g., in a promoter sequence, a ⁇ -lactoglobulin protein coding sequence, a secretion signal sequence, a termination sequence, and/or an additional regulatory element).
  • the recombinant host cell may comprise a recombinant expression construct that is stably integrated within the genome of the recombinant host cell (e.g., via targeted (e.g., via homologous recombination) or random (i.e,, non-targeted) integration), and/or a recombinant expression construct that is not stably integrated but rather maintained extra-chromosomally (e.g., on an autonomously replicating recombinant vector provided herein).
  • a recombinant expression construct that is stably integrated within the genome of the recombinant host cell (e.g., via targeted (e.g., via homologous recombination) or random (i.e,, non-targeted) integration), and/or a recombinant expression construct that is not stably integrated but rather maintained extra-chromosomally (e.g., on an autonomously replicating recombinant vector provided herein).
  • a recombinant host cell according to any of the above may be derived from any organism, including any bacterium, fungus (e.g., yeast, filamentous fungus), archaea, protista, animal (including any unicellular animal), plant (including any unicellular plant), algae, protozoan, and chromista, or from a genetic variant (e.g., mutant) thereof, as well as from any generally recognized as safe (GRAS) industrial host cell.
  • fungus e.g., yeast, filamentous fungus
  • archaea e.g., protista
  • animal including any unicellular animal
  • plant including any unicellular plant
  • algae including any unicellular plant
  • chromista or from a genetic variant (e.g., mutant) thereof, as well as from any generally recognized as safe (GRAS) industrial host cell.
  • GRAS generally recognized as safe
  • Non-limiting examples of suitable plants include cycad, ginkgo biloba, conifer, cypress, juniper, thuja, cedarwood, pine, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, helichrysum, marigold, mugwort, safflower, camomile, lettuce, wormwood, calendula, citronella, sage, thyme, chia seed, mustard, olive, coffee, capsicum, eggplant, paprika, cranberry, kiwi, vegetables (e.g., carrot, celery), tagete, tansy, tarragon, sunflower, wintergreen, basil, hyssop, lavender, lemon verbena, marjoram, melissa, patchouli, pennyroyal, peppermint, rosemary, sesame, spearmint, primrose, samara, pepper, pi
  • moss e.g., Phy scomitrella patens
  • Non-limiting examples of suitable yeast include members of any of the following genera, and derivatives and crosses thereof: Candida (e.g., Candida albicans, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida orthopsilosis, Candida palmioleophila, Candida pseudotropicalis, Candida sp., Candida utilis, Candida versatilis), Cladosporium, Cryptococcus (e.g., Cryptococcus terricolus, Cryptococcus curvatus), Debaryomyces (e.g., Debaryomyces hansenii), Endomyces (e.g., Endomyces vemalis)' , Endomycopsis (e.g., Endomycopsis vemalis), Eremothecium (e.g., Eremothecium ashbyii), Hansenula (e.g., Hansenula sp., Hansenula poly
  • Trichosporon cacaoliposimilis sp. nov. Trichosporon gracile, Trichosporon dulcitum, Trichosporon jirovecii, Trichosporon insectorum), Xanthophyllomyces (e.g., Xanthophyllomyces dendrorhous), Yarrowia (e.g., Yarrowia lipolytica), and Zygosaccharomyces (e.g., Zygosaccharomyces rouxii).
  • Xanthophyllomyces e.g., Xanthophyllomyces dendrorhous
  • Yarrowia e.g., Yarrowia lipolytica
  • Zygosaccharomyces e.g., Zygosaccharomyces rouxii).
  • Non-limiting examples of suitable filamentous fungi include any holomorphic, teleomorphic, and anamorphic forms of fungi, including members of any of the following genera, and derivatives and crosses thereof: Acremonium (e.g., Acremonium alabamense), Aspergillus (e.g., Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus niger var.
  • Acremonium e.g., Acremonium alabamense
  • Aspergillus e.g., Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus
  • awamori Aspergillus ochraceus, Aspergillus oryzae, Aspergillus sojae, Aspergillus terreus, as well as Emericella. Neosartorya, and Petromyces species), Aureobasidium, Canariomyces, Chaetomium, Chaetomidium, Corynascus, Chrysosporium (e.g., Chrysosporium botryoides, Chrysosporium carmichaeli, Chrysosporium crassitunicatum, Chrysosporium.
  • Chrysosporium e.g., Chrysosporium botryoides, Chrysosporium carmichaeli, Chrysosporium crassitunicatum, Chrysosporium.
  • Chrysosporium medium Chrysosporium medium var. spissescens, Chrysosporium mephiticum
  • Chrysosporium merdarium Chrysosporium merdarium var. roseum, Chrysosporium minor,
  • Chrysosporium synchronum Chrysosporium tropicum, Chrysosporium undulatum.
  • Chrysosporium vallenaren.se Chrysosporium vespertilium, Chrysosporium. zonatum
  • Cunninghamella e.g., Cunninghamella ehinulata
  • Dactylomyces e.g., Emericella
  • Fusarium e.g., Fusarium moniliforme, Fusarium venenatum, Fusarium oxysporum
  • Fusarium graminearum Fusarium proliferatum, Fusarium verticiollioides, Fusarium culmorum, Fusarium crookwellense, Fusarium poae, Fusarium sporotrichioides, Fusarium sambuccinum, Fusarium torulosum, as well as associated Gibberella teleomorphic forms thereof), Gibberella, Humicola, Hypocrea, Lentinula, Malbranchea (e.g., Malbranchea filamentosa), Magnaporthe, Malbranchium, Melanocarpus, Mortiere.Ua (e.g., Mortierella alpina 1S-4, Mortieralla isabelline, Mortierrla vinacea, Mortieralla vinaceae var.
  • Mortierella alpina 1S-4 Mortieralla isabelline
  • Mortierrla vinacea Mortieralla vinaceae var.
  • Mucor e.g., Mucor miehei Cooney et Emerson (Rhizomucor miehei (Cooney & R. Emerson)) Schipper, Mucor pusillus Lindt, Mucor circinelloides Mucor mucedo
  • Myceliophthora e.g., Myceliophthora thermophila
  • Myrothecium Neocallimastix
  • Neurospora e.g., Neurospora crassa
  • Paecilomyces Penicillium (e.g., Penicillium.
  • chrysogenum Pennicillium iilacinum, Penicillium roquefortii), Phenerochaete, Phlebia, Piromyces, Pythium, Rhizopus (e.g., Rhizopus niveus), Schizophyllum, Scytalidium, Sporotrichum (e.g., Sporotrichum.
  • Trichoderma e.g., Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichoderma virens, Trichoderma cit rinoviride , Trichoderma viride).
  • Non-limiting examples of suitable bacteria include firmicutes, cyanobacteria (blue- green algae), osciilatoriophcideae, bacillales, lactobacillales, oscillatoriales, bacillaceae, lactobacillaceae, and members of any of the following genera, and deri vatives and crosses thereof: Acinetobacter, Acetobacter (e.g., Acetobacter suboxydans , Acetobacter xylinum), Actinoplane (e.g., Actinoplane missouriensis), Arthrospira (e.g., Arthrospira platensis, Arthrospira maxima), Bacillus (e.g., Bacillus cercus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis), Escherichia (e.g., Escherichia colt), Lactobacillus (e.g., Lactobacill
  • Streptococcus lactis Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus
  • Streptomyces e.g., Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus
  • Tetrahymena e.g., Tetrahymena thermophile, Tetrahymena hegewischi, Tetrahymena hyperangularis.
  • Tetrahymena malaccensis Tetrahymena pigmentosa, Tetrahymena pyriformis, Tetrahymena vorax
  • Xanthomonas e.g., Xanthomonas campestris
  • Non-limiting examples of suitable algae include members of any of the following genera, and derivatives and crosses thereof: red algae, brown algae, green algae, microalgae, Achnanthes (e.g., Achnanthes orientalis), Agmenellum, Alaria (e.g., Alaria marginata), Amphiprora (e.g., Amphiprora hyaline), Amphora (e.g., Amphora coffeiformis, Amphora coffeiformis tinea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora americanissima, Amphora strigissima, Amphora americanissima capitata, Amphora sp.), Anabaena.
  • red algae brown algae, green algae, microalgae
  • Achnanthes e.g., Achnanthes orientalis
  • Agmenellum e.g., Al
  • Analipus e.g., Analipus japonicus
  • Ankistrodesmus e.g., Ankistrodesmus falcatus
  • Ascophyllum e.g., Ascophyllum nodosum
  • Boekelovia e.g., Boekelovia hooglandii
  • Borodinella e.g., Borodinella sp.
  • Botryococcus e.g., Botryococcus braunii, Botryococcus sudeticus
  • Carteria Chaetoceros (e.g., Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp.)
  • Chlorella e.g., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellips
  • Chlorella miniata Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoid.es var. acidicola, Chlorealla, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var.
  • Chlorella salina Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f terlia, Chlorella vulgaris var. autotrophica, Chlorella. vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var.
  • Chlorococcum e.g., Chlorococcum infusionum, Chlorococcum sp.
  • Chlorogonium Chondrus (e.g., Chondrus crispus, Chondrus ocellatus), Chroomonas (e.g., Chroomonas sp.), Chrysosphaera (e.g., Chrysosphaera sp.), Cricosphaera (e.g., Cricosphaera sp.), Cryptomonas (e.g., Cryptomonas sp.), Cyclotella (e.g., Cyclotella cryptica, Cyclotella meneghiniana , Cyclotella sp.), Dunaliella (e.g., Dunaliella sp., Dunaliella bardaw
  • Furcellaria e.g., Furcellaria fastigiate
  • Gigartina e.g., Gigartina acicularis, Gigartina bursa-pastoris, Gigartina pistillata, Gigartina radula, Gigartina skottsbergii, Gigartina stellate
  • Gleocapsa e.g., Gleocapsa sp.
  • Gloeothamnion e.g., Gloeothamnion sp.
  • Gloiopeltis e.g., Gloiopeltis furcate
  • Gracilaria e.g., Gracilaria bursa-pastoris, Gracilaria lichenoides
  • Hizikia e.g., Hizikia fusiforme
  • Hymenomonas e.g., Hymenomonas sp.
  • Isochrysis
  • Prototheca zopfii Pyramimonas (e.g., Pyramimonas sp.), Pyrobotrys, Rhodella (e.g., Rhodella maculate, Rhodella reticulata, Rhodella violacea), Rhodymenia (e.g., Rhodymenia palmata), Sarcinoid (e.g., Sarcinoid chrysophyte), Scenedesmus (e.g., Scenedesmus armatus), Scytosiphon (e.g., Scytosiphon lome), Spirogyra, Spirulina (e.g., Spirulina platensis), Stichococcus (e.g., Stichococcus sp.), Synechococcus (e.g., Synechococcus sp.), Tetraedron, Tetraselmis (e.g., Tetraselmis sp., Tetrasel
  • a recombinant host cell may comprise a genetic modification that improves production of the recombinant ⁇ -lactoglobulin protein.
  • suitable genetic modifications include altered kinase activities, altered phosphatase activities, altered protease activities, altered gene expression induction pathways, altered production and/or activity of a protein involved in protein folding, and altered production and/or activity of a protein involved in protein secretion (e.g., vesicular transport).
  • ⁇ -lactoglobulin protein according to any of the above with bound hydrophobic bioactive agent (e.g., hydrophobic bioactive agent according to any of the above).
  • composition comprising Recombinant Milk Protein
  • composition that comprises a recombinant ⁇ - lactoglobulin protein according to any of the above, or a recombinant ⁇ -lactoglobulin protein wi th bound hydrophobic bioactive agent according to any of the above.
  • the composition may comprise between 0.001% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%. 8%, 7%, 6%. 5%, 4%, 3%, 2%, 1%. 0.9%. 0.8%. 0.7%. 0.6%.
  • 0.5%, 0.4%, or 0.3% between 0.3% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, or 0.4%; between 0.4% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%. 45%, 40%, 35%, 30%, 25%, 20%, 15%.
  • 2% 1%, or 0,9%: between 0.9% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%;, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%; between 1% and 100%, 95%. 90%, 85%. 80%, 75%, 70%, 65%, 60%. 55%, 50%, 45%, 40%, 35%. 30%, 25%.
  • the composition according to any of the above may be a fluid, semi-solid (e.g., gelatinous), solid, or powder.
  • the powder may comprise a moisture content of less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, or less than 1%; or between 0.1% and 20%, 15%, 10%, 5%, or 1%; between 1% and 20%, 15%, 10%, or 5%; between 5% and 20%, 15%, or 10%; between 10% and 20%, or 15%; or between 15% and 20%.
  • the powder may be used in powder form, or the powder may be reconstituted with a hydrating agent prior to use, or the powder may be mixed with other dry components (e.g., flour, sugar, minerals, pH or ionic strength adjusting agents) before a hydrating agent is added to the mixture.
  • suitable hydrating agents include water, milk (e.g., animal milk, nut milk, plant-based milk), juice (e.g., vegetable juice, fruit juice, other plant juice), brine (e.g., fluid or liquid used to soak beans or legumes), and mixtures thereof.
  • composition according to any of the above may comprise or consist of a recombinant ⁇ -lactoglobulin protein according to any of the above, or of a recombinant ⁇ - lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above, as a monomer, and/or may comprise a recombinant ⁇ -lactoglobulin protein according to any of the above, or of a recombinant ⁇ -lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above, as a polymer comprising linked repeated protein monomers, wherein the repeated protein monomers comprise or consist of a recombinant ⁇ -lactoglobulin protein according to any of the above, or of a recombinant ⁇ -lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above, or of dimers, trimers, or tetramers of a recombinant ⁇ -lactoglobulin protein according to any of any of any of
  • compositions comprising linked repeated protein monomers that comprise or consist of recombinant ⁇ -lactoglobulin proteins, and methods for producing such polymers, are disclosed in patent publication US20210235714, filed August 15, 2012.
  • the composition according to any of the above may comprise the recombinant ⁇ -lactoglobulin protein, or the recombinant ⁇ -lactoglobulin protein with bound hydrophobic bioactive agent, as a monomer or as a polymer, comprised (e.g., covalently bound, non-covalently bound, encapsulated, physically trapped) in another polymer.
  • Non-limiting examples of suitable polymers in which the recombinant ⁇ -lactoglobulin protein monomer or polymer according to any of the above may be comprised include a polymer comprising linked repeated monomers of a polysaccharide (e.g., cellulose, cellulose derivatives [e.g., hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose], heparin, hyaluronic acid, pectin, chondroitin sulfate, pullulan, dextrin, dextran), alginate [e.g., calcium alginate], alginate derivatives, chitosan, chitin) or a protein (e.g., collagen, gelatin, elastin, fibrinogen, fibrin, keratin).
  • a polysaccharide e.g., cellulose, cellulose derivatives [e.g., hydroxyethyl cellulose, hydroxymethyl cellulose
  • composition according to any of the above may comprise between 0.001% and 100% (e.g., between 0.001% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1 %, or 0.01 %; between 0.01% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1%; between 0.1% and 100%, 99%, 95%.
  • the protein polymer may comprise between about 0.001% and about 100% (e.g., between 0.001% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01%; between 0.01% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1%; between 0.1 % and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 1%; between 1% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1%; between 0.1 % and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 1%; between 1% and 100%,
  • composition according to any of the above may be essentially free of any protein other than the recombinant protein contained in the composition according to any of the above.
  • composition according to any of the above may be essentially tree of any recombinant protein other than the recombinant protein contained in the composition according to any of the above.
  • composition according to any of the above may be essentially free of any recombinant milk protein other than the recombinant ⁇ -lactoglobulin protein contained in die composition according to any of the above.
  • composition according to any of the above may be essentially free of a component found in a mammal-produced milk (e.g., cow milk, goat milk, sheep milk, human milk, buffalo milk, yak milk, camel milk, llama milk, alpaca milk, horse milk, donkey milk), or may comprise a lower concentration of al least one component found in a mammal-produced milk.
  • a mammal-produced milk e.g., cow milk, goat milk, sheep milk, human milk, buffalo milk, yak milk, camel milk, llama milk, alpaca milk, horse milk, donkey milk
  • components found in mammal -derived milk include lactose, saturated fat. cholesterol, native milk proteins, and native milk lipids.
  • composition according to any of the above may be essentially free of a component obtained from an animal (i.e., a component that is native to an animal, including animal products [i.e., parts of an animal that are consumables or typically prepared for consumption by humans; e.g., animal meat, animal fat, animal blood]] animal byproducts [i.e., products that are typically not consumable by themselves but are the byproducts of slaughtering animals for consumption; e.g., animal bones, animal carcasses, and constituents isolated therefrom], products produced by an animal [e.g., mammal -derived milk, chicken eggs, bee honey], and consumables produced therefrom [e.g., gelatin, rennet, whey proteins extracted from mammal-derived milk, casein extracted from mammal-derived milk, milk lipid extracted from mammal-derived milk, animal lipids, animal proteins]), or comprise 2% or less by mass of such component.
  • an animal i.e., a component that is native to an animal, including
  • composition according to any of the above may be a food product.
  • the term "food product” as used herein refers to a composition that can be ingested by a human or an animal for dietary purposes (i.e., without ill health effects but with significant nutritional and/or caloric intake due to uptake of digested material in the gastrointestinal tract), including a domesticated animal (e.g., dog, cat), farm animal (e.g., cow, pig, horse), and wild animal (e.g., non-domesticated predatory animal).
  • the term includes compositions that can be combined with or added to one or more other ingredients to make a food product that can be ingested by a human or an animal.
  • Food products comprising ⁇ -lactoglobulin are desirable, particularly for athletes, as ⁇ - lactoglobulin protein has a high content of essential and branched-chain amino acids, which are thought to aide production of muscle tissue.
  • ⁇ -lactoglobulin is desirable as a food additive as it has good water binding ability, which property makes ⁇ -lactoglobulin suitable for managing water activity of food products.
  • ⁇ -lactoglobulin is desirable as a food additive as it has anti-microbial activity, which property makes ⁇ -lactoglobulin suitable for extending the shelf life of food products.
  • ⁇ -lactoglobulin is desirable as a food additive as it can readily absorb at interfaces, which property makes ⁇ -lactoglobulin suitable for producing highly stable dispersions in food products.
  • food products comprising ⁇ -lactoglobulin protein with bound hydrophobic bioactive agents are desirable as the ⁇ -lactoglobulin protein may solubilize, stabilize, and/or protect from degradation such hydrophobic bioactive agents until they are released by the ⁇ -lactoglobulin protein to exert their bioactivity.
  • the food product may be a food product, or may resemble a food product (i.e., may be a “substitute food product”), selected from any of the food product categories defined by the National Health and Nutrition Examination Survey (NHANES).
  • NHANES food product categories include snack foods and gums (e.g., snack bars, crackers, salty snacks from grain products, chewing gums); breads, grains, and pastas (e.g., oat breads and rolls, cornbread, corn muffins, tortillas, flour and dry mixes, biscuits, multi-grain breads and rolls, whole wheat breads and rolls, pastas, rye breads and rolls, cracked wheat breads and rolls, white breads and rolls): beverages (e.g., beers and ales, beverage concentrates, beverages, energy drinks, sports drinks, fluid replacements, soft drinks, carbonated beverages, juices, wines, beers, cocktails, nutrition drinks, nutrition powders, protein-enriched beverages, coffee, tea); sweets and desserts (e.g.,
  • the food product according to any of the above may be a dairy product, a supplemented dairy' product (i.e., a conventional dairy product that is supplemented with the recombinant ⁇ - lactoglobulin protein according to any of the above), or substitute dairy product (i.e., a food product that resembles a conventional dairy product).
  • a supplemented dairy' product i.e., a conventional dairy product that is supplemented with the recombinant ⁇ - lactoglobulin protein according to any of the above
  • substitute dairy product i.e., a food product that resembles a conventional dairy product
  • dairy product refers to milk (e.g., whole milk [at least 3.25% milk fat], partly skimmed milk [from 1% to 2% milk fat], skim milk [less than 0.2% milk fat], cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk, evaporated milk, milk foam), and products derived from milk, including but not limited to yogurt (e.g., whole milk yogurt [at least 6 grams of fat per 170 g], low- fat yogurt [between 2 and 5 grams of fat per 170 g], nonfat yogurt [0.5 grams or less of fat per 170 g], greek yogurt [strained yogurt with whey removed], whipped yogurt, goat milk yogurt, Labneh [labne], sheep milk yogurt, yogurt drinks [e.g..
  • yogurt e.g., whole milk yogurt [at least 3.25% milk fat], partly skimmed milk [from 1% to 2% milk fat], skim milk [less than 0.2% milk fat], cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk
  • cheese e.g., whey cheese such as ricotta; pasta filata cheese such as mozzarella; semi-soft cheese such as Havarti and Muenster: medium-hard cheese such as Swiss and Jarlsberg and halloumi; hard cheese such as Cheddar and Parmesan; washed curd cheese such as Colby and Monterey Jack; soft ripened cheese such as Brie and Camembert; fresh cheese such as cottage cheese, feta cheese, cream cheese, paneer, and curd), processed cheese, processed cheese food, processed cheese product, processed cheese spread, enzyme -modulated cheese; cold-pack cheese), dairy-based sauces (e.g., salad dressing, bechamel sauce, fresh sauces, frozen sauces, refrigerated sauces, shelf stable sauces), dairy spreads (e.g., low-fat spread, low-fat butter), cream (e.g., dry cream, heavy cream, light cream, whipping cream, half-and-half, coffee whitener,
  • cheese-based sauces e.g., salad
  • the food product according to any of the above may be an animal meat or animal meat product, a supplemented animal meat or animal meat product (i.e., a conventional animal meal or animal meat product that is supplemented with a recombinant ⁇ -lactoglobulin protein according to any of the above), or substitute animal meat or animal meat product (i.e., a food product that resembles a conventional animal meat or animal meat product).
  • animal meats and animal meat products include flesh obtained from skeletal muscle or from other organs (e.g., kidney, heart, liver, gallbladder, intestine, stomach, bone marrow, brain, thymus, lung, tongue), or parts thereof, obtained from an animal.
  • the animal meat may be dark or white meat.
  • Non-limiting examples of animals from which animal meat or animal meat product can be obtained include cattle, lamb, mutton, horse, poultry (e.g., chicken, duck, goose, turkey), fowl (e.g., pigeon, dove, grouse, partridge, ostrich, emu, pheasant, quail), fresh or salt water fish (e.g., catfish, tuna, spearfish, shark, halibut, sturgeon, salmon, bass, muskie, pike, bowfin, gar, eel, paddlefish, bream, carp, trout, walleye, snakehead, crappie, sister, mussel, scallop, abalone, squid, octopus, sea urchin, cuttlefish, tunicate), crustacean (e.g., crab, lobster, shrimp, barnacle), game animal (e.g., deer, fox, wild pig, elk, moose, reindeer, caribou,
  • the animal meat or animal meat product may be ground, chopped, shredded, or otherwise processed, and uncooked, cooking, or cooked.
  • the food product according to any of the above may be an egg or egg product, a supplemented egg product (i.e., a conventional egg or egg product that is supplemented with the recombinant ⁇ -lactoglobulin protein according to any of the above), or substitute egg or egg product (i.e., a food product that resembles a conventional egg or egg product).
  • eggs or egg products include whole egg (e.g., liquid whole egg, spray-dried whole egg, frozen whole egg), egg white (e.g., liquid egg white, spray-dried egg white, frozen egg white), egg yolk, egg dishes, egg soups, mixtures made with egg whites, mixtures made with egg substitutes, mayonnaise, custard, and salad dressings.
  • Resemblance of a substitute food product provided herein to a conventional food product may be due to any physical, mechanical, chemical/biological, sensory, and/or functional attribute.
  • composition according to any of the above may be a cosmetic or personal care composition.
  • cosmetic or personal care composition refers to a composition that upon application to a body surface (i.e., an exposed area of a human body, such as skin, hair, nail, tooth, and tissues of the oral cavity [e.g., gums]) confers a perceived or actual beautifying or hygienizing effect.
  • Non-limiting examples of cosmetic or personal care compositions include anti-wrinkling treatments (i.e., compositions used for tensioning [e.g., smoothing out of skin, reducing wrinkles in skin, removing fine lines in skin]), anti-aging treatments (i.e., compositions used for removing signs of aging [e.g., wrinkles, fine lines, manifestations of photodamage (e.g., sun spots)]), sun protection (i.e., compositions used to protect against UV exposure), anti-burn treatments (i.e., compositions used for soothing burns [e.g., sunburns]), anti-acne treatments (i.e., compositions that are effective in the treatment of acne and/or the symptoms associated therewith), skin cleansers (i.e., compositions used for cleaning skin and/or skin pores [e.g., nose strips for pore cleaning]), anti-dandruff treatments (i.e., compositions used for reducing or eliminating dandruff), anti-body odor treatments (i.
  • composition according to any of the above may be a therapeutic product that can be used for treating a disorder, disease, or injury in a subject.
  • Non-limiting examples of disorders, diseases, or injuries include cancer, microbial infections or contaminations, viral infections, skin defects (e.g., rashes, sores, abrasions, burns, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers. diabetic ulcers, diabetic foot ulcers, decubitus ulcers, itchy skin), or pain (e.g., acute pain, chronic pain).
  • cancer microbial infections or contaminations
  • viral infections e.g., rashes, sores, abrasions, burns, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers. diabetic ulcers, diabetic foot ulcers, decubitus ulcers, itchy skin
  • skin defects e.g., rashes, sores, abrasions, burns, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers. diabetic ulcers, diabetic foot
  • a method for obtaining a recombinant host cell according to any of the above comprises; obtaining a polynucleotide that encodes a recombinant ⁇ -lactoglobulin protein (and optional secretion signal) according to any of the above, or a recombinant expression construct according to any of the above, or a recombinant vector according to any of the above; and introducing the polynucleotide, recombinant expression construct, or recombinant vector into a host cell (e.g., any of the host cells disclosed herein) to obtain a recombinant host cell according to any of the above,
  • a host cell e.g., any of the host cells disclosed herein
  • polynucleotide, recombinant expression construct, and/or recombinant vector may- be obtained by any suitable method known in the art, including, without limitation, direct chemical synthesis and cloning.
  • Methods for introducing a polynucleotide, recombinant expression construct, or recombinant vector into a host cell are well-known in the art.
  • Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, DEAE-dextran transfection, cell squeezing, sonoporation, optical transfection, protoplast fusion, protoplast transformation, impalefection, hyrodynamic delivery, gene gun, magnetofection, viral transduction, electroporation, and chemical transformation (e.g., using PEG).
  • Methods for identifying a recombinant host cell include screening for expression of a drug resistance or auxotrophic marker encoded by the polynucleotide, recombinant expression construct, or recombinant vector that permits selection for or against growth of cells, or by other means (e.g., detection of light emitting peptide comprised in the polynucleotide, recombinant expression construct, or recombinant vector, molecular analysis of individual recombinant host cell colonies [e.g., by restriction enzyme mapping, PCR amplification, Southern analysis, or sequence analysis of isolated extrachromosomal vectors or chromosomal integration sites]).
  • a drug resistance or auxotrophic marker encoded by the polynucleotide, recombinant expression construct, or recombinant vector that permits selection for or against growth of cells, or by other means (e.g., detection of light emitting peptide comprised in the polynucleotide, recombinant expression construct
  • Production of the recombinant ⁇ -lactoglobulin protein by a recombinant host cell according to any of the above may be evaluated using any suitable method known in the art, such as assays that are carried out at the RNA level and. most suitable, at the protein level, or by use of functional bioassays that measure the production or activity of the recombinant ⁇ -lactoglobulin protein.
  • Non-limiting examples of such assays include Northern blotting, dot blotting (DNA or RNA), RT-PCR (reverse transcriptase polymerase chain reaction), RNA-Seq, in situ hybridization, Southern blotting, enzyme activity assays, immunological assays (e.g., immunohistochemical staining, immunoassays, Western blotting, ELISA), and free thiol assays (e.g., for measuring production of protein comprising free cysteine residues).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RNA-Seq in situ hybridization
  • Southern blotting enzyme activity assays
  • immunological assays e.g., immunohistochemical staining, immunoassays, Western blotting, ELISA
  • free thiol assays e.g., for measuring production of protein comprising free cysteine residues.
  • a method for producing a recombinant ⁇ - lactoglobulin protein according to any of the above comprises: fermenting a recombinant host cell according to any of the above in a culture medium under conditions suitable for production of the recombinant ⁇ -lactoglobulin protein.
  • the method may further comprise: purifying the recombinant ⁇ -lactoglobulin protein from the fermentation broth to obtain a preparation comprising the recombinant ⁇ -lactoglobulin protein; and/or post-processing the recombinant ⁇ -lactoglobulin protein.
  • the recombinant ⁇ -lactoglobulin protein may be obtained using in vitro methods (e.g., using cell-free transcription and/or translation systems)
  • Suitable conditions for producing the recombinant ⁇ -lactoglobulin protein are typically those under which a recombinant host ceil according to any of the above can grow and/or remain viable, and produce the recombinant ⁇ -lactoglobulin protein.
  • Non-limiting examples of suitable conditions include a suitable culture medium (e.g., a culture medium having a suitable nutrient content [e.g., a suitable carbon content, a suitable nitrogen content, a suitable phosphorus content], a suitable supplement content, a suitable trace metal content, a suitable pH), a suitable temperature, a suitable feed rate, a suitable pressure, a suitable level of oxygenation, a suitable fermentation duration (i.e., volume of culture media comprising the recombinant host cells), a suitable fermentation volume (i.e., volume of culture media comprising the recombinant host cells), and a suitable fermentation vessel.
  • a suitable culture medium e.g., a culture medium having a suitable nutrient content [e.g., a suitable carbon content, a suitable nitrogen content, a suitable phosphorus content], a suitable supplement content, a suitable trace metal content, a suitable pH
  • a suitable temperature e.g., a suitable feed rate, a suitable pressure, a suitable level of
  • Suitable culture media include all culture media in which the recombinant host cell can grow and/or remain viable, and produce the recombinant ⁇ -lactoglobulin protein.
  • the culture medium is an aqueous medium that comprises a carbon source, an assimilable nitrogen source (i.e., a nitrogen-containing compound capable of releasing nitrogen in a form suitable for metabolic utilization by the recombinant host cell ), and a phosphate source.
  • Non-limiting examples of carbon sources include monosaccharides, disaccharides, polysaccharides, acetate, ethanol, methanol, glycerol, methane, and combinations thereof.
  • Non-limiting examples of monosaccharides include dextrose (glucose), fructose, galactose, xylose, arabinose, and combinations thereof.
  • Non-limiting examples of disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.
  • Non-limiting examples of polysaccharides include starch, glycogen, cellulose, amylose, hemicellulose, maltodextrin, and combinations thereof.
  • Non-limiting examples of assimilable nitrogen sources include anhydrous ammonia, ammonium sulfate, ammonium hydroxide, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium pyrophosphate, ammonium chloride, sodium nitrate, urea, peptone, protein hydrolysates, corn steep liquor, corn steep solids, spent grain, spent grain extract, and yeast extract.
  • Use of ammonia gas is convenient for large scale operations, and may- be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia may also be employed to assist in pH control.
  • the culture medium may further comprise an inorganic salt, a mineral (e.g., magnesium, calcium, potassium, sodium; e.g., in suitable soluble assimilable ionic and combined forms), a metal or transition metal (e.g., copper, manganese, molybdenum, zinc, iron, boron, iodine; e.g., in suitable soluble assimilable form), a vitamin, and any other nutrient or functional ingredient (e.g., a protease [e.g., a plant-based protease] that can prevent degradation of the recombinant ⁇ -iactoglobulin protein, a protease inhibitor that can reduce the activity of a protease that can degrade the recombinant ⁇ -lactoglobulin protein, and/or a sacrificial protein that can siphon away protease activity, an anti-foaming agent, an anti-microbial agent, a surfactant
  • Suitable culture media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).
  • a suitable pH may be a pH of between about 2 and about 8 (e.g., a pH of between 2 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4, 3.5, 3, or 2.5; between 2.5 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2. 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4, 3.5, or 3: between 3 and 8,
  • a suitable temperature may be a temperature of between about 20°C and about 46°C (e.g., between 20°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C, 28°C, 26°C, 24°C, or 22°C; between 22°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C.
  • a suitable feed rate may be a feed rate of between about 0.01 g and about 0.2 g glucose equivalent per g dry cell weight (DCW) per hour.
  • a suitable pressure may be a pressure of between 0 psig and about 50 psig (e.g., between 0 psig and 50 psig, 40 psig, 30 psig, 20 psig, or 10 psig; between 10 psig and 50 psig, 40 psig, 30 psig, or 20 psig; between 20 psig and 50 psig, 40 psig, or 30 psig; between 30 psig and 50 psig, or 40 psig; or between 40 psig and 50 psig).
  • a suitable oxygenation may be an aeration rate of between about 0.1 volumes of oxygen per liquid volume in the fermentor per minute (vvm) and about 2.1 vvm (e.g., between 0.1 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1 ,5 vvm, 1.3 vvm, 1.1 vvm, 0.9 vvm, 0.7 vvm, 0.5 vvm, or 0.3 vvm; between 0.3 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, 1.3 vvm, 1.1 vvm, 0.9 vvm, 0.7 vvm, or 0.5 vvm; between 0.5 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, 1.3 vvm, 1.1 vvm, 0.9 vv
  • a suitable fermentation duration may be a fermentation duration of between about 10 hours and about 500 hours (e.g., between 10 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, 40 hours, 30 hours, or 20 hours; between 20 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, 40 hours, or 30 hours; between 30 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, or 40 hours; between 40 hours and 500 hours, 400 hours, 300 hours, 200 hours.
  • 10 hours and 500 hours e.g., between 10 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, 40 hours, 30 hours, or 20 hours; between 20 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, 40 hours, or 30 hours; between 30 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, or 40 hours; between 40 hours and 500 hours, 400 hours, 300 hours, 200 hours.
  • a suitable fermentation volume may be between about 1 L and about 10,000,000 L (e.g., between 1 L and 10,000,000 L, 5,000,000 L, 1 ,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, 500 L, 100 L, 50 L, or 10 L; between 10 L and 10,000,000 L, 5,000,000 L, 1,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, 500 L, 100 L, or 50 L; between 50 L and 10.000.000 L, 5,000,000 L, 1.000.000 L, 500,000L, 100.000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, 500 L, or 100 L; between 100 L and 10,000,000 L, 5,000,000 L, 1,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, or 500 L; between 500 L and 10,000,000 L, 5,000,000 L, 1,000,000 I.,, 500,0001.., 100,000 L, 50,000 L, 10, 000 L, 5,000 L, or 500 L;
  • a suitable fermentation vessel may be any fermentation vessel known in the art.
  • Non- limiting examples of suitable fermentation vessels include culture plates, shake flasks, fermenters (e.g., stirred tank fermenters, airlift fermenters, bubble column fermenters, fixed bed bioreactors, laboratory fermenters, industrial fermenters, or any combination thereof), used at any suitable scale (e.g., small-scale, large-scale) and in any process (e.g., solid culture, submerged culture, batch, fed-batch, or continuous-flow).
  • fermenters e.g., stirred tank fermenters, airlift fermenters, bubble column fermenters, fixed bed bioreactors, laboratory fermenters, industrial fermenters, or any combination thereof
  • suitable scale e.g., small-scale, large-scale
  • process e.g., solid culture, submerged culture, batch, fed-batch, or continuous-flow.
  • Methods for purifying a recombinant protein e.g., from a fermentation broth to obtain a preparation comprising the recombinant protein are well-known in the art (see, for example, Protein Purification, JC Janson and L Ryden, Eds., VCH Publishers, New York, 1989; Protein Purification Methods: A Practical Approach, ELV Harris and S Angel. Eds., IRL Press, Oxford, England, 1989, respectively), and may be adapted to purify a recombinant ⁇ -lactoglobulin protein according to any of the above.
  • a recombinant ⁇ -lactoglobulin protein according to any of the above may be purified on the basis of its molecular weight, for example, by size exclusion/exchange chromatography, ultrafiltration through membranes, gel penneation chromatography (e.g., preparative disc-gel electrophoresis), or density centrifugation.
  • a recombinant ⁇ -lactoglobulin protein according to any of the above also may be purified on the basis of its surface charge or hydrophobicity/hydrophilicity, for example, by isoelectric precipitation, anion/cation exchange chromatography, isoelectric focusing (IEF), or reverse phase chromatography.
  • a recombinant ⁇ -lactoglobulin protein according to any of the above also may be purified on the basis of its solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents, or solvent extraction.
  • a recombinant ⁇ -lactoglobulin protein according to any of the above also may be purified on the basis of its affinity to another molecule, for example, by affinity chromatography, reactive dyes, or hydroxyapatite.
  • Affinity chromatography may include the use of an antibody having a specific binding affinity for the recombinant ⁇ -lactoglobulin protein, or a lectin to bind to a sugar moiety on the recombinant ⁇ -lactoglobulin protein, or any other molecule that specifically binds the recombinant ⁇ -lactoglobulin protein.
  • the recombinant ⁇ -lactoglobulin protein may comprise a purification tag operably linked to its C -terminus, N -terminus, or both to facilitate affinity-based purification of the recombinant ⁇ -lactoglobulin protein.
  • suitable purification tags include affinity tags (i.e.
  • peptides or polypeptides that bind to certain agents or matrices solubilization tags (i.e., peptides or polypeptides that assist in proper folding of proteins and prevent precipitation), chromatography tags (i.e., peptides or polypeptides that alter the chromatographic properties of a protein to afford different resolution across a particular separation techniques), epitope tags (i.e., peptides or polypeptides that are bound by antibodies), fluorescence tags, chromogenic tags, enzyme substrate tags (i.e., peptides or polypeptides that are the substrates for specific enzymatic reactions), chemical substrate tags (i.e., peptides or polypeptides that are the substrates for specific chemical modifications), self-cleaving tags (peptides or polypeptides that possess inducible proteolytic activity; e.g., Sortase tag, Npro tag, FrpC module, CPD), hydrophobic tags (proteins or polypeptides that are highly
  • Non-limiting examples of suitable affinity tags include maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag, poly (His) tag, hexa(His) tag, S BP-tag, Strep-tag, and calmodulin-tag.
  • suitable solubility tags include thioredoxin (TRX) tag, poly(NANP) tag, MBP tag, SUMO tag, GB1 tag, NUSA CBD tag, and GST tag.
  • Non-limiting examples of chromatography tags include polyanionic amino acid tags (e.g., FLAG-tag) and polyglutamate tag.
  • Non-limiting examples of epitope tags include V5-tag, VSV-tag, E-tag, NE- tag, hemagglutinin (Ha)-tag, Myc-tag, and FLAG-tag.
  • Non-limiting examples of fluorescence tags include green fluorescent protein (GFP) tag, blue fluorescent protein (BFP) tag, cyan fluorescent protein (CFP) tag, yellow fluorescent protein (YFP) tag, orange fluorescent protein (OFP) tag, red fluorescent protein (RFP) tag, and derivatives thereof.
  • Non-limiting examples of enzyme substrate tags include peptides or polypeptides comprising a lysine within a sequence suitable for biotinilation (e.g., AviTag, Biotin Carboxyl Carrier Protein [BCCP]).
  • Non-limiting examples of chemical substrate tags include substrates suitable for reaction with FIAsH-EDT2.
  • the tag peptide or polypeptide may be removed following isolation of the recombinant ⁇ -lactoglobulin protein (e.g., via protease cleavage).
  • the recombinant ⁇ -lactoglobulin protein may be purified directly from the culture medium. In other embodiments, the recombinant ⁇ -lactoglobulin protein may be purified from a cell lysate.
  • the recombinant ⁇ -lactoglobulin protein may be purified to obtain a preparation comprising the recombinant ⁇ -lactoglobulin protein at a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%', greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% relative to other components comprised in the fermentation broth; or to at least 2 -fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater abundancy relative to other components comprised in the fermentation broth; or to a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%', greater than 65%, greater than 70%, greater than 75%,
  • the identity of the recombinant ⁇ -lactoglobulin protein may be confirmed and/or quantified by high performance liquid chromatography (HPLC), Western blot analysis. Eastern blot analysis, polyacrylamide gel electrophoresis, capillary electrophoresis, formation of an enzyme product, disappearance of an enzyme substrate, and 2-dimensional mass spectroscopy (2D-MS/MS) sequence identification.
  • HPLC high performance liquid chromatography
  • Western blot analysis Eastern blot analysis, polyacrylamide gel electrophoresis, capillary electrophoresis, formation of an enzyme product, disappearance of an enzyme substrate, and 2-dimensional mass spectroscopy (2D-MS/MS) sequence identification.
  • Post-processing may alter certain chemical and/or physical properties of the recombinant ⁇ -lactoglobulin protein, including but not limited to size, charge, hydrophobicity, hydrophilicity, solvation, protein folding, and chemical reactivity.
  • Post-processing may comprise refolding of the recombinant ⁇ -lactoglobulin protein (e.g., by removing a denaturant); fragmenting of the recombinant ⁇ -lactoglobulin protein (e.g., by chemical means or by exposure to proteases [e.g., trypsin, pepsin]); heating of the recombinant ⁇ - lactoglobulin protein (e.g., to remove protein aggregates); removing reactive sites of the recombinant ⁇ -lactoglobulin protein (e.g., removing reactive sites of methionine and/or tryptophan residues by oxidation); modulating of the recombinant ⁇ -lactoglobulin protein (e.g...
  • demineralizing of a preparation comprising the recombinant ⁇ -lactoglobulin protein by, e.g., dialysis, ultrafiltration, reverse osmosis, ion exchange chromatography); removing tags and/or fusion polypeptides from the recombinant ⁇ - lactoglobulin protein (e.g., by exposure to site- specific proteases); biotinylating of the recombinant ⁇ -lactoglobulin protein (i.e., attaching biotin); and/or conjugating of the recombinant ⁇ - lactoglobulin protein to other elements (e.g., poly-ethylene-glycol, antibodies, liposomes, phospholipids, DNA, RNA, polynucleotides, sugars, disaccharides, polysaccharides, starches, cellulose, detergents, cell walls).
  • elements e.g., poly-ethylene-glycol, antibodies, liposomes, phospholipids, DNA,
  • Post-processing may occur in a random manner or in a site-specific manner (e.g., at sulfhydryl groups of cysteine residues [e.g., for aminoethylation, formation of iodoacetamides, formation of maleimides, formation of Dha, covalent attachment via disulfide bonds, and desulfurization], at primary amine groups of lysine residues [e.g., for attachment of activated esters, sulfonyl chlorides, isothiocyanates, unsaturated aldehyde esters, and aldehydes], at phenolic hydroxyl groups of tyrosine residues, at specific allergenic epitopes [e.g., glycan groups]).
  • sulfhydryl groups of cysteine residues e.g., for aminoethylation, formation of iodoacetamides, formation of maleimides, formation of Dha, covalent attachment via disulfide bonds, and
  • the recombinant ⁇ -lactoglobulin protein may be dried (e.g., via spray drying or lyophilization) or concentrated (e.g., via precipitation or evaporation) (e.g., to obtain a powder).
  • a method for producing a composition according to any of the above e.g., a food product according to any of the above
  • the method comprises the step of obtaining a recombinant ⁇ -lactoglobulin protein according to any of the above.
  • composition is a food product (e.g., the food product according to any of the above), a variety of recipes known in the art may be used to prepare the food product.
  • composition comprises or consists of a polymer or polymer network according to any of the above
  • a variety of methods for polymerizing protein monomers known in the art may be used to polymerize the recombinant ⁇ -lactoglobulin protein (see, for example, patent publication US20210235714, filed October 30, 2020).
  • the hydrophobic bioactive agent may be bound to the recombinant ⁇ -lactoglobulin protein prior or post polymerization.
  • conditions for polymerization are most suitably selected to not disrupt binding of the hydrophobic bioactive agent to the recombinant ⁇ -lactoglobulin protein and/or to not denature a tertiary or quaternary structure of the recombinant ⁇ -lactoglobulin protein.
  • Such conditions may include polymerization under non- or mildly denaturing conditions, such as disclosed, for example, in PCT publication WO2021168343, published August 26, 2021.
  • a method for administering a hydrophobic bioactive agent to a subject comprises administering to the subject a composition according to any of the above.
  • the subject may be a human or other animal (e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig]).
  • a mammal e.g., cow, sheep, goat, rabbit, pig
  • Administering the composition to the subject may occur via any route, including via oral administration.
  • Example 1 Production of Recombinant ⁇ -lactoglobulin Proteins Having Improved Binding of Bioactive Agent
  • the recombinant vector is constructed using genetic engineering methods known in the art.
  • the recombinant vector comprises an expression construct comprising a protein coding sequence encoding ⁇ -lactoglobulin protein (“recombinant ⁇ -lactoglobulin protein ORF”; e.g., any of SEQ ID NOs: 1-9); codon- optimized for expression in the host cell, and operably linked to an N-terminal secretion signal sequence (e.g., pre or pre-pro signal peptide of proteins CBH1 or XYN1 for Trichoderma host cell; pre or pre -pro signal peptide of protein GLAA for Aspergillus host cell; pre or pre-pro signal peptide of Saccharomyces cerevisiae alpha mating factor for Pichia pastor is host cell): and under control of a suitable promoter sequence (e.g., promoter sequence of cbhl,
  • the recombinant vector further comprises a polynucleotide that can direct integration of the expression construct into the genome of the host cell (e.g., into the cbh1 or egl1 locus for Trichoderma host cells; into the glaA locus for Aspergillus host cells; into the aoxl locus for Pichia host cells), selection markers for selection of bacterial and/or fungal transformants, and a bacterial origin of replication.
  • the bacterial selection markers and origin of replication are removed from the recombinant vector via restriction enzyme digestion prior to transformation of the recombinant vector into the host cell.
  • site-directed mutagenesis is performed according to the manufacturer’s direction on the recombinant vectors above to introduce the amino acid substitutions listed in Table 1 into the encoded ⁇ -lactoglobulin protein, as well as any combination of two or more of such amino acid substitutions.
  • the recombinant vectors are transformed into the host cell (e.g., Trichoderma reesei, Aspergillus niger, Pichia pastoris ( Komagataella phaffii; e.g., strain BG12 [(Biogrammatics, Carlsbad, CA]), and transformants are selected by growth on minimal media or antibiotics for positive selection.
  • the transformants are grown in expression media, in 24-well plates, and culture supernatants are harvested for identification of recombinant host cells comprising an integrated copy of the expression construct and secreting a recombinant ⁇ -lactoglobulin protein by SDS- PAGE gel analyses.
  • the recombinant host cells are fermented in a stirred fermentation vessel under conditions that permit cell growth and production of the recombinant ⁇ -lactoglobulin protein.
  • the fermentation is harvested after at least 100 hours, at a biomass concentration of between about 20 g and about 50 g dry cell weight (DCW) per L.
  • DCW dry cell weight
  • the biomass is removed from the broth by centrifugation at 5,000 x g.
  • the culture supernatant is concentrated over a membrane with suitable molecular weight cutoff.
  • the concentrate retentate is diafiltered over 5 kda MWCO membranes into 50 mM Imidazole, pH 6.8.
  • the concentrated retentate is passed over a Q sepharose FF column.
  • the mobile phase is 50 mM Imidazole, pH 6.8, and the recombinant ⁇ -lactoglobulin protein is eluted on a 2M NaCl gradient. The gradient is run from 0-30% over 30 column volumes. Peak fractions are collected and analyzed on RP-HPLC.
  • Example 2 Production of Polymer Comprising Recombinant ⁇ -lactoglobulin Protein Monomers
  • a recombinant ⁇ -lactoglobulin protein of Example 1 is combined with a weak acid (e.g., 5% acetic acid) or base (e.g., 5% sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide) alone or in combination with an other ingredient (e.g., any of the other ingredients disclosed herein) to a final concentration of between about 2% and about 18% by mass of the recombinant ⁇ -lactoglobulin protein, a final pH of between about 4 and about 11, and a final conductivity of between about 10 ms/cm and about 300 mS/cm.
  • a weak acid e.g., 5% acetic acid
  • base e.g., 5% sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide
  • an other ingredient e.g., any of the other ingredients disclosed herein
  • the mixture is heated for 20 minutes at a temperature and a pH at which the recombinant ⁇ -lactoglobulin protein is mildly denatured (e.g.. at which less than 20% of the recombinant ⁇ -lactoglobulin protein is denatured), such that the free thiol group( s ) of the recombinant ⁇ -lactoglobulin protein are exposed but existing intra-molecular disulfide bonds are not broken (e.g., at between 10°C and 20°C below the Tm of the recombinant pdactoglobulin protein [as determined in Example 3]).
  • the mixture is then cooled to 21°C or ambient temperature.
  • the polymer is captured by centrifugation (e.g., at 4,000g for 20 min), filtration, solvent extractions, chromatography, or other method.
  • the polymer is dried to a moisture content that still permits shaping. After shaping, the polymer is further dried to sei a final form.
  • a cylindrical (10-mm inner diameter, 40-mm length) stainless steel tube is filled with a solution comprising between about 2% and about 18% by mass of a recombinant ⁇ -lactoglobulin protein of Experiment 1 at a final pH of between about 4 and about 11 .
  • the tube is closed with rubber stoppers, sealed with vinyl electrical tape, and placed vertically in a water bath.
  • the solution is heated as described in Example 2 to obtain mildly denatured recombinant ⁇ -lactoglobulin protein.
  • the gel is cooled to room temperature, and then left at 4 °C overnight, before being removed from the tube and cut into 10-mm diameter, 2-mm thick tablets, and dried in an enclosed desiccator until the tablets reach a constant mass (i.e., within ⁇ 0.001 g).
  • a solution comprising between about 20% and about 40% by weight of a recombinant ⁇ -lactoglobulin protein of Example 1 is heated as described in Example 2 to obtain mildly denatured recombinant ⁇ -lactoglobulin protein.
  • the mildly denatured recombinant ⁇ -lactoglobulin protein is optionally combined with a synthetic co-polymer (e.g., polyvinyl acetate [PVAC], polyvinyl alcohol [PVA], polyvinyl pyrollidone [PVP]).
  • PVAC polyvinyl acetate
  • PVA polyvinyl alcohol
  • PVP polyvinyl pyrollidone

Abstract

Provided is a recombinant milk protein for delivery of a hydrophobic bioactive agent, a composition comprising such recombinant milk protein, and a method for using such composition.

Description

COMPOSITIONS AND METHODS FOR DELIVERING HYDROPHOBIC BIOACTIVE AGENTS
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/306,423, filed on February 3, 2022, which is incorporated herein by reference, in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the use of recombinant milk proteins for delivery of hydrophobic bioactive agents. In particular, the present invention relates to recombinant β-lactoglobulin proteins having improved binding of such hydrophobic bioactive agents, to compositions comprising such recombinant β-lactoglobulin proteins, and to methods for producing and using such compositions.
BACKGROUND OF THE INVENTION
[0003] The nutritional value of milk and its derivative products (e.g., yogurt, cheese, cream, butter) lies not just in the high- quality protein they comprise. Rather, milk proteins evolved to bind valuable nutrients, such as, for example, minerals and hydrophobic compounds, and thereby to help solubilize, stabilize, and/or protect from degradation or inactivation of such nutrients, and to carry them from mother to neonate. Based on this capability of milk proteins, strategies may be developed for using milk proteins as high-quality, natural food components that deliver bioactive agents to promote health and treat disorders (see, for example, Chen et al. 2006. Trends Food Sci Technol. 17:272).
[0004] A major shortcoming of some milk proteins as vehicles for delivery of bioactive agents is that their binding of certain bioactive agents in not stringent enough to prevent premature release of the bioactive agents (e.g., release prior to having reached the site of action of the bioactive agents, due to, for example, early temporal release or release under certain environmental conditions).
[0005] Therefore, there exists a need for milk proteins that can bind bioactive agents with greater affinity and/or stability, and compositions that comprise such milk proteins. SUMMARY OF THE INVENTION
[0006] Provided herein is a recombinant β-lactoglobulin protein comprising a modification compared to a native β-lactoglobulin protein that improves binding of a hydrophobic bioactive agent to the recombinant β-lactoglobulin protein.
[0007] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is native Bos taurus β- lactoglobulin protein (SEQ ID NO: 1), native Capra hircus β-lactoglobulin protein (SEQ ID NO: 3), or native Ovis aries β-lactoglobulin protein (SEQ ID NO: 5), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I A37M, A37V, A37Q, A37D, A37G, A37S. L39I, L39M, L39W, L39F, L39V, V41L V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H. I71 L, I71 F. I71 Y. I71 W, I71 V. I84L. I84M, I84F, I84V, I84 Y, I84W, A86G, A86H, A86Y, N90I, N90K, N90R, N90Q, N90S, N90T, N90D, N90E, V92I, V92L, V92M, V92F, V92Y, F105I, F105V, M107I, M107V, L122I, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R, K138H, K138E, K138D, K138L, K138I, K141Q, K141L, K141M, K141R, K141F, K141H, A142V, A142M, A142L A142G, A142T, L 1431, L143F, L143M, L143R, L143V, M145L, M145I, M145V, M145G, H146R, H146K, H146D, I147L, I147V, I147M, 1147K, I147F. R148Q, R148W, R148H. R148K, and R148E.
[0008] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is native Homo sapiens P~ lactoglobulin protein (SEQ ID NO: 2), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: A37L, A37L A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41 L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I84L, I84M, I84F, I84V, I84Y, I84W, N90I, N90K, N90R, N90Q, N90S, N90T, N90D, N90E, L122I, L122V, LI 22 Y, L122F, L143I. L143F, L143M, L143R, L143V, H146R, H146K, and H146D.
[0009] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is Equus asinus β - lactoglobulin protein (SEQ ID NO: 4), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43L V43L, L46I, L46F. L46M, L46V, L46Y, L46T. I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71F, I71Y, I71W, I71 V, F105I, F105V, L 123I, L123V, L123Y, L123F, L144I, L144F, L144M, L144R, L144V, H147R, H147K, and H147D.
[0010] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is native Equus caballus β-lactoglobulin protein (SEQ ID NO: 6), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W. L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71 Y, I71 W, I71 V, I84L, I84M, I84F, I84 V, I84Y, I84W, V92I, V92L, V92M, V92F, V92Y, M107I, M107V, L122I, L122V, L122Y, L122F, L1431, L143F, L143M, L143R, and L143V.
[0011] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is native Equus caballus β-lactoglobulin protein (SEQ ID NO: 7), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T. I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71F, I71 Y, I71W, I71 V, F105I, F105V, L1231, L123V, L123Y, L123F, L144I, L144F, L144M, L144R, and L144V.
[0012] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is native Equus asinus β- lactoglobulin protein (SEQ ID NO: 8), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L391, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71 Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, V92I, V92L, V92M, V92F. V92Y, M107I, M107V, L1221, L122V, L122Y, L122F, L143I, L143F, L143M, L143R, and L143V.
[0013] In some instances, the present invention comprises the recombinant β-lactoglobulin protein of paragraph [0006], wherein the native β-lactoglobulin protein is native Ovis aries β- lactoglobulin protein (SEQ ID NO: 9), and wherein the modification comprises or consists of one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43L V43L, L46I, L46F, L46M, L46V, L46Y, L46T. I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R. K69H, I71L, I71F, I71 Y, I71W, I71 V, I84L, I84M, I84F, I84V, I84Y, I84W, A86G, A86H, A86Y, N90I, N90K, N90R, N90Q, N90S, N90T, N90D, N90E, V92I, V92L, V92M, V92F, V92Y, F105I, F105V, M107I, M 107V, L122I, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R, K138H, K138E, K138D, K138L, K 138I, K141Q, K141L, K141M, K141R, K141F, K141H, A142V, A142M, A 142I, A142G, A142T, L143I, L143F, L143M, L143R, L143V, M145L, M145I, M145V, M145G, H146R, H146K, H146D, I147L, I 147V, I147M, I147K, I147F, R148Q, R148W, R148H, R148K, and R148E.
[0014] Further provided herein is a compositing comprising or consisting of the recombinant β-lactoglobulin protein of paragraphs [0006] through [0013].
[0015] In some instances, the present invention comprises the composition of paragraph [0014], wherein the composition further comprises a bioactive agent bound to the recombinant β - lactoglobulin protein .
[0016] In some instances, the present invention comprises the composition of paragraph [0014] or [0015], wherein the bioactive agent is bound to the recombinant β-lactoglobulin protein via covalent bonding.
[0017] In some instances, the present invention comprises the composition of paragraph [0014] or [0015], wherein the bioactive agent is bound to the recombinant β-lactoglobulin protein via non-covalent bonding. [0018] In some instances, the present invention comprises the composition of paragraph [0014] or [0015], wherein the bioactive agent is bound to the recombinant β-lactoglobulin protein via a linker peptide.
[0019] In some instances, the present invention comprises the composition of paragraphs [0014] through [0018], wherein the bioactive agent is bound to a solvent-exposed region of the recombinant β-lactoglobulin protein.
[0020] In some instances, the present invention comprises the composition of paragraphs [0014] through [0018], wherein the bioactive agent is bound to a not-solvent-exposed region of the recombinant β-lactoglobulin protein.
[0021] In some instances, the present invention comprises the composition of paragraphs [0014] through [0020], wherein the composition comprises between 0.1% and 100% by mass of a recombinant β-lactoglobulin protein.
[0022] In some instances, the present invention comprises the composition of paragraphs [0014] through [0021], wherein the recombinant β-lactoglobulin protein is a monomer.
[0023] In some instances, the present invention comprises the composition of paragraphs [0014] through [0021], wherein the recombinant β-lactoglobulin protein is a polymer or polymer network comprising linked repeated protein monomers, wherein the repeated protein monomers comprise or consist of the recombinant β-lactoglobulin protein of paragraphs [0006] through [0013], or of dimers, trimers, or tetramers of the recombinant β-lactoglobulin protein of paragraphs [0006] through [0013],
[0024] Further provided herein is a recombinant expression construct consisting of a polynucleotide comprising: a promoter sequence, an optional secretion signal sequence, a protein coding sequence encoding the recombinant β-lactoglobulin protein of paragraphs [0006] through [0013], and a termination sequence; wherein: the promoter sequence is operably linked in sense orientation to the optional secretion signal sequence and the protein coding sequence, the optional secretion signal sequence is operably linked in sense orientation to the protein coding sequence, and the one or more terminator sequences are operably linked to the protein coding sequence.
[0(125] Further provided herein is a recombinant vector comprising the recombinant expression construct of paragraph [0024].
[0026] Further provided herein is a recombinant host cell comprising the recombinant expression construct of paragraph [0024]. [0027] The recombinant host cell of paragraph [0026], wherein the recombinant host cell is derived from a bacterium.
[0028] The recombinant host cell of paragraph [0026], wherein the recombinant host cell is derived from a fungus.
[0029] The recombinant host cell of paragraph [0026], wherein the recombinant host cell is derived from a yeast.
[0030] The recombinant host cell of paragraph [0026], wherein the recombinant host cell is derived from a filamentous fungus.
[0031] Further provided herein is a method for obtaining the recombinant host cell of paragraphs [0026] through [0030], wherein the method comprises: obtaining a polynucleotide encoding the recombinant β-lactoglobulin protein of paragraphs [0003] through [0013], or the recombinant expression construct of paragraph [0024], or the recombinant vector of paragraph [0024]; and introducing the polynucleotide, recombinant expression construct, or recombinant vector, respectively, into a host cell.
[0032] Further provided herein is a method for producing the recombinant β-lactoglobulin protein of paragraphs [0006] through [0013], wherein the method comprises: fermenting the recombinant host cell of paragraphs [0026] through [0030] in a culture medium under conditions suitable for production of the recombinant β-lactoglobulin protein.
[0033] In some instances, the present invention comprises the method of paragraph [0032], wherein the method further comprises purifying the recombinant β-lactoglobulin protein from the fermentation broth to obtain a preparation comprising the recombinant β-lactoglobulin protein.
[0034] In some instances, the present invention comprises the method of paragraph [0032] or [0033], wherein the method further comprises post-processing the recombinant β-lactoglobulin protein.
[0035] Further provided herein is a method for producing the composition of paragraphs [0014] through [0021], wherein the method comprises obtaining the recombinant β-lactoglobulin protein of paragraphs [0006] through [0013],
[0036] In some instances, the present invention comprises the method of paragraph [0035], wherein the method further comprises polymerizing the recombinant β-lactoglobulin protein to obtain a polymer or polymer network comprising or consisting of the recombinant β-lactoglobulin protein. [0037] In some instances, the present invention comprises the method of paragraph [0035] or [0036], wherein the method further comprises binding the bioactive agent to the recombinant β - lactoglobulin protein.
INCORPORATION BY REFERENCE
[0038] All publications, patents, patent applications, sequences, database entries, scientific publications, and other references mentioned herein are incorporated by reference in their entireties to the same extent as if each individual publication, patent, patent application, sequence, database entry, scientific publication, or other reference was specifically and individually indicated to be incorporated by reference. To the extent the material incorporated by reference contradicts or is inconsistent with the present disclosure, the present disclosure, including definitions, will supersede any such material.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The terminology and description used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.
Definitions
[0040] Amino acids can be referred to herein by their single-letter codes, amino acid names, or three-letter codes. The single-letter codes, amino acid names, and three-letter codes are as follows: G Glycine (Gly), P - Proline (Pro), A - Alanine (Ala), V Valine (Vai), L - Leucine
(Leu), I - Isoleucine (Ile), M - Methionine (Met), C - Cysteine (Cys), F - Phenylalanine (Phe), Y -
Tyrosine (Tyr), W -Tryptophan (Trp), H - Histidine (His), K - Lysine (Lys), R - Arginine (Arg),
Q - Glutamine (Gin), N - Asparagine (Asn), E - Glutamic Acid (Glu), D - Aspartic Acid (Asp), S - Serine (Ser), T -Threonine (Thr). Amino acid residues are denoted by a first letter for the amino acid, followed by a number that specifies the position of the amino acid in a reference sequence (e.g., any of SEQ ID NOs: 1-9). Amino acid substitutions are denoted by a first letter for the amino acid that is to be replaced, followed by a number that specifies the position of the amino acid to be replaced in a reference sequence (e.g., any of SEQ ID NOs: 1-9), and a second leter that is to be substituted at the position in place of the amino acid that is to be replaced,
[0041] The terms "a" and "an" and "the" and similar references as used herein refer to both the singular and the plural (e.g., meaning "at least one" or "one or more"), unless otherwise indicated herein or clearly contradicted by context. For example, the term "a compound" is synonymous with the terms "at least one compound" and “one or more compounds”, and may refer to a single compound or to a plurality of compounds, including mixtures thereof.
[0042] The term "and/or" as used herein refers to multiple components in combination with or exclusive of one another. For example, "x, y, and/or z” may refer to "x” alone, "y” alone, "z" alone, "x, y, and z", "(x and y) or z", "(x and z) or y", "(y and z) or x", "x and y" alone, "x and z" alone, "y and z" alone, or “x or y or z".
[0043] The term “at least” or “one or more” as used herein refers to one, two, three, four, five, six, seven, eight, nine, ten, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more, or all of the elements subsequently listed.
[0044] The term “corresponding native β-lactoglobulin protein” as used herein refers to a native β-lactoglobulin protein that is identical to a recombinant β-lactoglobulin protein that is compared to the “corresponding native β-lactoglobulin protein” except that it does not comprise a modification as provided herein in the recombinant β-lactoglobulin protein.
[0045] The term "encoding" as used herein in context of a polynucleotide refers to a polynucleotide that comprises a coding sequence that when placed under the control of appropriate regulatory sequences is transcribed into mRNA that may be translated into a polypeptide. A coding sequence generally starts at a start codon (e.g., ATG) and ends at a stop codon (e.g., UAA, UAG and UGA). A coding sequence may contain a single open reading frame, or several open reading frames (e.g., separated by introns).
[0046] The term "essentially free of" as used herein refers to the indicated component being either not detectable in the indicated composition by common analytical methods, or to the indicated component being present in such trace amount as to not be functional. The term "functional" as used in this context refers to not materially contributing to properties of the composition comprising the trace amount of the indicated component, or to not having material activity (e.g., chemical activity, enzymatic activity) in the indicated composition comprising the trace amount of the indicated component, or to not having health-adverse effects upon use or consumption of the composition comprising the trace amount of the indicated component, The term “materially contributing’' as used herein refers to the indicated component contributing to an attribute of a composition to such extent that in the absence of the component (e.g., in a reference composition that is identical to the composition except that it lacks the indicated component) the attribute is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less present/active/measurable.
[0047] The term "filamentous fungus" as used herein refers to an organism from the filamentous form of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge. UK). A filamentous fungus is distinguished from a yeast by its hyphal elongation during vegetative growth.
[0048] The term "fungus" as used herein refers to organisms of the phyla Ascomycotas, Basidiomycota, Zygomycota, and Chythridiomycota, Oomycota, and Glomeromycota. It is understood, however, that fungal taxonomy is continually evolving, and therefore this specific definition of the fungal kingdom may be adjusted in the future. The term “fungal host cell” as used herein refers to a host cell that is obtained from a fungus.
[0049] The term "homolog" as used herein refers to a protein that comprises an amino acid sequence that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%) identical to a sequence of amino acids of a similar length (i.e., a length that is within +/- 20% of the length of the query amino acid sequence) comprised in a reference protein, and that has a functional property that is similar (e.g., is within 50%, within 40%, within 30%, within 20%, or within 10% of) or identical to that of the reference protein. The term includes polymorphic variants, interspecies homologs (e.g., orthologs), paralogs, and alleles of a protein, as well as variants that are man-made using genetic engineering techniques.
[0050] The term "host cell" as used herein refers not only to the particular subject cell but to the progeny of such cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the subject cell, but are still included within the scope of the term "host cell" as used herein. [0051] The terms “identity” or “identical” in the context of two or more polynucleotide or polypeptide sequences as used herein refer to the nucleotide or amino acid residues that are the same when the two or more polynucleotide or polypeptide sequences, respectively , are aligned for maximum correspondence. Depending on the application, the "identity” may exist over a region of the sequences being compared (e.g.. over the length of a functional domain) or over the full length of the sequences. A “region” is considered to be a continuous stretch of at least 6, 9, 14, 19, 24, 29, 34, 39, or more nucleotides, or of at least 2, 6, 10, 14, 18, 22, 26, 30, or more amino acids. For comparison, typically one sequence acts as a reference sequence to which one or more test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman (1981. Adv Appl Math. 2:482), by the homology alignment algorithm of Needleman & Wunsch (1970. J Mol Biol. 48:443), by the search for similarity method of Pearson & Lipman (1988. Proc Natl Acad Sci USA. 85:2444), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, which can be used with default parameters), or by visual inspection (see generally Altschul et al. 1990. J Mol Biol. 215:403). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm (see, for example, Altschul et al. 1990. J Mol Biol. 215:403: Gish & States. 1993. Nature Genet. 3:266; Madden et al. 1996. Meth Enzymol. 266:131; Altschul et al. 1997. Nucleic Acids Res. 25:3389; Zhang & Madden. 1997. Genome Res. 7:649). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
[0052] The terms “including,” “includes,” “having,” “has,” “with,” or variants thereof as used herein are intended to be inclusive in a manner similar to the term “comprising”.
[0053] The term “mammal-produced milk” as used herein refers to a milk produced by a mammal. Non-limiting examples of mammals include cow, human, sheep, wild sheep, goat, buffalo, camel, horse, donkey, alpaca, yak, llama, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, and woolly mammoth.
[0054] The term "native" as used herein refers to what is found in nature in its unmodified state (e.g.. a cell that is not genetically modified by a human, and that is maintained under conditions [e.g., level of oxygenation, pH, salt concentration, temperature, and nutrient (e.g., carbon, nitrogen, sulfur) availability] that are not defined by a human).
[0055] The term "operably linked" as used herein refers to an arrangement of elements that allows them to be functionally related. For example, a promoter sequence is operably linked to a protein coding sequence if it controls the transcription of the protein coding sequence, and a secretion signal sequence is operably linked to a protein if the secretion signal sequence directs the protein through the secretion system of a cell. An "operably linked" element may be in contiguous linkage with another element, or act in trans or at a distance to another element. Non- limiting examples of functions that may be operably linked include control of transcription, control of translation, protein folding, and protein secretion.
[0056] The terms "optional" or "optionally" as used herein refer to a feature or structure being present or not, or an event or circumstance occurring or not. The description includes instances in which a feature or structure is present, instances in which a feature or structure is absent, instances in which an event or circumstance occurs, and instances in which an event or circumstance does not occur.
[0057] The term "polymer" as used herein refers to a molecule that is composed of repeated molecular units that are covalently linked, either directly with each other or via intermediary molecules.
[0058] The term "polymer network" as used herein refers to a network of polymers that are crosslinked with each other via covalent bonds. Non-limiting examples of suitable covalent crosslinks include amide bonds (e.g., lactam bridges, native chemical ligation bonds, Staudinger ligation bonds) and disulfide bonds.
[0059] The term "polynucleotide” as used herein refers to a polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1,000) nucleotides. The term includes both sense and antisense strands of DNA molecules (e.g., cDNA, genomic DNA, synthetic DNA) and RNA molecules (e.g., mRNA, synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, and/or chemical modifications. A polynucleotide may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels; methylation; substitution of one or more of the naturally occulting nucleotides with an analog; internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Examples of modified nucleotides are described in the art (see, for example, Malyshev et al. 2014. Nature 509:385; Li et al. 2014. J. Am. Chem. Soc. 136:826). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding or other chemical interaction. Such molecules are known in the art and include, for example, molecules in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications may include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" polynucleotides. A polynucleotide may be in any topological conformation. For instance, a polynucleotide may be single- stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular', or in a padlocked conformation. The term “polynucleotide sequence” as used herein refers to a sequence of nucleotides that are comprised in a polynucleotide or of which a polynucleotide consists.
[0060] The term "protease" as used herein refers to a protein that can hydrolyze (i.e., cleave) a peptide bond (e.g., members of enzyme classification groups EC 3.4).
[0061] The terms "polypeptide" and "protein" as used herein can be interchanged, and refer to a naturally -occurring or a naturally not occurring polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100) amino acids. A “polypeptide” or “protein” may have an active structure or lack a functional structure, comprise coded and/or non-coded amino acids, comprise amino acids that occur in nature and/or amino acids that do not occur in nature, comprise chemically modified and/or biochemically modified and/or derivatized amino acids, comprise unmodified and/or modified peptide backbones, and/or be monomeric (i.e., having a single chain) or polymeric (i.e., having of two or more chains, which may be covalently or non-covalently associated). The term “amino acid sequence” as used herein refers to a sequence of amino acids that is comprised in a "polypeptide” or “protein”, or of which a “polypeptide” or “protein” consists, Amino acids comprised in an amino acid sequence are sequentially numbered by counting from the N-terminus of the polypeptide, A “polypeptide” or “protein may be isolated from naturally occurring systems (e.g., plant or animal lifeforms), synthesized in solution or on solid support, or produced recombinantly.
[0062] The term "promoter sequence" as used herein refers to a polynucleotide that directs transcription of a downstream polynucleotide in a cell. A promoter sequence may include necessary nucleotides near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter sequence may also optionally include distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
[0063] The term "protease recognition or cleavage sequence" or “recognition or cleavage sequence for a protease” as used herein refers to an amino acid sequence in a polypeptide that is preferably recognized by a protease and in which a peptide bond is cleaved by the protease. The general nomenclature of positions in protease recognition or cleavage sequences are defined as described by Schechter & Berger (1967. Biochem Biophys Res Common. 27(2):157; 1968. Biochem Biophys Res Commun. 32(5):898), which designate the cleavage site as being located between amino acid residues P1 and P1', and incrementing numbering in the N-terminal direction of the cleaved peptide bond (i.e., P1, P2, P3, P4, etc.) and in the C -terminal (i.e., P1', P2', P3', P4', etc.).
[0064] The term "recombinant β-lactoglobulin" as used herein refers to a recombinantly produced polypeptide (i.e., a polypeptide that is produced in a recombinant host cell, or to a polypeptide that is synthesized from a recombinant polynucleotide) that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a native β-lactoglobulin protein (e.g., a native Bos taurus β-lactoglobulin protein [amino acids 17 to 178 of UniProt sequence P02754, SEQ ID NO: 1], a native Homo sapiens β-lactoglobulin protein [amino acids 19 to 180 of UniProt sequence P09466, SEQ ID NO: 2] , a native Capra hircus β-lactoglobulin protein [e.g., amino acids 19 to 180 of UniProt sequence P02756, SEQ ID NO: 3], a native Equus asinus β-lactoglobulin protein [UniProt sequence P19647, SEQ ID NO: 4], a native Ovis aries β-lactoglobulin protein [e.g., amino acids 19 to 180 of UniProt sequence P67976, SEQ ID NO: 5], a native Equus eabaUus β-lactoglobulin protein [amino acids 19-180 of UniProt sequence P02758. SEQ ID NO: 6], a native a native Equus caballus β-lactoglobulin protein [amino acids 19 to 181 of UniProt sequence P07380, SEQ ID NO: 7], a native Equus asinus β- lactoglobulin protein [UniProt sequence P13613, SEQ ID NO: 8], a native Ovis aries musimon β- lactoglobulin protein [e.g., UniProt sequence P67975, SEQ ID NO: 9] ).
[0065] The term "recombinant host cell" as used herein refers to a host cell that comprises a recombinant polynucleotide. Thus, for example, a recombinant host cell may produce a polynucleotide or polypeptide not found in the native (non -recombinant) form of the host cell, or a recombinant host cell may produce a polynucleotide or polypeptide at a level that is different from that in the native (non-recombinant) form of the host cell. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the subject cell, but are still included within the scope of the term "recombinant host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture, or may be a cell which resides in a living tissue or organism.
[0066] The term "recombinant polynucleotide" as used herein refers to a polynucleotide that is removed from its naturally occurring environment, or a polynucleotide that is not associated with all or a portion of a polynucleotide abutting or proximal to the polynucleotide when it is found in nature, or a polynucleotide that is operatively linked to a polynucleotide that it is not linked to in nature, or a polynucleotide that does not occur in nature, or a polynucleotide that contains a modification that is not found in that polynucleotide in nature (e.g., insertion, deletion, or point mutation introduced artificially, e.g., by human intervention), or a polynucleotide that is integrated into a chromosome at a heterologous site. The term may be used, e.g., to describe cloned DNA isolates, or a polynucleotide comprising a chemically synthesized nucleotide analog. A polynucleotide is also considered "recombinant" if it contains a genetic modification that does not naturally occur. For instance, an endogenous polynucleotide is considered a "recombinant polynucleotide" if it contains an insertion, deletion, or substitution of one or more nucleotides that is introduced artificially (e.g., by human intervention). Such modification may introduce into the polynucleotide a point mutation, substitution mutation, deletion mutation, insertion mutation, missense mutation, frameshift mutation, duplication mutation, amplification mutation, translocation mutation, or inversion mutation. The term includes a polynucleotide in a host cell's chromosome, as well as a polynucleotide that is not in a host cell’s chromosome (e.g., a polynucleotide that is comprised in an episome). A recombinant polynucleotide in a host cell or organism may replicate using the in vivo cellular machinery of the host cell; however, such recombinant polynucleotide, although subsequently replicated intracellularly, is still considered recombinant for purposes of this invention.
[0067] The term "regulatory element” as used herein refers a polynucleotide sequence that mediates, modulates, or regulates expression (e.g., transcription, post-transcriptional events, translation) of a polynucleotide to which the regulatory element is operably linked.
[0068] The term "secretion signal” as used herein refers to a peptide that is operably linked to the N -terminus of a protein, and that mediates the delivery of the protein via the intracellular secretory pathway of a host cell in which the protein is produced (i.e,, synthesized) to the exterior of the host cell. Typically, operable linkage of a recombinant protein with a secretion signal requires removal of a start codon of the polynucleotide sequence encoding the recombinant protein.
[0069] The term “treating” as used herein includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
[0070] The term "two or more” as used herein refers to two, three, four, five, six, seven, eight, nine, ten, or more, or all of the elements subsequently li sted.
[0071] The term "vector" as used herein refers to a nucleic acid that can carry a polynucleotide sequence to be introduced into a host cell. Non-limiting examples of vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, viral vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), vims particles (e.g., comprising heterologous polynucleotides), DNA constructs (e.g., produced by cloning or PCR amplification), and linear double- stranded molecules (e.g., PCR fragments). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors may be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
[0072] The term "yeast" as used herein refers to any organism of the order Saccharomycetales. Vegetative growth of yeast is by budding /b lebbing of a unicellular thallus, and carbon catabolism may be fermentative.
[0073] The term “% by mass” as used herein refers to a percentage value for a mass as determined in a hydrated composition, such that the composition includes the mass of powder as well as the mass of the hydrating agent, with 100% fixed as the percentage value for the entire hydrated composition. In embodiments in which the composition provided herein is in powder form (to which the mass of the hydrating agent will be added at a later time), the term refers to a percentage value for a mass as determined relative to the eventual entire hydrated composition (with 100% fixed as the percentage value for that entire eventual hydrated composition).
[0074] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value (fractional or integral) falling within the range inclusive of the recited minimum and maximum value, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub- ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of less than or equal to 10. It should further be understood that all ranges and quantities described below are approximations and are not intended to limit the invention.
[0075] It further should be understood that in any method disclosed herein the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.
Recombinant β-Lactoglobulin Protein Having Improved Binding of Bioactive Agent
[0076] In various aspects, provided herein is a recombinant β-lactoglobulin protein that comprises a modification compared to a corresponding native β-lactoglobulin protein, wherein the modification improves binding of a hydrophobic bioactive agent to the recombinant β- lactoglobulin protein. [0077] Without wishing to be bound by theory, a recombinant β-lactoglobulin protein according to the above may be loaded with one or more of a number of desirable hydrophobic bioactive agents, and may then be administered to a subject (e.g., a human or other animal) where it can provide the one or more hydrophobic bioactive agents such that they can exert their bioactivity. The rate of binding of the one or more hydrophobic bioactive agents may be controlled by the type and/or number of modifications comprised in the recombinant β-lactoglobulin protein (e.g., the more modifications, the greater the binding affinity for the one or more hydrophobic bioactive compounds by the recombinant β-lactoglobulin protein).
[0078] The modification that improves binding of a hydrophobic bioactive compound to the recombinant β-lactoglobulin protein according to the above may be a modification that increases binding affinity of the hydrophobic bioactive agent to the recombinant β-lactoglobulin protein. The modification may, for example, comprise or consist of a substitution of an amino acid in a region of the β-lactoglobulin protein that can bind the hydrophobic bioactive agent with an amino acid that can provide for a stronger, non-covalent interaction (e.g., electrostatic interaction, van der Waals bonding, hydrogen bonding) with the hydrophobic bioactive agent.
[0079] The modification typically has minimal impact on protein structure of the recombinant β-lactoglobulin protein compared to that of the corresponding native β-lactoglobulin protein. Such minimal impact may be achieved by creating the modification on a solvent-exposed loop, avoiding changing amino acids in beta-sheet or alpha-helixes, selecting conservative amino acid substitutions (i.e., substitutions of amino acids having similar biochemical properties), and/or selecting amino acid deletions, substitutions, and/or additions that do not create steric hindrances between side chains of amino acids in a three-dimensional conformation of the recombinant β- lactoglobulin protein (as determined, for example, by examination using PyMol [Schrodinger, New York, NY] and multi -sequence alignments [e.g., of orthologs of native β-lactoglobulin proteins; for example, using MUSCLE (Edgar, 2004, Nucleic Acids Res, 32: 1792-1797)]).
Modified Recombinant β-Lactoglobulin Protein
[0080] The modification that improves binding of a hydrophobic bioactive agent to a recombinant β-lactoglobulin protein according to any of the above may be comprised in or in the vicinity (e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) of a region of a corresponding native β-lactoglobulin protein that can bind hydrophobic compounds. Non-limiting examples of regions of native β-lactoglobulin protein that can bind hydrophobic compounds include regions spanning from approximately amino acid 21 to approximately amino acid 124 (i.e., “central binding cavity” or “beta-barrel region”), and from approximately amino acid 135 to approximately amino acid 150 (“interface region”) of native Bos taurus β-lactoglobulin protein (SEQ ID NO: 1), and corresponding regions in orthologs (e.g., SEQ ID NOs: 2-9).
[0081] The modification that improves binding of a hydrophobic bioactive agent to a recombinant β-lactoglobulin protein according to any of the above may consist of a single modification (e.g., a single amino acid substitution, deletion, or addition).
[0082] Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L. V43L V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71 L, I71 F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, A86G, A86H, A86Y, N90L N90K, N90R, N90Q, N90S, N90T, N90D, N90E, V92I, V92L, V92M, V92F, V92Y, F105I, F105V, M107I, M107V, L122I, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R, K138H, K138E, K138D, K138L, K 138I, K141Q, K141L, K141M, K141R, K141F, K141H, A142V, A142M, A142I, A142G, A142T, L143I, L143F, L143M, L143R, L143V, M145L, M145I, M145V, M145G, H146R, H146K, H146D, I147L, H47V. I147M, I147K, I147F, R148Q, R148W, R148H. R148K, and R148E of native Bos taunts β-lactoglobulin protein (SEQ ID NO: 1), native Capra hircus β-lactoglobulin protein (SEQ ID NO: 3), or native Ovis aries β-lactoglobulin protein (SEQ ID NO: 5).
[0083] Such single modification may be an amino acid substitution selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I84L, I84M, I84F, I84V, I84Y, I84W, N90L N90K, N90R, N90Q, N90S, N90T, N90D, N90E, L122L L122V, I.122Y, L122F, L143I, L143F, L143M, L143R, L143V, H146R, H146K, and H146D of native Homo sapiens β-lactoglobulin protein (SEQ ID NO: 2).
[0084] Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y. I56A, L58I, L58W. L58Y, L58M, I71L, I71F, I71Y, I71W, I71 V, F105I, F105V, L123L L123V, L123Y, L123F, L144I, L144F, L144M, L144R, L144V, H147R, H147K, and H147D of native Equus asinus P -lactoglobulin protein (SEQ ID NO: 4).
[0085] Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41L V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58L L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69L K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W. V92I, V92L. V92M, V92F, V92Y, M107I, M107V, L122I, L122V, L122Y, L122F, L143I, L143F. L143M, L143R, and L143V of native Equus caballus β-lactoglobulin protein (SEQ ID NO: 6).
[0086] Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39I, L39M, L39W, L39F, L39V, V411, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71 F, I71 Y, I71W, I71 V, F105I, F105V, L123L L123V, L123Y, L123F, L144I, L144F, L144M, L144R, and L144V of native Equus caballus β- lactoglobulin protein (SEQ ID NO: 7).
[0087] Such single modification may be an amino acid substitution selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y. L58M, E62A. E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I841F, I84V, I84Y, I84W, Y92I, V92L, V92M, V92F, V92Y, M107I, M107V, L122I, L122V, L122Y, L122F, L143I, L143F, L143M, L143R, and L143V of native Equus asinus β-lactoglobulin protein (SEQ ID NO: 8).
[0088] Such single modification may be an amino acid substitution selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71 Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, A86G, A86H, A86Y, N90L N90K. N90R, N90Q, N90S, N90T, N90D, N90E. V92I, V92L, V92M, V92F, V92Y, F105I, F105V, M107I, M107V, L122I, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R. K138H. K138E, K138D, K138L, K138I, K141Q, K141L, K141M. K141R, K141F, K141H, A142V, A142M, A142I, A142G, A142T, L143I, L143F, L143M, L143R, L143V, M145L, M 1451, M145V, M145G, H146R, H146K, H146D, I147L, L147V. I147M, I147K, I147F, R148Q, R148W, R148H, R148K, and R148E of native Ovis aries β-lactoglobulin protein (SEQ ID NO: 9).
[0089] Alternatively, the modification that improves binding of a hydrophobic bioactive agent to a recombinant β-lactoglobulin protein according to any of the above may consist of two or more modifications (e.g., two or more amino acid substitutions, deletions, or additions, or combinations thereof).
[0090] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84 W, A86G, A86H, A86Y, N90I, N90K, N90R, N90Q, N90S, N90T, N90D. N90E, V92I, V92L, V92M, V92F, V92Y, F105L F105V, M107I, M107V, L122I, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R, K138H, K138E, K138D, K138L, K 1381, K141Q, K141L, K141M, K141R. K141F, K141H, A142V, A142M, A142I, A142G. A142T, L143L L143F, L143M, L143R, L143V, M145L, M145I, M145V, M145G, H146R, H146K, H146D, I147L, 1147V, I147M, I147K, I147F, R148Q, R148W, R148H, R148K, and R148E of native Bos taurus β-lactoglobulin protein (SEQ ID NO: 1 OR 10), native Capra hircus β-lactoglobulin protein (SEQ ID NO: 3), or native Ovis aries β-lactoglobulin protein (SEQ ID NO: 5).
[0091] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, L461. L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I84L, I84M. I84F, I84V, I84Y, I84W, N90I, N90K, N90R, N90Q, N90S, N90T, N90D, N90E, L122I, L122V, L122Y. L122F, L143I, L143F, L143M, L143R, L143V, H146R, H146K, and H146D of native Homo sapiens β- lactoglobulin protein (SEQ ID NO; 2).
[0092] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L.39W, L39F, L39V, V411, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71F, I71Y, I71W, I71V, F105I, F105V, L123I, L123V, L123Y, L123F, L144I, L144F, L144M, L144R, L144V, H147R, H147K, and H147D of native Equus asinus β-lactoglobulin protein (SEQ ID NO: 4).
[0093] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M. L39W. L39F, L39V. V41I, V41 M. V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71 V, I84L, I84M, I84F, I84V, I84Y, 184W, V92I, V92L, V92M, V92F, V92Y, M107I, M107V, L122I, L122V, L122Y, L122F, L143I, L143F, L143M, L143R, and L143V of native Equus caballus β-lactoglobulin protein (SEQ ID NO: 6).
[0094] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39I, L39M, L39W, L39F, L39V, V41L V41M, V41L. V43I, V43L, L46I, L46E L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W. L58Y, L58M, I71L, I71F, I71Y. I71 W, I71 V, F105I, F105V, L123I, L123V, L123Y, L123F, L144I, L144F, L144M, L144R, and L144V of native Equus caballus β-lactoglobulin protein (SEQ ID NO: 7).
[0095] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: A37L, A371, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41 L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58L L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69L K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, V92I, V92L, V92M, V92F, V92Y, M107L M107V, L122I, L122V, L122Y, L122F, L143I, L143F, L143M, L143R, and L143V of native Equus asinus β- lactoglobulin protein (SEQ ID NO: 8).
[0096] Such two or more modifications may comprise or consist of two or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M. L39W. L39F, L39V. V41I, V41 M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71 F, I71 Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W. A86G, A86H, A86Y, N90I, N90K, N90R, N90Q, N90S, N90T, N90D, N90E, V92I, V92L, V92M, V92F, V92Y, Fl 051, F105V, M107I, M 107V, L122I, L122V, L122Y, L122F, D137N, D137Q. D137E, D137S, K138R, K138H, K138E, K138D, K138L, K138I, K141Q, K141L, K14IM, K141R, K141F, K141 H, A142V, A142M, A142I, A142G, A142T, L143I, L143F, L143M, L143R, L143V, M145L, M145I, M145V, M145G, H146R, H146K, H146D, I147L, I147V, I147M, I147K, I147F, R148Q, R148W, R148H, R148K, and R148E of native Ovis aries β-lactoglobulin protein (SEQ ID NO: 9).
[0097] The recombinant β-lactoglobulin protein according to any of the above may comprise a further modification. For example, the recombinant β-lactoglobulin protein may comprise a modification that introduces a non-native protease recognition or cleavage sequence in or in the vicinity (e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids) of a solvent- exposed region of the corresponding native β-lactoglobulin protein (i.e., a region that is on the surface of a three-dimensional structure of the corresponding native β-lactoglobulin protein), a lipid-binding region of the corresponding native β-lactoglobulin protein (i.e., a region in a three- dimensional structure of the corresponding native β-lactoglobulin protein that can bind a lipid); and/or an allergenic epitope comprised in the corresponding native β-lactoglobulin protein. Such further modification may facilitate cleavage and/or denaturation of the recombinant β- lactoglobulin protein in the gastrointestinal tract of a subject (e.g., (e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig, human])), and thereby attenuate or essentially eliminate allergenicity of the recombinant β-lactoglobulin protein, and/or facilitate in vi vo release of the bound hydrophobic bioactive agent from the recombinant β-lactoglobulin protein. Non-limiting examples of suitable modifications that introduce a non-native protease recognition or cleavage sequence are disclosed, for example, in PCT publication WO2021168343, published August 26, 2021. Hydrophobic Bioactive Agent
[0098] The hydrophobic bioactive agent of which binding to the recombinant β-lactoglobulin protein according to any of the above (e.g.. the recombinant β-lactoglobulin protein according to any of the above) is improved may be any hydrophobic bioactive agent.
[0099] Non-limiting examples of suitable hydrophobic bioactive agents include hydrophobic nutraceuticals (i.e., hydrophobic compounds that have physiological benefit or provide protection against disease), hydrophobic compounds that have micro-/biocidal activity, hydrophobic compounds that have anti-viral activity, lipids, and hydrophobic therapeutics (i.e., hydrophobic compounds that treat disease, disorder, or injury: e.g., hydrophobic compounds that have anti- cancer activity, treat pain [e.g., acute pain, chronic pain], heal or soothe skin defects [i.e., defects in skin that disrupt the continuity of epithelium, such as, for example rashes, sores, abrasions, bums, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers, diabetic ulcers, diabetic fool ulcers, and decubitus ulcers, itchy skin], or have anesthetic effects).
[0100] Non-limiting examples of hydrophobic nutraceuticals include lipid- soluble pro- vitamins and vitamins and lipid-soluble antioxidants.
[0101] Non-limiting examples of lipid-soluble vitamins include vitamin A (retinoids), vitamin D (e.g., vitamin D2, vitamin D3), vitamin E (e.g., tocopherols [e.g., alpha- tocopherol, beta- tocopherol, gamma- tocopherol, delta-tocopherol], tocotrienols [e.g., alpha-tocotrienol, beta- tocotrienol, gamma-tocotrienol, delta-tocotrienol]), vitamin K (e.g., vitamin K1 (phylloquinone), vitamin K2 (menaquinone), vitamin K3 (menadione), vitamin K4 (menadiol diacetate), vitamin K5), and derivatives.
[0102] Non-limiting examples of lipid-soluble antioxidants include fatty acids (e.g., linolenic, linoleic, oleic, palmitic) and derivatives (e.g., lipoic acid), carotenoids (e.g., a-carotene, β- carotene, γ-carotene, lutein, zeaxanthin, astaxanthin), fat-soluble polyphenols (e.g., astaxanthin, olive oil), coenzyme Q10, and derivatives and mixtures thereof.
[0103] Non-limiting examples of hydrophobic compounds that have micro-fbiocidal activity include 4-aminododecylpyridinium chloride, 4-acetylaminododecylpyridinium chloride, 4- benzoylaminododecylpyridinium bromide and 4-( 1 -naphthoyl) aminododecylpyridinium bromide (Zhao & Sun. 2008. J Appl Microbiol. 104(3):824), hydrophobic compounds extracted from garlic (e.g., allicin, vinyldithiin, ajoene, and diallyl polysulfides; see, for example, Nakamoto et al. 2020. Exp Ther Med. 19(2): 1550).
[0104] Non-limiting examples of hydrophobic compounds that have anti-viral activity are known in the art (see, for example, Hadfield et al. 1999. Proc Natl Acad Sci USA. 96(26): 14730: Anasir et al. 2021. J Biomed Sci. 28, 10; and Linnakoski et al. 2018. Front Microbiol. 9:2325).
[0105] Non-limiting examples of lipids include octanoic acid (caprylic acid), lionoleic acid, lauric acid, myristic acid, palmitic acid, succinic acid, hepta-decanoic acid, 12-bromododecanoic acid, and oleic acid.
[0106] Non-limiting examples of hydrophobic compounds that have anti-cancer activity include all-trans-retinol, all-trans retinoic acid [ATR.A], all-trans-retinyl acetate, curcumin, epigallocatechin gallate (EGCG), geinistein, 3,3-diindolylmethane (DII), indole-3-carbinol, and resveratrol.
[0107] The hydrophobic bioactive agent may be bound to the recombinant β-lactoglobulin protein according to any of the above (e.g,, a recombinant β-lactoglobulin protein according to any of the above) via non-covalent bonding (e.g., electrostatic interaction, van der Waals bonding, hydrogen bonding).
Recombinant Expression Construct
[0108] In various aspects, provided herein is a recombinant expression construct consisting of a polynucleotide comprising: i) a promoter sequence (e.g., a polynucleotide sequence for any of the promoters disclosed herein), ii) an optional secretion signal sequence (i.e., a sequence that encodes a peptide that mediates the delivery of a nascent protein attached to the peptide to the exterior of the cell in which the nascent protein is synthesized: e.g., a polynucleotide sequence encoding any of the secretion signals disclosed herein), iii) a recombinant β-lactoglobulin protein coding sequence (i.e., a polynucleotide sequence encoding a recombinant β-lactoglobulin protein according to any of the above, optionally comprising a tag polypeptide (e.g., any of the tag polypeptides disclosed herein)), and iv) a termination sequence (e.g., a polynucleotide sequence for any of the terminators disclosed herein); wherein: a) the promoter sequence is operably linked in sense orientation to the optional secretion signal sequence and the recombinant β-lactoglobulin protein coding sequence (i.e., the promoter sequence and the optional secretion signal sequence and the recombinant β -lactoglobulin protein coding sequence are positioned such that the promoter sequence is effective in mediating or regulating transcription of the optional secretion signal sequence and the recombinant β-lactoglobulin protein coding sequence), b) the optional secretion signal sequence is operably linked in sense orientation to the recombinant β-lactoglobulin protein coding sequence (i.e., the optional secretion signal sequence and the recombinant β-lactoglobulin protein coding sequence are positioned such that transcription and translation produces a recombinant β-lactoglobulin protein comprising the optional secretion signal), and c) the one or more terminator sequences are operably linked to the recombinant β-lactoglobulin protein coding sequence (i.e., the recombinant β-lactoglobulin protein coding sequence and the one or more terminator sequences are positioned such that the one or more terminator sequences are effective in terminating transcription of the recombinant β-lactoglobulin protein coding sequence).
[0109] The recombinant expression construct may further comprise an operably linked sequence encoding for an affinity purification tag, such that the expressed recombinant β- lactoglobulin protein includes a peptide sequence for affinity purification. Such affinity purification tag may be operably linked such that when expressed the affinity purification tag is present either at or toward the amino terminus, the carboxy terminus, or both. Such affinity purification tag may comprise a maltose binding protein (MBP) tag, a glutathione-S-transferase (GST) tag, a poly(His) tag, a hexa(His) tag, a FLAG-tag, a V5-tag, a VSV-tag, an E-tag, an NE- tag, a hemagglutinin (Ha)-tag, Strep-tag, and a Myc-tag.
[0110] The recombinant expression construct may further comprise a sequence for integration by homologous (i.e., targeted integration) or nonhomologous recombination into the genome of a host cell. The recombinant expression construct may comprise at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1,000, or at least 10,000 base pairs that have sufficient identity with a target sequence in the genome of the host cell to enhance the probability of homologous recombination of the recombinant expression construct. Such homologous sequence may be non-coding or coding.
[0111] The optional secretion signal sequence and/or recombinant β-lactoglobulin protein coding sequence comprised in a recombinant expression construct according to any of the above may be codon-optimized for expression in a recombinant host cell according to any of the above. [0112] A recombinant expression construct according to any of the above may be isolated.
[0113] A recombinant expression construct according to any of the above may be generated upon integration of a fragment of the recombinant expression construct into the genome of a host cell (e.g., the genome of a recombinant host cell according to any of the above). For example, a polynucleotide comprising a recombinant β-lactoglobulin protein coding sequence (optionally operably linked to a secretion signal sequence) may be stably integrated within the genome of a host cell such that one or more regulatory elements of an endogenous gene locus become operably linked to the recombinant β-lactoglobulin protein coding sequence, thereby generating a recombinant expression construct according to any of the above.
Promoter Sequence
[0114] A recombinant expression construct according to any of the above may comprise any promoter sequence that is active in a recombinant host cell according to any of the below.
[0115] The promoter sequence may be a constitutive promoter sequence (i.e., a promoter sequence that is active under most environmental and developmental conditions), or an inducible or repressible promoter sequence (i.e., a promoter sequence that is active only under certain environmental or developmental conditions [e.g., in presence or absence of certain factors, such as, but not limited to, carbon (e.g., glucose, galactose, lactose, sucrose, cellulose, sophorose, gentiobiose, sorbose, disaccharides that induce the cellulase promoters, starch, tryptophan, thiamine, methanol), phosphate, nitrogen, or other nutrient; temperature; pH; osmolarity; heavy metals or heavy metal ions; inhibitors; stress; catabolites; and combinations thereof]).
[0116] The promoter sequence may consist of a single promoter sequence, or of two or more promoter sequences (e.g., combination of two or more promoters or functional parts thereof arranged in sequence, combination of an inducible and a constitutive promoter). The two or more promoter sequences may be identical, or at least two of the two or more promoter sequences may not be identical.
[0117] The promoter sequence may comprise or consist of a bidirectional promoter sequence (i.e., a polynucleotide that initiates transcription in both orientations by recruiting transcription factors, for example generated by fusing two identical or different promoters in opposite directions).
[0118] Non-limiting examples of suitable promoter sequences include promoter sequences that are functional in a bacterial host cell, including T7 promoter, T5 promoter, Tac promoter, pL/pR promoter, phoA promoter, lacUV5 promoter, trc promoter, trp promoter, cstA promoter, xylA promoter, manP promoter, malA promoter, lacA promoter, aprE promoter, AaprE promoter, srfA promoter, p43 promoter, ylbA promoter, oB promoter, veg promoter, PG1 promoter, PG6 promoter, λ PL promoter, λPR promoter, and spa promoter, and functional parts and combinations thereof,
[0119] Non-limiting examples of suitable promoter sequences include promoter sequences that are functional in a fungal host cell, including xlnA promoter, xynl promoter, xyn2 promoter, xyn3 promoter, xyn4 promoter, bxll promoter, cbhl promoter, cbh2 promoter, egl1 promoter, egl2 promoter, egl3 promoter, egl4 promoter, egl5 promoter, glaA promoter, agdA promoter, gpdA promoter, gpdl promoter, AOX 1 promoter, GAP1 promoter, MET3 promoter, ENO1 promoter, GPD1 promoter, PDC1 promoter, TEF1 promoter, AXE1 promoter. CIP1 promoter, GH61 promoter, PKI1 promoter, RP2 promoter, ADH1 promoter, CUP1 promoter, GAL1 promoter, PGK1 promoter, YPT1 promoter, LAC4 promoter, LAC4-PB 1 promoter, FLD1 promoter, MOX promoter. DAS1 promoter, DAS2 promoter, GAP1 promoter, STR3 promoter, ADH3 promoter, GUT2 promoter, CYC1 promoter, TDH3 promoter, PGL1 promoter, ADH2 promoter, HXT7 promoter, CLB 1 promoter, and PHO5 promoter, and functional parts and combinations thereof. Secretion Signal Sequence
[0120] A recombinant expression construct according to any of the above may optionally comprise any secretion signal sequence that is active in a recombinant host cell according to any of the below.
[0121] The optional secretion signal sequence may encode a secretion signal that mediates translocation of the nascent recombinant β-lactoglobulin protein into the ER post-translationally (i.e., protein synthesis precedes translocation such that the nascent recombinant β-lactoglobulin protein is present in the cell cytosol prior to translocating into the ER) or co-translationally (i.e., protein synthesis and translocation into the ER occur simultaneously).
[0122] Non-limiting examples of suitable secretion signal sequences include secretion signal sequences that are functional in a bacterial host cell, including secretion signal sequences of genes encoding any of the following proteins: PelB, OmpA, Bla, PhoA, PhoS, MalE, LivK, LivJ, MglB, AraF, AmpC, RbsB, MerP, CpdB, Lpp, LamB, OmpC, PhoE, OmpF, TolC, BtuB, and LutA, and functional parts and combinations thereof.
[0123] Non-limiting examples of suitable secretion signal sequences include secretion signal sequences that are functional in a fungal host cell, including secretion signal sequences of genes encoding any of the following proteins: CBH1, CBH2, EGL1, EGL2, XYN1, XYN2, BXL1, HFB1, HFB2, GLAA, AMYA, AMYC, AAMA, alpha mating factor, SUC2, PHO5, INV, AMY, LIP, PIR, OST1, and β- glucosidase, and functional parts and combinations thereof.
Termination Sequence
[0124] A recombinant expression construct according to any of the above may comprise any termination sequence that is active in a recombinant host cell according to any of the below.
[0125] Non-limiting examples of suitable termination sequences include termination sequences include termination sequences of the adhl , amaA, amdS, amyA, aox1, cbh1, cbh2, cyc1 , egl1 , egl2, gal1, gap1, glaA, gpd1 , gpdA, pdc1, pgk1 tef1 , tps1 , trpC, xyn1, xyn2, xyn3, and xyn4 genes, and functional parts and combinations thereof.
[0126] The termination sequence may consist of a single termination sequence, or of two or more termination sequences, wherein the two or more termination sequences may be identical, or at least two of the two or more termination sequences may be not identical. The termmation sequence may consist of a bidirectional termination sequence.
Additional Regulatory Elements
[0127] A recombinant expression construct according to any of the above may further comprise additional regulatory elements.
[0128] Non-limiting examples of regulatory elements include promoter sequences, termination sequences, transcriptional start sequences, translational start sequences, translation stop sequences, enhancer sequences, activator sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions, upstream activation sequences (UAS), introns, operators (i.e., sequences of nucleic acids adjacent to a promoter that comprise a protein- binding domain where a repressor protein can bind and reduce or eliminate activity of the promoter), efficient RNA processing signals (e.g., splicing signals, polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., ribosome binding sites [e.g., Shine-Dalgamo sequences]), sequences that enhance protein stability, sequences that enhance protein secretion, and combinations thereof.
Recombinant Vector
[0129] In various aspects, provided herein is a recombinant vector that comprises a recombinant expression construct according to any of the above or a fragment thereof (e.g., a polynucleotide that comprises a recombinant β-lactoglobulin protein coding sequence and optional secretion signal sequence, which upon integration into the genome of a host cell creates a recombinant expression construct according to any of the above),
[0130] The recombinant vector may comprise a single recombinant expression construct according to any of the above, or two or more recombinant expression constructs according to any of the above, which may be identical or at least two of which may be not identical (e.g., differ from each other in a promoter sequence, a secretion signal, a β-lactoglobulin protein coding sequence, a termination sequence, and/or an additional regulatory element). In embodiments in which the recombinant vector comprises two or more recombinant expression constructs, the two or more recombinant expression constructs may encode the same recombinant β-lactoglobulin protein. In some such embodiments, the two or more recombinant expression constructs encoding the same recombinant β-lactoglobulin protein differ from each other in a promoter sequence, secretion signal sequence, termination sequence, and/or additional regulatory element.
[0131] The recombinant vector may further comprise one or more other elements suitable for propagation of the recombinant vector in a recombinant host cell. Non-limiting examples of such other elements include origins of replication and selection markers. Origins of replication and selection markers are known in the art, and include bacterial and fungal origins of replication (e.g., AMA1, ANSI). Selection markers may be resistance genes (i.e., polynucleotides that encode proteins that enable host cells to detoxify an exogenously added compound [e.g., an antibiotic compound]), auxotrophic markers (i.e., polynucleotides that encode proteins that permit a host cell to synthesize an essential component (usually an amino acid) while grown in media that lacks that essential component), or color markers (i.e., genes that encode proteins that can produce a color). Non-limiting examples of suitable selection markers include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine 5' -phosphate decarboxylase), sC
(sulfate adenyltransferase), trpC (anthranilate synthase), and ble (bleomycin-type antibiotic resistance), and derivatives thereof. The selection marker may comprise an alteration that decreases production of the selective marker, thus increasing the number of copies needed to permit a recombinant host cell comprising the recombinant vector to survive under selection. Selection may also be accomplished by co-transformation, wherein the transformation is carried out with a mixture of two vectors and the selection is made for one vector only. [0132] The recombinant vector may further comprise sequences for integration by homologous (i.e,, targeted integration) or nonhomologous recombination into the genome of a host cell. The recombinant expression construct may comprise at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1,000, or at least 10,000 base pairs that have sufficient identity with a target sequence in the genome of the host cell to enhance the probability of homologous recombination of the recombinant expression construct. Such homologous sequence may be non-coding or coding.
[0133] A recombinant vector according to any of the above may be isolated.
Recombinant Host Cell
[0134] In various aspects, provided herein is a recombinant host cell that is capable of producing a recombinant β-lactoglobulin protein according to any of the above, wherein the recombinant host cell comprises a recombinant expression construct according to any of the above. [0135] The recombinant host cell may comprise a single recombinant expression construct according to any of the above, or comprise two or more recombinant expression constructs according to any of the above. In embodiments in which the recombinant host cell comprises two or more recombinant expression constructs, the two or more recombinant expression constructs may be identical, or at least two of the two or more recombinant expression constructs may differ from each other (e.g., in a promoter sequence, a β-lactoglobulin protein coding sequence, a secretion signal sequence, a termination sequence, and/or an additional regulatory element).
[0136] The recombinant host cell may comprise a recombinant expression construct that is stably integrated within the genome of the recombinant host cell (e.g., via targeted (e.g., via homologous recombination) or random (i.e,, non-targeted) integration), and/or a recombinant expression construct that is not stably integrated but rather maintained extra-chromosomally (e.g., on an autonomously replicating recombinant vector provided herein).
[0137] A recombinant host cell according to any of the above may be derived from any organism, including any bacterium, fungus (e.g., yeast, filamentous fungus), archaea, protista, animal (including any unicellular animal), plant (including any unicellular plant), algae, protozoan, and chromista, or from a genetic variant (e.g., mutant) thereof, as well as from any generally recognized as safe (GRAS) industrial host cell. [0138] Non-limiting examples of suitable plants include cycad, ginkgo biloba, conifer, cypress, juniper, thuja, cedarwood, pine, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, helichrysum, marigold, mugwort, safflower, camomile, lettuce, wormwood, calendula, citronella, sage, thyme, chia seed, mustard, olive, coffee, capsicum, eggplant, paprika, cranberry, kiwi, vegetables (e.g., carrot, celery), tagete, tansy, tarragon, sunflower, wintergreen, basil, hyssop, lavender, lemon verbena, marjoram, melissa, patchouli, pennyroyal, peppermint, rosemary, sesame, spearmint, primrose, samara, pepper, pimento, potato, sweet potato, tomato, blueberry, nightshade, petunia, morning glory, lilac, jasmin, honeysuckle, snapdragon, psyllium, wormseed, buckwheat, amaranth, chard, quinoa, spinach, rhubarb, jojoba, cypselea, chlorella, marula, hazelnut, canola, kale, bok choy, rutabaga, frankincense, myrrh, elemi, hemp, pumpkin, squash, curcurbit, manioc, dalbergia, legume plants (e.g., alfalfa, lentil, bean, clover, pea, fava coceira, frijole bola roja, frijole negro, lespedeza, licorice, lupin, mesquite, carob, soybean, peanut, tamarind, wisteria, cassia, chickpea/garbanzo, fenugreek, green pea, yellow pea, snow pea, lima bean, fava bean), geranium, flax, pomegranate, cotton, okra, neem, fig, mulberry, clove, eucalyptus, tea tree, niaouli, fruiting plant (e.g., apple, apricot, peach, plum, pear, nectarine), strawberry, blackberry, raspberry, cherry, prune, rose, tangerine, citrus (e.g., grapefruit, lemon, lime, orange, bitter orange, mandarin, tangerine), mango, citrus bergamot, buchu, grape, broccoli, brussels sprout, camelina, cauliflower, rape, rapeseed (canola), turnip, cabbage, cucumber, watermelon, honeydew melon, zucchini, birch, walnut, cassava, baobab, allspice, almond, breadfruit, sandalwood, macadamia, taro, tuberose, aloe vera, garlic, onion, shallot, vanilla, yucca, vetiver, galangal, barley, corn, curcuma aromatica, ginger, lemon grass, oat, palm, pineapple, rice, rye, sorghum, triticale, turmeric, yam, bamboo, barley, cajuput, canna, cardamom, maize, oat, wheat, cinnamon, sassafras, lindera benzoin, bay laurel, avocado, ylang-ylang, mace, nutmeg, moringa. horsetail, oregano, cilantro, chervil, chive, aggregate fruit, grain plant, herbal plant, leafy vegetable, non-grain legume plant, nut plant, succulent plant, land plant, water plant, delbergia, millet, drupe, schizocarp, flowering plant, non-flowering plant, cultured plant, wild plant, tree, shrub, flower, grass, herbaceous plant, brush, lianas, cacti, tropical plant, subtropical plant, temperate plant, and moss (e.g., Phy scomitrella patens).
[0139] Non-limiting examples of suitable yeast include members of any of the following genera, and derivatives and crosses thereof: Candida (e.g., Candida albicans, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida orthopsilosis, Candida palmioleophila, Candida pseudotropicalis, Candida sp., Candida utilis, Candida versatilis), Cladosporium, Cryptococcus (e.g., Cryptococcus terricolus, Cryptococcus curvatus), Debaryomyces (e.g., Debaryomyces hansenii), Endomyces (e.g., Endomyces vemalis)' , Endomycopsis (e.g., Endomycopsis vemalis), Eremothecium (e.g., Eremothecium ashbyii), Hansenula (e.g., Hansenula sp., Hansenula polymorpha), Kluyveromyces (e.g., Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans), Lipomyces (e.g,, Lipomyces starkeyi, Lipomyecs lipofer), Ogataea (e.g., Ogataea minuta), Pichia (e.g., Pichia sp., Pichia pastoris (Komagataella phaffii), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum. Pichia pijperi, Pichia stiptis, Pichia methanolica), Rhodosporidium (e.g., Rhodosporidium toruloides), Rhodotorula (e.g., Rhodotorula sp., Rhodotorula gracilis, Rhodotorula glutinis, Rhodotorula graminis), Saccharomyces (e.g., Saccharomyces sp., Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces potnbe, Saccharomyces sake, Saccharomyces uvarum), Sporobolomyces (e.g., Sporobolomyces roseus), Sporidiobolus (e.g., Sporidiobolus johnsonii, Sporidiobolus salmonicolor), Trichosporon (e.g., Trichosporon cacaoliposimilis, Trichosporon oleaginosus sp. nov., Trichosporon cacaoliposimilis sp. nov., Trichosporon gracile, Trichosporon dulcitum, Trichosporon jirovecii, Trichosporon insectorum), Xanthophyllomyces (e.g., Xanthophyllomyces dendrorhous), Yarrowia (e.g., Yarrowia lipolytica), and Zygosaccharomyces (e.g., Zygosaccharomyces rouxii).
[0140] Non-limiting examples of suitable filamentous fungi include any holomorphic, teleomorphic, and anamorphic forms of fungi, including members of any of the following genera, and derivatives and crosses thereof: Acremonium (e.g., Acremonium alabamense), Aspergillus (e.g., Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus sojae, Aspergillus terreus, as well as Emericella. Neosartorya, and Petromyces species), Aureobasidium, Canariomyces, Chaetomium, Chaetomidium, Corynascus, Chrysosporium (e.g., Chrysosporium botryoides, Chrysosporium carmichaeli, Chrysosporium crassitunicatum, Chrysosporium. europae, Chrysosporium evolceannui, Chrysosporium farinicola, Chrysosporium fastidium, Chrysosporium filiforme, Chrysosporium georgiae, Chrysosporium globiferum, Chrysosporium globiferum. var. articuiatum, Chrysosporium globiferum var. niveum, Chrysosporium hirundo, Chrysosporium hispanicum, Chrysosporium holmii, Chrysosporium indicum, Chrysosporium tops, Chrysosporium. keratinophilum,
Chrysosporium kreiselii, Chrysosporium kuzurovianum, Chrysosporium lignorum,
Chrysosporium obatum, Chrysosporium. lucknowense, Chrysosporium lucknowense Garg 27K,
Chrysosporium medium, Chrysosporium medium var. spissescens, Chrysosporium mephiticum,
Chrysosporium merdarium, Chrysosporium merdarium var. roseum, Chrysosporium minor,
Chrysosporium. pannicola, Chrysosporium parvum, Chrysosporium parvum var. crescens,
Chrysosporium pilosum, Chrysosporium pseudomerdarium, Chysosporium pyriformis,
Chrysosporium queenshmdicum, Chrysosporium sigleri, Chrysosporium sulfureum,
Chrysosporium synchronum, Chrysosporium tropicum, Chrysosporium undulatum.
Chrysosporium vallenaren.se, Chrysosporium vespertilium, Chrysosporium. zonatum),
Coonemeria, Cunninghamella (e.g., Cunninghamella ehinulata), Dactylomyces, Emericella,
Filibasidium., Fusarium, (e.g., Fusarium moniliforme, Fusarium venenatum, Fusarium oxysporum,
Fusarium graminearum, Fusarium proliferatum, Fusarium verticiollioides, Fusarium culmorum, Fusarium crookwellense, Fusarium poae, Fusarium sporotrichioides, Fusarium sambuccinum, Fusarium torulosum, as well as associated Gibberella teleomorphic forms thereof), Gibberella, Humicola, Hypocrea, Lentinula, Malbranchea (e.g., Malbranchea filamentosa), Magnaporthe, Malbranchium, Melanocarpus, Mortiere.Ua (e.g., Mortierella alpina 1S-4, Mortieralla isabelline, Mortierrla vinacea, Mortieralla vinaceae var. raffinoseutilizer), Mucor (e.g., Mucor miehei Cooney et Emerson (Rhizomucor miehei (Cooney & R. Emerson)) Schipper, Mucor pusillus Lindt, Mucor circinelloides Mucor mucedo), Myceliophthora (e.g., Myceliophthora thermophila), Myrothecium, Neocallimastix, Neurospora (e.g., Neurospora crassa), Paecilomyces, Penicillium (e.g., Penicillium. chrysogenum, Pennicillium iilacinum, Penicillium roquefortii), Phenerochaete, Phlebia, Piromyces, Pythium, Rhizopus (e.g., Rhizopus niveus), Schizophyllum, Scytalidium, Sporotrichum (e.g., Sporotrichum. celhdophilum), Stereum, Talarornyces, Thermoascus, Thermomyces, Thielavia (e.g., Thielavia terrestris), Tolypocladium, and Trichoderma (e.g., Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichoderma virens, Trichoderma cit rinoviride , Trichoderma viride).
[0141] Non-limiting examples of suitable bacteria include firmicutes, cyanobacteria (blue- green algae), osciilatoriophcideae, bacillales, lactobacillales, oscillatoriales, bacillaceae, lactobacillaceae, and members of any of the following genera, and deri vatives and crosses thereof: Acinetobacter, Acetobacter (e.g., Acetobacter suboxydans , Acetobacter xylinum), Actinoplane (e.g., Actinoplane missouriensis), Arthrospira (e.g., Arthrospira platensis, Arthrospira maxima), Bacillus (e.g., Bacillus cercus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis), Escherichia (e.g., Escherichia colt), Lactobacillus (e.g., Lactobacillus acidophilus, Lactobacillus bulgaricus), Lactococcus (e.g., Lactococcus lactis, Lactococcus lactis Lancefield Group N, Lactobacillus reuteri), Leuconostoc (e.g., Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides), Micrococcus (e.g., Micrococcus lysodeikticus), Rhodococcus (e.g., Rhodococcus opacus, Rhodococcus opacus strain PD630), Spirulina, Streptococcus (e.g., Streptococcus cremoris. Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus), Streptomyces (e.g., Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus), Tetrahymena (e.g., Tetrahymena thermophile, Tetrahymena hegewischi, Tetrahymena hyperangularis. Tetrahymena malaccensis, Tetrahymena pigmentosa, Tetrahymena pyriformis, Tetrahymena vorax ), and Xanthomonas (e.g., Xanthomonas campestris).
[0142] Non-limiting examples of suitable algae include members of any of the following genera, and derivatives and crosses thereof: red algae, brown algae, green algae, microalgae, Achnanthes (e.g., Achnanthes orientalis), Agmenellum, Alaria (e.g., Alaria marginata), Amphiprora (e.g., Amphiprora hyaline), Amphora (e.g., Amphora coffeiformis, Amphora coffeiformis tinea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp.), Anabaena. Analipus (e.g., Analipus japonicus), Ankistrodesmus (e.g., Ankistrodesmus falcatus), Ascophyllum (e.g., Ascophyllum nodosum), Boekelovia (e.g., Boekelovia hooglandii), Borodinella (e.g., Borodinella sp.), Botryococcus (e.g., Botryococcus braunii, Botryococcus sudeticus), Carteria, Chaetoceros (e.g., Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp.), Chlorella (e.g., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fitsca var. vacuolata, Chlorella glucotropha, Chlorella infusionum. Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella Iuteoviridis , Chlorella luteoviridis var. aureoviridis, Chlorella luieoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoid.es var. acidicola, Chlorealla, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f terlia, Chlorella vulgaris var. autotrophica, Chlorella. vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris), Chlorococcum (e.g., Chlorococcum infusionum, Chlorococcum sp.), Chlorogonium, Chondrus (e.g., Chondrus crispus, Chondrus ocellatus), Chroomonas (e.g., Chroomonas sp.), Chrysosphaera (e.g., Chrysosphaera sp.), Cricosphaera (e.g., Cricosphaera sp.), Cryptomonas (e.g., Cryptomonas sp.), Cyclotella (e.g., Cyclotella cryptica, Cyclotella meneghiniana , Cyclotella sp.), Dunaliella (e.g., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella. salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta), Ecklonia (e.g., Ecklonia sp), Eisenia (e.g., Eisenia bicyclis), Ellipsoidon (e.g., Ellipsoidon sp.), Eremosphaera (e.g., Eremosphaera viridis, Eremosphaera sp.), Eucheuma (e.g., Eucheuma cottonii, Eucheuma spinosum), Euglena, Fragilaria (e.g., Fragilaria crotonensis, Fragilaria sp.), Franceia (e.g., Franceia sp.). Furcellaria (e.g., Furcellaria fastigiate), Gigartina (e.g., Gigartina acicularis, Gigartina bursa-pastoris, Gigartina pistillata, Gigartina radula, Gigartina skottsbergii, Gigartina stellate), Gleocapsa (e.g., Gleocapsa sp.), Gloeothamnion (e.g., Gloeothamnion sp.), Gloiopeltis (e.g., Gloiopeltis furcate), Gracilaria (e.g., Gracilaria bursa-pastoris, Gracilaria lichenoides), Hizikia (e.g., Hizikia fusiforme), Hymenomonas (e.g., Hymenomonas sp.), Isochrysis (e.g., Isochrysis aff. galbana, Isochrysis galbana), Kjellmaniella (e.g., Kjellmaniella gyrate), Laminaria (e.g., Laminaria angustata, Laminaria longirruris, Laminaria Longissima, Laminaria ochotensis, Laminaria claustonia, Laminaria saccharina, Laminaria digitata, Laminaria japonicd), Lepocinclis, Macrocystis (e.g., Macrocystis pyrifera), Micractinium, Monoraphidium (e.g., Monoraphidium minutum, Monoraphidium sp.), Nannochloris (e.g., Nannochloris sp.), Nannochloropsis (e.g., Nannochloropsis salina, Nannochloropsis sp.), Navicula (e.g., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp.), Nephrochloris (e.g., Nephrochloris sp.), Nephroselmis (e.g., Nephroselmis sp.), Nitzschia (e.g., Nitzschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilia, Nitzschia. pus ilia elliptica, Nitzschia pusilia monoensis, Nitzschia quadrangular, Nitzschia sp.), Ochromonas (e.g., Ochromonas sp.), Oocystis (e.g., Oocystis parva, Oocystis pusilia, Oocystis sp.), Oscillatoria (e.g., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis), Palmaria (e.g., Palmaria palmata), Pascheria (e.g., Pascheria acidophild), Pavlova (e.g., Pavlova sp.), Petalonia (e.g., Petalonia fascia), Phagus, Phomudium, Platymonas (e.g., Platymonas sp.), Pleurochrysis (e.g., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp.), Porphyra (e.g., Porphyra colum.bina, Porphyra crispata, Porhyra deutata, Porhyra perforata, Porhyra suborbictdata, Porphyra tenerd), Porphyridium (e.g., Porphyridium cruentum, Porphyridium purpureum, Porphyridium aerugineum), Prototheca (e.g., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis. Prototheca zopfii), Pyramimonas (e.g., Pyramimonas sp.), Pyrobotrys, Rhodella (e.g., Rhodella maculate, Rhodella reticulata, Rhodella violacea), Rhodymenia (e.g., Rhodymenia palmata), Sarcinoid (e.g., Sarcinoid chrysophyte), Scenedesmus (e.g., Scenedesmus armatus), Scytosiphon (e.g., Scytosiphon lome), Spirogyra, Spirulina (e.g., Spirulina platensis), Stichococcus (e.g., Stichococcus sp.), Synechococcus (e.g., Synechococcus sp.), Tetraedron, Tetraselmis (e.g., Tetraselmis sp., Tetraselmis suecica), Thalassiosira (e.g., Thalassiosira weissflogii) and Viridiella (e.g., Viridiellafridericiand).
[0143] A recombinant host cell according to any of the above may comprise a genetic modification that improves production of the recombinant β-lactoglobulin protein. Non-limiting examples of suitable genetic modifications include altered kinase activities, altered phosphatase activities, altered protease activities, altered gene expression induction pathways, altered production and/or activity of a protein involved in protein folding, and altered production and/or activity of a protein involved in protein secretion (e.g., vesicular transport).
Recombinant Milk Protein With Bound Hydrophobic Bioactive Agent
[0144] In various aspects, provided herein is a recombinant β-lactoglobulin protein according to any of the above with bound hydrophobic bioactive agent (e.g., hydrophobic bioactive agent according to any of the above).
Composition Comprising Recombinant Milk Protein
[0145] In various aspects, provided herein is a composition that comprises a recombinant β- lactoglobulin protein according to any of the above, or a recombinant β-lactoglobulin protein wi th bound hydrophobic bioactive agent according to any of the above.
[0146] The composition may comprise between 0.001% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%. 8%, 7%, 6%. 5%, 4%, 3%, 2%, 1%. 0.9%. 0.8%. 0.7%. 0.6%. 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%; between 0.1% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, or 0.2%; between 0.2% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%. 0.8%, 0.7%, 0.6%. 0.5%, 0.4%, or 0.3%; between 0.3% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, or 0.4%; between 0.4% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%. 45%, 40%, 35%, 30%, 25%, 20%, 15%. 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%. 0.6%, or 0.5%; between 0.5% and 100%, 95%, 90%, 85%, 80%. 75%, 70%. 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%. 20%, 15%, 14%, 13%, 12%, 11%. 10%. 9%. 8%. 7%. 6%. 5%. 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, or 0.6%: between 0.6% and 100%, 95%. 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%, 1%. 0.9%. 0.8%. or 0.7%; between 0.7% and 100%, 95%, 90%, 85%, 80%, 75%. 70%, 65%, 60%, 55%, 50%, 45%, 40%. 35%, 30%, 25%, 20%, 15%. 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.9%, or 0.8%: between 0.8% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%. 2%, 1%, or 0,9%: between 0.9% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%;, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%; between 1% and 100%, 95%. 90%, 85%. 80%, 75%, 70%, 65%, 60%. 55%, 50%, 45%, 40%, 35%. 30%, 25%. 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%; between 2% and 100%, 95%, 90%, 85%. 80%, 75%, 70%, 65%, 60%. 55%, 50%. 45%, 40%, 35%, 30%, 25%. 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%; between 3% and 100%, 95%, 90%, 85%, 80%. 75%, 70%, 65%, 60%, 55%, 50%, 45%. 40%, 35%, 30%, 25%, 20%. 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%; between 4% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%. 35%, 30%, 25%, 20%, 15%, 14%, 13%. 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5%; between 5% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, or 6%; between 6% and 100%, 95%, 90%, 85%, 80%, 75%, 70%. 65%, 60%. 55%, 50%. 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, or 7%; between 7% and 100%, 95%, 90%. 85%, 80%, 75%, 70%, 65%, 60%, 55%. 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, or 8%; between 8% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, or 9%; between 9% and 100%, 95%, 90%. 85%, 80%, 75%. 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%. 11%, or 10%; between 10% and 100%, 95%, 90%, 85%. 80%, 75%, 70%, 65%, 60%. 55%, 50%. 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, or 11%; between 11% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%. 45%, 40%, 35%, 30%, 25%, 20%, 15%. 14%, 13%, or 12%; between 12% and 100%, 95%, 90%. 85%, 80%, 75%, 70%, 65%, 60%, 55%. 50%, 45%, 40%, 35%, 30%. 25%, 20%, 15%, 14%, or 13%; between 13% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%. 30%, 25%, 20%, 15%, or 14%; between 14% and 100%, 95%, 90%, 85%. 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15%; between 15% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20%; between 20% and 100%. 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25%; between 25% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30%; between 30% and 100%, 95%, 90%, 85%. 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, or 35%; between 35% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%; between 40% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45%; between 45% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%; between 50% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 55%; between 55% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%. or 60%; between 60% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, or 65%; between 65% and 100%, 95%, 90%, 85%, 80%, 75%, or 70%; between 70% and 100%, 95%, 90%, 85%, 80%, or 75%; between 75% and 100%, 95%, 90%, 85%-, or 80%; between 80% and 100%, 95%, 90%, or 85%'; or between 85% and 100%, 95%, 90%; between 90% and 100% or 95%, or between 95% and 100% by mass of the recombinant β-lactoglobulin protein according to any of the above, or of the recombinant β- lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above.
[0147] At standard ambient temperature and conditions (i.e., 20-30°C and 0.95-1 .05 atm), the composition according to any of the above may be a fluid, semi-solid (e.g., gelatinous), solid, or powder. The powder may comprise a moisture content of less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, or less than 1%; or between 0.1% and 20%, 15%, 10%, 5%, or 1%; between 1% and 20%, 15%, 10%, or 5%; between 5% and 20%, 15%, or 10%; between 10% and 20%, or 15%; or between 15% and 20%. The powder may be used in powder form, or the powder may be reconstituted with a hydrating agent prior to use, or the powder may be mixed with other dry components (e.g., flour, sugar, minerals, pH or ionic strength adjusting agents) before a hydrating agent is added to the mixture. Non-limiting examples of suitable hydrating agents include water, milk (e.g., animal milk, nut milk, plant-based milk), juice (e.g., vegetable juice, fruit juice, other plant juice), brine (e.g., fluid or liquid used to soak beans or legumes), and mixtures thereof.
[0148] The composition according to any of the above may comprise or consist of a recombinant β-lactoglobulin protein according to any of the above, or of a recombinant β- lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above, as a monomer, and/or may comprise a recombinant β-lactoglobulin protein according to any of the above, or of a recombinant β-lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above, as a polymer comprising linked repeated protein monomers, wherein the repeated protein monomers comprise or consist of a recombinant β-lactoglobulin protein according to any of the above, or of a recombinant β-lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above, or of dimers, trimers, or tetramers of a recombinant β-lactoglobulin protein according to any of the above, or of a recombinant β- lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above. Polymers comprising linked repeated protein monomers that comprise or consist of recombinant β-lactoglobulin proteins, and methods for producing such polymers, are disclosed in patent publication US20210235714, filed August 15, 2012. The composition according to any of the above may comprise the recombinant β-lactoglobulin protein, or the recombinant β-lactoglobulin protein with bound hydrophobic bioactive agent, as a monomer or as a polymer, comprised (e.g., covalently bound, non-covalently bound, encapsulated, physically trapped) in another polymer. Non-limiting examples of suitable polymers in which the recombinant β-lactoglobulin protein monomer or polymer according to any of the above may be comprised include a polymer comprising linked repeated monomers of a polysaccharide (e.g., cellulose, cellulose derivatives [e.g., hydroxyethyl cellulose, hydroxymethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose], heparin, hyaluronic acid, pectin, chondroitin sulfate, pullulan, dextrin, dextran), alginate [e.g., calcium alginate], alginate derivatives, chitosan, chitin) or a protein (e.g., collagen, gelatin, elastin, fibrinogen, fibrin, keratin).
[0149] The composition according to any of the above may comprise between 0.001% and 100% (e.g., between 0.001% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1 %, or 0.01 %; between 0.01% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1%; between 0.1% and 100%, 99%, 95%. 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 1%; between 1% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; between 10% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%. 30%, or 20%; between 20% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, or 30%; between 30% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, or 40% ; between 40% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, or 50%; between 50% and 100%. 99%, 95%, 90%, 80%, 70%, or 60%; between 60% and 100%, 99%, 95%, 90%, 80%, or 70%; between 70% and 100%', 99%, 95%, 90%, or 80%; between 80% and 100%;, 99%, 95%, or 90%; between 90% and 100%, 99%, or 95%; between 95%' and 100% or 99%; or between 99% and 100%) by mass of a protein polymer, wherein the protein polymer may comprise between about 0.001% and about 100% (e.g., between 0.001% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01%; between 0.01% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, or 0.1%; between 0.1 % and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 1%; between 1% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%; between 10% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%; between 20% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%. or 30%; between 30% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, or 40%; between 40% and 100%, 99%, 95%, 90%, 80%, 70%, 60%, or 50%; between 50% and 100%, 99%, 95%, 90%, 80%, 70%, or 60%; between 60% and 100%, 99%, 95%, 90%, 80%, or 70%; between 70% and 100%, 99%, 95%, 90%, or 80%; between 80% and 100%, 99%, 95%, or 90%; between 90% and 100%, 99%, or 95%; between 95% and 100% or 99%; or between 99% and 100%) by mass of a recombinant β-lactoglobulin protein according to any of the above, or of a recombinant β-lactoglobulin protein with bound hydrophobic bioactive agent according to any of the above.
[0150] The composition according to any of the above may be essentially free of any protein other than the recombinant protein contained in the composition according to any of the above.
[0151] The composition according to any of the above may be essentially tree of any recombinant protein other than the recombinant protein contained in the composition according to any of the above.
[0152] The composition according to any of the above may be essentially free of any recombinant milk protein other than the recombinant β-lactoglobulin protein contained in die composition according to any of the above.
[0153] The composition according to any of the above may be essentially free of a component found in a mammal-produced milk (e.g., cow milk, goat milk, sheep milk, human milk, buffalo milk, yak milk, camel milk, llama milk, alpaca milk, horse milk, donkey milk), or may comprise a lower concentration of al least one component found in a mammal-produced milk. Non-limiting examples of components found in mammal -derived milk include lactose, saturated fat. cholesterol, native milk proteins, and native milk lipids.
[0154] The composition according to any of the above may be essentially free of a component obtained from an animal (i.e., a component that is native to an animal, including animal products [i.e., parts of an animal that are consumables or typically prepared for consumption by humans; e.g., animal meat, animal fat, animal blood]] animal byproducts [i.e., products that are typically not consumable by themselves but are the byproducts of slaughtering animals for consumption; e.g., animal bones, animal carcasses, and constituents isolated therefrom], products produced by an animal [e.g., mammal -derived milk, chicken eggs, bee honey], and consumables produced therefrom [e.g., gelatin, rennet, whey proteins extracted from mammal-derived milk, casein extracted from mammal-derived milk, milk lipid extracted from mammal-derived milk, animal lipids, animal proteins]), or comprise 2% or less by mass of such component.
Food Product
[0155] The composition according to any of the above may be a food product.
[0156] The term "food product" as used herein refers to a composition that can be ingested by a human or an animal for dietary purposes (i.e., without ill health effects but with significant nutritional and/or caloric intake due to uptake of digested material in the gastrointestinal tract), including a domesticated animal (e.g., dog, cat), farm animal (e.g., cow, pig, horse), and wild animal (e.g., non-domesticated predatory animal). The term includes compositions that can be combined with or added to one or more other ingredients to make a food product that can be ingested by a human or an animal.
[0157] Food products comprising β-lactoglobulin are desirable, particularly for athletes, as β- lactoglobulin protein has a high content of essential and branched-chain amino acids, which are thought to aide production of muscle tissue. Moreover, β-lactoglobulin is desirable as a food additive as it has good water binding ability, which property makes β-lactoglobulin suitable for managing water activity of food products. Moreover, β-lactoglobulin is desirable as a food additive as it has anti-microbial activity, which property makes β-lactoglobulin suitable for extending the shelf life of food products. Moreover, β-lactoglobulin is desirable as a food additive as it can readily absorb at interfaces, which property makes β-lactoglobulin suitable for producing highly stable dispersions in food products. Moreover, food products comprising β-lactoglobulin protein with bound hydrophobic bioactive agents are desirable as the β-lactoglobulin protein may solubilize, stabilize, and/or protect from degradation such hydrophobic bioactive agents until they are released by the β-lactoglobulin protein to exert their bioactivity.
[0158] The food product may be a food product, or may resemble a food product (i.e., may be a “substitute food product”), selected from any of the food product categories defined by the National Health and Nutrition Examination Survey (NHANES). Non-limiting examples of NHANES food product categories include snack foods and gums (e.g., snack bars, crackers, salty snacks from grain products, chewing gums); breads, grains, and pastas (e.g., oat breads and rolls, cornbread, corn muffins, tortillas, flour and dry mixes, biscuits, multi-grain breads and rolls, whole wheat breads and rolls, pastas, rye breads and rolls, cracked wheat breads and rolls, white breads and rolls): beverages (e.g., beers and ales, beverage concentrates, beverages, energy drinks, sports drinks, fluid replacements, soft drinks, carbonated beverages, juices, wines, beers, cocktails, nutrition drinks, nutrition powders, protein-enriched beverages, coffee, tea); sweets and desserts (e.g., cakes, candies, chips, cookies, cobblers, pastries, ices or popsicles, muffins, pies, sugar replacements or substitutes, syrups, honey, jellies, jams, preserves, salads, crepes, Danish, breakfast pastries, doughnuts); breakfast foods (e.g,, cereal grains, cereal, rice, French toast, pancakes, waffles, coffee cake); salad dressings, oils, sauces, condiments (e.g., cooking fats, vegetable oils, salad dressings, tomato sauces, gravies); potatoes (e.g., potato salad, potato soups, chips and sticks, fried potatoes, mashed potatoes, stuffed potatoes, puffs); and soups (e.g., vegetable soups, vegetable broths), meals, main dishes, proteins (e.g., meat substitutes), and seafoods.
[0159] The food product according to any of the above may be a dairy product, a supplemented dairy' product (i.e., a conventional dairy product that is supplemented with the recombinant β- lactoglobulin protein according to any of the above), or substitute dairy product (i.e., a food product that resembles a conventional dairy product). The term "dairy product" as used herein refers to milk (e.g., whole milk [at least 3.25% milk fat], partly skimmed milk [from 1% to 2% milk fat], skim milk [less than 0.2% milk fat], cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk, evaporated milk, milk foam), and products derived from milk, including but not limited to yogurt (e.g., whole milk yogurt [at least 6 grams of fat per 170 g], low- fat yogurt [between 2 and 5 grams of fat per 170 g], nonfat yogurt [0.5 grams or less of fat per 170 g], greek yogurt [strained yogurt with whey removed], whipped yogurt, goat milk yogurt, Labneh [labne], sheep milk yogurt, yogurt drinks [e.g.. whole milk Kefir, low-fat milk Kefir], Lassi), cheese (e.g., whey cheese such as ricotta; pasta filata cheese such as mozzarella; semi-soft cheese such as Havarti and Muenster: medium-hard cheese such as Swiss and Jarlsberg and halloumi; hard cheese such as Cheddar and Parmesan; washed curd cheese such as Colby and Monterey Jack; soft ripened cheese such as Brie and Camembert; fresh cheese such as cottage cheese, feta cheese, cream cheese, paneer, and curd), processed cheese, processed cheese food, processed cheese product, processed cheese spread, enzyme -modulated cheese; cold-pack cheese), dairy-based sauces (e.g., salad dressing, bechamel sauce, fresh sauces, frozen sauces, refrigerated sauces, shelf stable sauces), dairy spreads (e.g., low-fat spread, low-fat butter), cream (e.g., dry cream, heavy cream, light cream, whipping cream, half-and-half, coffee whitener, coffee creamer, sour cream, crème fraiche), frozen confections (e.g., ice cream, smoothie, milk shake, frozen yogurt, sundae, gelato, custard), dairy desserts (e.g., fresh, refrigerated, or frozen), butter (e.g., whipped butter, cultured butter), dairy powders (e.g., whole milk powder, skim milk powder, fat-filled milk powder (i.e., milk powder comprising plant fat in place of all or some animal fat), infant formula, milk protein concentrate (i.e., protein content of at least 80% by weight; e.g., milk protein concentrate, whey protein concentrate, demineralized whey protein concentrate, β-lactoglobulin concentrate, a-lactalbumin concentrate, glycomacropeptide concentrate, casein concentrate), milk protein isolate (i.e., protein content of at least 90% by weight; e.g., milk protein isolate, whey protein isolate, demineralized whey protein isolate, β-lactoglobulin isolate, a-lactalbumin isolate, glycomacropeptide isolate, casein isolate), nutritional supplements, texturizing blends, flavoring blends, coloring blends, ready-to-drink or ready-to-mix products (e.g., fresh, refrigerated, or shelf stable dairy protein beverages, weight loss beverages, nutritional beverages, sports recovery beverages, and energy drinks), puddings, gels, chewables, crisps, bars (e.g., nutrition bars, protein bars), and fermented dairy products (e.g., yoghurt, cheese, sour cream, cultured buttermilk, cultured butter, cultured butter oil).
[0160] The food product according to any of the above may be an animal meat or animal meat product, a supplemented animal meat or animal meat product (i.e., a conventional animal meal or animal meat product that is supplemented with a recombinant β-lactoglobulin protein according to any of the above), or substitute animal meat or animal meat product (i.e., a food product that resembles a conventional animal meat or animal meat product). Non-limiting examples of animal meats and animal meat products include flesh obtained from skeletal muscle or from other organs (e.g., kidney, heart, liver, gallbladder, intestine, stomach, bone marrow, brain, thymus, lung, tongue), or parts thereof, obtained from an animal. The animal meat may be dark or white meat. Non-limiting examples of animals from which animal meat or animal meat product can be obtained include cattle, lamb, mutton, horse, poultry (e.g., chicken, duck, goose, turkey), fowl (e.g., pigeon, dove, grouse, partridge, ostrich, emu, pheasant, quail), fresh or salt water fish (e.g., catfish, tuna, spearfish, shark, halibut, sturgeon, salmon, bass, muskie, pike, bowfin, gar, eel, paddlefish, bream, carp, trout, walleye, snakehead, crappie, sister, mussel, scallop, abalone, squid, octopus, sea urchin, cuttlefish, tunicate), crustacean (e.g., crab, lobster, shrimp, barnacle), game animal (e.g., deer, fox, wild pig, elk, moose, reindeer, caribou, antelope, zebra, squirrel, marmot, rabbit, bear. beaver, muskrat, opossum, raccoon, armadillo, porcupine, bison, buffalo, boar, lynx, bobcat, bat), reptile (e.g., snakes, turtles, lizards, alligators, crocodiles), any insect or other arthropod, rodent (nutria, guinea pig, rat, mice, vole, groundhog, capybara), kangaroo, whale, and seal. The animal meat or animal meat product may be ground, chopped, shredded, or otherwise processed, and uncooked, cooking, or cooked.
[0161] The food product according to any of the above may be an egg or egg product, a supplemented egg product (i.e., a conventional egg or egg product that is supplemented with the recombinant β-lactoglobulin protein according to any of the above), or substitute egg or egg product (i.e., a food product that resembles a conventional egg or egg product). Non-limiting examples of eggs or egg products include whole egg (e.g., liquid whole egg, spray-dried whole egg, frozen whole egg), egg white (e.g., liquid egg white, spray-dried egg white, frozen egg white), egg yolk, egg dishes, egg soups, mixtures made with egg whites, mixtures made with egg substitutes, mayonnaise, custard, and salad dressings.
[0162] Resemblance of a substitute food product provided herein to a conventional food product may be due to any physical, mechanical, chemical/biological, sensory, and/or functional attribute.
Cosmetic or Personal Care Product
[0163] The composition according to any of the above may be a cosmetic or personal care composition.
[0164] The term "cosmetic or personal care composition" as used herein refers to a composition that upon application to a body surface (i.e., an exposed area of a human body, such as skin, hair, nail, tooth, and tissues of the oral cavity [e.g., gums]) confers a perceived or actual beautifying or hygienizing effect. Non-limiting examples of cosmetic or personal care compositions include anti-wrinkling treatments (i.e., compositions used for tensioning [e.g., smoothing out of skin, reducing wrinkles in skin, removing fine lines in skin]), anti-aging treatments (i.e., compositions used for removing signs of aging [e.g., wrinkles, fine lines, manifestations of photodamage (e.g., sun spots)]), sun protection (i.e., compositions used to protect against UV exposure), anti-burn treatments (i.e., compositions used for soothing burns [e.g., sunburns]), anti-acne treatments (i.e., compositions that are effective in the treatment of acne and/or the symptoms associated therewith), skin cleansers (i.e., compositions used for cleaning skin and/or skin pores [e.g., nose strips for pore cleaning]), anti-dandruff treatments (i.e., compositions used for reducing or eliminating dandruff), anti-body odor treatments (i.e., compositions used for reducing or eliminating body odor), self-tanning treatments (i.e., compositions used for darkening skin color), skin whitening treatments (i.e., compositions used for bleaching/depigmenting skin color), hair coloring treatments (i.e., compositions used for coloring hair), lotions (e.g., skin lotions, body care lotions, wash lotions, moisture retention lotions, pre-shave lotions, after-shave lotions), pastes (e.g., washing pastes), ointments, balms, salves, masks, creams (e.g., water in oil creams, oil in water creams, day creams, night creams, eye creams, skin creams, face creams, anti-wrinkle creams, sun protection creams, moisture retention creams, after-shave creams, skin bleaching creams, self-tanning creams, vitamin creams, moisturizing creams, massage creams), milks (e.g., body milks, cleansing milks), gels (e.g., anhydrous gels, shower gels), Eau de Toilette, soaps (e.g., transparent soaps, luxurious soaps, deodorant soaps, cream soaps, baby soaps, skin protection soaps, abrasive soaps, syndets, pasty soaps, soft soaps, peeling soaps), skin peeling treatments, liquid washes, shower and bath preparations (e.g., wash lotions, shower baths, shower gels, foam baths, oil baths, scrub preparations), foams (e.g., shaving foams, foam baths), deodorants, hair care products (e.g., shampoos, conditioners, hair mousses, hair colorants, hair sprays, rinse-off lotions, hair gels, hair emulsions, hair laquers, hair tonics), lip glosses, sprays (e.g., hair sprays, pump sprays, sprays containing blowing agents), treatments for skin defects (e.g., dermatitis, weals, chaps, blemishes, cracks, scars, freckles, moles, rashes, blisters, pustules), toners, cleaning tissues, sanitary towels, tampons, nappies, repellents, make-up products (e.g., studio pigments, mascara, eye shadows, eyeliners, eye liner pens, rouges, face powders, eyebrow pencils, lipsticks, foundations, tinted creams, concealer sticks, blemish sticks, blushes), sticks (e.g., lipsticks, concealer sticks, blemish sticks), hair removing agents, hand cleaning products, intimate hygiene products, foot care products, baby care products, and oral hygiene products (e.g., chewing gums, mouthwashes, toothpastes, gum-cleaning agents, denture adhesives, denture fixatives).
Therapeutic Product
[0165] The composition according to any of the above may be a therapeutic product that can be used for treating a disorder, disease, or injury in a subject.
[0166] Non-limiting examples of disorders, diseases, or injuries include cancer, microbial infections or contaminations, viral infections, skin defects (e.g., rashes, sores, abrasions, burns, blisters, cuts, acute wounds, chronic wounds, postoperative surgical wounds, venous ulcers. diabetic ulcers, diabetic foot ulcers, decubitus ulcers, itchy skin), or pain (e.g., acute pain, chronic pain).
Method for Obtaining Recombinant Host Cell
[0167] In various aspects, provided herein is a method for obtaining a recombinant host cell according to any of the above, wherein the method comprises; obtaining a polynucleotide that encodes a recombinant β-lactoglobulin protein (and optional secretion signal) according to any of the above, or a recombinant expression construct according to any of the above, or a recombinant vector according to any of the above; and introducing the polynucleotide, recombinant expression construct, or recombinant vector into a host cell (e.g., any of the host cells disclosed herein) to obtain a recombinant host cell according to any of the above,
[0168] The polynucleotide, recombinant expression construct, and/or recombinant vector may- be obtained by any suitable method known in the art, including, without limitation, direct chemical synthesis and cloning.
[0169] Methods for introducing a polynucleotide, recombinant expression construct, or recombinant vector into a host cell are well-known in the art. Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, DEAE-dextran transfection, cell squeezing, sonoporation, optical transfection, protoplast fusion, protoplast transformation, impalefection, hyrodynamic delivery, gene gun, magnetofection, viral transduction, electroporation, and chemical transformation (e.g., using PEG).
[0170] Methods for identifying a recombinant host cell are well-known in the art, and include screening for expression of a drug resistance or auxotrophic marker encoded by the polynucleotide, recombinant expression construct, or recombinant vector that permits selection for or against growth of cells, or by other means (e.g., detection of light emitting peptide comprised in the polynucleotide, recombinant expression construct, or recombinant vector, molecular analysis of individual recombinant host cell colonies [e.g., by restriction enzyme mapping, PCR amplification, Southern analysis, or sequence analysis of isolated extrachromosomal vectors or chromosomal integration sites]).
[0171] Production of the recombinant β-lactoglobulin protein by a recombinant host cell according to any of the above may be evaluated using any suitable method known in the art, such as assays that are carried out at the RNA level and. most suitable, at the protein level, or by use of functional bioassays that measure the production or activity of the recombinant β-lactoglobulin protein. Non-limiting examples of such assays include Northern blotting, dot blotting (DNA or RNA), RT-PCR (reverse transcriptase polymerase chain reaction), RNA-Seq, in situ hybridization, Southern blotting, enzyme activity assays, immunological assays (e.g., immunohistochemical staining, immunoassays, Western blotting, ELISA), and free thiol assays (e.g., for measuring production of protein comprising free cysteine residues).
Method for Producing Recombinant β-Lactoglobulin Protein
[0172] In various aspects, provided herein is a method for producing a recombinant β - lactoglobulin protein according to any of the above, wherein the method comprises: fermenting a recombinant host cell according to any of the above in a culture medium under conditions suitable for production of the recombinant β-lactoglobulin protein.
[0173] The method may further comprise: purifying the recombinant β-lactoglobulin protein from the fermentation broth to obtain a preparation comprising the recombinant β-lactoglobulin protein; and/or post-processing the recombinant β-lactoglobulin protein.
[0174] Alternatively, the recombinant β-lactoglobulin protein may be obtained using in vitro methods (e.g., using cell-free transcription and/or translation systems)
Fermenting
[0175] Suitable conditions for producing the recombinant β-lactoglobulin protein are typically those under which a recombinant host ceil according to any of the above can grow and/or remain viable, and produce the recombinant β-lactoglobulin protein.
[0176] Non-limiting examples of suitable conditions include a suitable culture medium (e.g., a culture medium having a suitable nutrient content [e.g., a suitable carbon content, a suitable nitrogen content, a suitable phosphorus content], a suitable supplement content, a suitable trace metal content, a suitable pH), a suitable temperature, a suitable feed rate, a suitable pressure, a suitable level of oxygenation, a suitable fermentation duration (i.e., volume of culture media comprising the recombinant host cells), a suitable fermentation volume (i.e., volume of culture media comprising the recombinant host cells), and a suitable fermentation vessel.
[0177] Suitable culture media include all culture media in which the recombinant host cell can grow and/or remain viable, and produce the recombinant β-lactoglobulin protein. Typically, the culture medium is an aqueous medium that comprises a carbon source, an assimilable nitrogen source (i.e., a nitrogen-containing compound capable of releasing nitrogen in a form suitable for metabolic utilization by the recombinant host cell ), and a phosphate source.
[0178] Non-limiting examples of carbon sources include monosaccharides, disaccharides, polysaccharides, acetate, ethanol, methanol, glycerol, methane, and combinations thereof. Non- limiting examples of monosaccharides include dextrose (glucose), fructose, galactose, xylose, arabinose, and combinations thereof. Non- limiting examples of disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, amylose, hemicellulose, maltodextrin, and combinations thereof.
[0179] Non-limiting examples of assimilable nitrogen sources include anhydrous ammonia, ammonium sulfate, ammonium hydroxide, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium pyrophosphate, ammonium chloride, sodium nitrate, urea, peptone, protein hydrolysates, corn steep liquor, corn steep solids, spent grain, spent grain extract, and yeast extract. Use of ammonia gas is convenient for large scale operations, and may- be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia may also be employed to assist in pH control.
[0180] The culture medium may further comprise an inorganic salt, a mineral (e.g., magnesium, calcium, potassium, sodium; e.g., in suitable soluble assimilable ionic and combined forms), a metal or transition metal (e.g., copper, manganese, molybdenum, zinc, iron, boron, iodine; e.g., in suitable soluble assimilable form), a vitamin, and any other nutrient or functional ingredient (e.g., a protease [e.g., a plant-based protease] that can prevent degradation of the recombinant β-iactoglobulin protein, a protease inhibitor that can reduce the activity of a protease that can degrade the recombinant β-lactoglobulin protein, and/or a sacrificial protein that can siphon away protease activity, an anti-foaming agent, an anti-microbial agent, a surfactant, an emulsifying oil).
[0181] Suitable culture media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).
[0182] A suitable pH may be a pH of between about 2 and about 8 (e.g., a pH of between 2 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4, 3.5, 3, or 2.5; between 2.5 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2. 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4, 3.5, or 3: between 3 and 8,
7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4, or 3.5; between 3.5 and 8, 7.5, 7,
6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, or 4: between 4 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, or 4.5; between 4.5 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, or 4.6; between 4.6 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, or 4.7; between 4.7 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, or 4.8; between 4.8 and 8, 7.5, 7,
6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, or 4.9; between 4.9 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, 5.1, or 5; between 5 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, 5.2, or 5.1; between 5.1 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, 5.3, or 5.2; between 5.2 and 8, 7.5, 7, 6.5, 6, 5.5, 5.4, or 5.3; between 5.3 and 8, 7,5, 7, 6.5, 6, 5.5, or 5.4; between 5.4 and 8, 7.5, 7, 6.5, 6, or 5.5; between 5.5 and 8, 7.5, 7, 6.5, or 6; between 6 and 8, 7.5, 7, or 6.5; between 6.5 and 8, 7.5, or 7; between 7 and 8, or 7.5; or between 7.5 and 8).
[0183] A suitable temperature may be a temperature of between about 20°C and about 46°C (e.g., between 20°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C, 28°C, 26°C, 24°C, or 22°C; between 22°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C. 28°C, 26°C, or 24°C; between 24°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C, 28°C, or 26°C; between 26°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, 30°C, or 28°C; between 28°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, 32°C, or 30°C; between 30°C and 46°C, 44°C, 42°C, 40°C, 38°C, 36°C, 34°C, or 32°C; between 32°C and 46°C, 44°C, 42°C, 40°C, 38CC, 36°C, or 34°C; between 36°C and 46°C, 44°C, 42°C, 40°C, or 38°C; between 38°C and 46°C, 44°C, 42°C, or 40°C; between 40°C and 46°C, 44°C, or 42°C, between 42°C and 46CC or 44°C; or between 44°C and 46°C
[0184] A suitable feed rate may be a feed rate of between about 0.01 g and about 0.2 g glucose equivalent per g dry cell weight (DCW) per hour.
[0185] A suitable pressure may be a pressure of between 0 psig and about 50 psig (e.g., between 0 psig and 50 psig, 40 psig, 30 psig, 20 psig, or 10 psig; between 10 psig and 50 psig, 40 psig, 30 psig, or 20 psig; between 20 psig and 50 psig, 40 psig, or 30 psig; between 30 psig and 50 psig, or 40 psig; or between 40 psig and 50 psig).
[0186] A suitable oxygenation may be an aeration rate of between about 0.1 volumes of oxygen per liquid volume in the fermentor per minute (vvm) and about 2.1 vvm (e.g., between 0.1 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1 ,5 vvm, 1.3 vvm, 1.1 vvm, 0.9 vvm, 0.7 vvm, 0.5 vvm, or 0.3 vvm; between 0.3 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, 1.3 vvm, 1.1 vvm, 0.9 vvm, 0.7 vvm, or 0.5 vvm; between 0.5 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, 1.3 vvm, 1.1 vvm, 0.9 vvm, or 0.7 vvm; between 0.7 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, 1.3 vvm, 1.1 vvm, or 0.9 vvm; between 0.9 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, 1.3 vvm, or 1.1 vvm; between 1.1 vvm and 2.1 vvm, 1.9 vvm, 1.7 vvm, 1.5 vvm, or 1.3 vvm; between 1.3 vvm and 2.1 vvm, 1.9 vvm, 1.7 wm, or 1.5 vvm; between 1.5 vvm and 2.1 vvm, 1.9 vvm, or 1.7 vvm; between 1.7 vvm and 2.1 vvm or 1.9 vvm; or between 1.9 vvm and 2.1 vvm).
[0187] A suitable fermentation duration may be a fermentation duration of between about 10 hours and about 500 hours (e.g., between 10 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, 40 hours, 30 hours, or 20 hours; between 20 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, 40 hours, or 30 hours; between 30 hours and 500 hours, 400 hours, 300 hours, 200 hours, 100 hours, 50 hours, or 40 hours; between 40 hours and 500 hours, 400 hours, 300 hours, 200 hours. 100 hours, or 50 hours; between 50 hours and 500 hours, 400 hours, 300 hours, 200 hours, or 100 hours; between 100 hours and 500 hours, 400 hours, 300 hours, or 200 hours; between 200 hours and 500 hours, 400 hours, or 300 hours; between 300 hours and 500 hours, or 400 hours; or between 400 hours and 500 hours).
[0188] A suitable fermentation volume may be between about 1 L and about 10,000,000 L (e.g., between 1 L and 10,000,000 L, 5,000,000 L, 1 ,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, 500 L, 100 L, 50 L, or 10 L; between 10 L and 10,000,000 L, 5,000,000 L, 1,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, 500 L, 100 L, or 50 L; between 50 L and 10.000.000 L, 5,000,000 L, 1.000.000 L, 500,000L, 100.000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, 500 L, or 100 L; between 100 L and 10,000,000 L, 5,000,000 L, 1,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, 5,000 L, 1,000 L, or 500 L; between 500 L and 10,000,000 L, 5,000,000 L, 1,000,000 I.,, 500,0001.., 100,000 L, 50,000 L, 10, 000 L, 5,000 L, or 1,000 L; between 1,000 L and 10.000,000 L, 5,000,000 L, 1,000,000 L, 500,000L, 100,000 L, 50,000 L, 10, 000 L, or 5,000 L; between 5,000 L and 10,000,000 L, 5,000,000 L, 1,000,000 L, 500,000L, 100,000 L, 50,000 L, or 10, 000 L; between 10,000 L and 10,000,000 L, 5,000,000 L. 1,000,000 L, 500,000L, 100,000 L, or 50,000 L; between 50,000 L and 10,000,000 L, 5,000,000 L, 1 ,000,000 L, 5()(),0()0L, or 100,000 L; between 100,000 L and 10,000,000 L, 5.000,000 L. 1,000.000 L, or 500.000L; between 500,000 L and 10,000,000 L, 5,000,000 L, or 1,000,000 L; between 1,000,000 L and 10,000,000 L, or 5,000,000 L; or between 5,000,000 L and 10,000,000 L). [0189] A suitable fermentation vessel may be any fermentation vessel known in the art. Non- limiting examples of suitable fermentation vessels include culture plates, shake flasks, fermenters (e.g., stirred tank fermenters, airlift fermenters, bubble column fermenters, fixed bed bioreactors, laboratory fermenters, industrial fermenters, or any combination thereof), used at any suitable scale (e.g., small-scale, large-scale) and in any process (e.g., solid culture, submerged culture, batch, fed-batch, or continuous-flow).
Purifying
[0190] Methods for purifying a recombinant protein (e.g., from a fermentation broth) to obtain a preparation comprising the recombinant protein are well-known in the art (see, for example, Protein Purification, JC Janson and L Ryden, Eds., VCH Publishers, New York, 1989; Protein Purification Methods: A Practical Approach, ELV Harris and S Angel. Eds., IRL Press, Oxford, England, 1989, respectively), and may be adapted to purify a recombinant β-lactoglobulin protein according to any of the above.
[0191] A recombinant β -lactoglobulin protein according to any of the above may be purified on the basis of its molecular weight, for example, by size exclusion/exchange chromatography, ultrafiltration through membranes, gel penneation chromatography (e.g., preparative disc-gel electrophoresis), or density centrifugation.
[0192] A recombinant β-lactoglobulin protein according to any of the above also may be purified on the basis of its surface charge or hydrophobicity/hydrophilicity, for example, by isoelectric precipitation, anion/cation exchange chromatography, isoelectric focusing (IEF), or reverse phase chromatography.
[0193] A recombinant β-lactoglobulin protein according to any of the above also may be purified on the basis of its solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents, or solvent extraction.
[0194] A recombinant β-lactoglobulin protein according to any of the above also may be purified on the basis of its affinity to another molecule, for example, by affinity chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography may include the use of an antibody having a specific binding affinity for the recombinant β-lactoglobulin protein, or a lectin to bind to a sugar moiety on the recombinant β-lactoglobulin protein, or any other molecule that specifically binds the recombinant β-lactoglobulin protein. The recombinant β-lactoglobulin protein may comprise a purification tag operably linked to its C -terminus, N -terminus, or both to facilitate affinity-based purification of the recombinant β-lactoglobulin protein. Non-limiting examples of suitable purification tags include affinity tags (i.e. , peptides or polypeptides that bind to certain agents or matrices), solubilization tags (i.e., peptides or polypeptides that assist in proper folding of proteins and prevent precipitation), chromatography tags (i.e., peptides or polypeptides that alter the chromatographic properties of a protein to afford different resolution across a particular separation techniques), epitope tags (i.e., peptides or polypeptides that are bound by antibodies), fluorescence tags, chromogenic tags, enzyme substrate tags (i.e., peptides or polypeptides that are the substrates for specific enzymatic reactions), chemical substrate tags (i.e., peptides or polypeptides that are the substrates for specific chemical modifications), self-cleaving tags (peptides or polypeptides that possess inducible proteolytic activity; e.g., Sortase tag, Npro tag, FrpC module, CPD), hydrophobic tags (proteins or polypeptides that are highly hydrophobic and direct the protein for inclusion body formation; e.g., KSI tag, TrpE tag), or combinations thereof. Non-limiting examples of suitable affinity tags include maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag, poly (His) tag, hexa(His) tag, S BP-tag, Strep-tag, and calmodulin-tag. Non-limiting examples of suitable solubility tags include thioredoxin (TRX) tag, poly(NANP) tag, MBP tag, SUMO tag, GB1 tag, NUSA CBD tag, and GST tag. Non-limiting examples of chromatography tags include polyanionic amino acid tags (e.g., FLAG-tag) and polyglutamate tag. Non-limiting examples of epitope tags include V5-tag, VSV-tag, E-tag, NE- tag, hemagglutinin (Ha)-tag, Myc-tag, and FLAG-tag. Non-limiting examples of fluorescence tags include green fluorescent protein (GFP) tag, blue fluorescent protein (BFP) tag, cyan fluorescent protein (CFP) tag, yellow fluorescent protein (YFP) tag, orange fluorescent protein (OFP) tag, red fluorescent protein (RFP) tag, and derivatives thereof. Non-limiting examples of enzyme substrate tags include peptides or polypeptides comprising a lysine within a sequence suitable for biotinilation (e.g., AviTag, Biotin Carboxyl Carrier Protein [BCCP]). Non-limiting examples of chemical substrate tags include substrates suitable for reaction with FIAsH-EDT2. The tag peptide or polypeptide may be removed following isolation of the recombinant β-lactoglobulin protein (e.g., via protease cleavage).
[0195] In embodiments in which a recombinant β-lactoglobulin protein according to any of the above is secreted by a recombinant host cell according to any of the above, the recombinant β- lactoglobulin protein may be purified directly from the culture medium. In other embodiments, the recombinant β-lactoglobulin protein may be purified from a cell lysate. [0196] The recombinant β-lactoglobulin protein may be purified to obtain a preparation comprising the recombinant β-lactoglobulin protein at a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%', greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% relative to other components comprised in the fermentation broth; or to at least 2 -fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater abundancy relative to other components comprised in the fermentation broth; or to a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%', greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% by mass of total protein.
[0197] The identity of the recombinant β-lactoglobulin protein may be confirmed and/or quantified by high performance liquid chromatography (HPLC), Western blot analysis. Eastern blot analysis, polyacrylamide gel electrophoresis, capillary electrophoresis, formation of an enzyme product, disappearance of an enzyme substrate, and 2-dimensional mass spectroscopy (2D-MS/MS) sequence identification.
Post-Processing
[0198] Post-processing may alter certain chemical and/or physical properties of the recombinant β-lactoglobulin protein, including but not limited to size, charge, hydrophobicity, hydrophilicity, solvation, protein folding, and chemical reactivity.
[0199] Post-processing may comprise refolding of the recombinant β-lactoglobulin protein (e.g., by removing a denaturant); fragmenting of the recombinant β-lactoglobulin protein (e.g., by chemical means or by exposure to proteases [e.g., trypsin, pepsin]); heating of the recombinant β- lactoglobulin protein (e.g., to remove protein aggregates); removing reactive sites of the recombinant β-lactoglobulin protein (e.g., removing reactive sites of methionine and/or tryptophan residues by oxidation); modulating of the recombinant β-lactoglobulin protein (e.g.. via chemical, photochemical, and/or enzymatic strategies); demineralizing of a preparation comprising the recombinant β-lactoglobulin protein (by, e.g., dialysis, ultrafiltration, reverse osmosis, ion exchange chromatography); removing tags and/or fusion polypeptides from the recombinant β- lactoglobulin protein (e.g., by exposure to site- specific proteases); biotinylating of the recombinant β-lactoglobulin protein (i.e., attaching biotin); and/or conjugating of the recombinant β- lactoglobulin protein to other elements (e.g., poly-ethylene-glycol, antibodies, liposomes, phospholipids, DNA, RNA, polynucleotides, sugars, disaccharides, polysaccharides, starches, cellulose, detergents, cell walls).
[0200] Post-processing may occur in a random manner or in a site-specific manner (e.g., at sulfhydryl groups of cysteine residues [e.g., for aminoethylation, formation of iodoacetamides, formation of maleimides, formation of Dha, covalent attachment via disulfide bonds, and desulfurization], at primary amine groups of lysine residues [e.g., for attachment of activated esters, sulfonyl chlorides, isothiocyanates, unsaturated aldehyde esters, and aldehydes], at phenolic hydroxyl groups of tyrosine residues, at specific allergenic epitopes [e.g., glycan groups]).
[0201] The recombinant β-lactoglobulin protein may be dried (e.g., via spray drying or lyophilization) or concentrated (e.g., via precipitation or evaporation) (e.g., to obtain a powder).
Method for Producing Composition
[0202] In various aspects, provided herein is a method for producing a composition according to any of the above (e.g., a food product according to any of the above), wherein the method comprises the step of obtaining a recombinant β-lactoglobulin protein according to any of the above.
[0203] When the composition is a food product (e.g., the food product according to any of the above), a variety of recipes known in the art may be used to prepare the food product.
[0204] When the composition comprises or consists of a polymer or polymer network according to any of the above, a variety of methods for polymerizing protein monomers known in the art may be used to polymerize the recombinant β-lactoglobulin protein (see, for example, patent publication US20210235714, filed October 30, 2020). The hydrophobic bioactive agent may be bound to the recombinant β-lactoglobulin protein prior or post polymerization. When the hydrophobic bioactive agent is bound prior to polymerization, conditions for polymerization are most suitably selected to not disrupt binding of the hydrophobic bioactive agent to the recombinant β-lactoglobulin protein and/or to not denature a tertiary or quaternary structure of the recombinant β-lactoglobulin protein. Such conditions may include polymerization under non- or mildly denaturing conditions, such as disclosed, for example, in PCT publication WO2021168343, published August 26, 2021.
5b Method for Administering Hydrophobic Bioactive Agent to Subject
[0205] In various aspects, provided herein is a method for administering a hydrophobic bioactive agent to a subject, wherein the method comprises administering to the subject a composition according to any of the above.
[0206] The subject may be a human or other animal (e.g., a mammal [e.g., cow, sheep, goat, rabbit, pig]).
[0207] Administering the composition to the subject may occur via any route, including via oral administration.
EXAMPLES
[0208] The following examples are included to illustrate specific embodiments of this disclosure. The techniques disclosed in the examples represent techniques discovered by the inventors to function well in the methods and processes of this disclosure; however, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Therefore, all matter set forth or shown in the examples is to be interpreted as illustrative and not in a limiting sense.
Example 1 : Production of Recombinant β-lactoglobulin Proteins Having Improved Binding of Bioactive Agent
[0209] For recombinant β-lactoglobulin protein expression in a host cell, the recombinant vector is constructed using genetic engineering methods known in the art. The recombinant vector comprises an expression construct comprising a protein coding sequence encoding β-lactoglobulin protein (“recombinant β-lactoglobulin protein ORF”; e.g., any of SEQ ID NOs: 1-9); codon- optimized for expression in the host cell, and operably linked to an N-terminal secretion signal sequence (e.g., pre or pre-pro signal peptide of proteins CBH1 or XYN1 for Trichoderma host cell; pre or pre -pro signal peptide of protein GLAA for Aspergillus host cell; pre or pre-pro signal peptide of Saccharomyces cerevisiae alpha mating factor for Pichia pastor is host cell): and under control of a suitable promoter sequence (e.g., promoter sequence of cbhl, xynl, or tefl gene for Trichoderma host cells; promoter sequence of glaA or gpdA gene for Aspergillus host cells; promoter sequence of gap or aoxl gene for Pichia host cells) and a terminator (e.g., terminator sequence of pdcl gene for Trichoderma host cells: terminator sequence of glaA or gpdA gene for Aspergillus host cells; terminator sequence of aoxl gene for Pichia host cells). The recombinant vector further comprises a polynucleotide that can direct integration of the expression construct into the genome of the host cell (e.g., into the cbh1 or egl1 locus for Trichoderma host cells; into the glaA locus for Aspergillus host cells; into the aoxl locus for Pichia host cells), selection markers for selection of bacterial and/or fungal transformants, and a bacterial origin of replication. The bacterial selection markers and origin of replication are removed from the recombinant vector via restriction enzyme digestion prior to transformation of the recombinant vector into the host cell.
[0210] Using QuikChange® Site-Directed Mutagenesis Kit (Agilent, Santa Clara, California, USA) and QuikChange Multi Site-Directed Mutagenesis Kit (Agilent, Santa Clara, California, USA), site-directed mutagenesis is performed according to the manufacturer’s direction on the recombinant vectors above to introduce the amino acid substitutions listed in Table 1 into the encoded β-lactoglobulin protein, as well as any combination of two or more of such amino acid substitutions.
Figure imgf000058_0001
Figure imgf000059_0001
[0211] The recombinant vectors are transformed into the host cell (e.g., Trichoderma reesei, Aspergillus niger, Pichia pastoris (Komagataella phaffii; e.g., strain BG12 [(Biogrammatics, Carlsbad, CA]), and transformants are selected by growth on minimal media or antibiotics for positive selection. The transformants are grown in expression media, in 24-well plates, and culture supernatants are harvested for identification of recombinant host cells comprising an integrated copy of the expression construct and secreting a recombinant β -lactoglobulin protein by SDS- PAGE gel analyses.
[0212] The recombinant host cells are fermented in a stirred fermentation vessel under conditions that permit cell growth and production of the recombinant β-lactoglobulin protein. The fermentation is harvested after at least 100 hours, at a biomass concentration of between about 20 g and about 50 g dry cell weight (DCW) per L. The biomass is removed from the broth by centrifugation at 5,000 x g.
[0213] The culture supernatant is concentrated over a membrane with suitable molecular weight cutoff. The concentrate retentate is diafiltered over 5 kda MWCO membranes into 50 mM Imidazole, pH 6.8. The concentrated retentate is passed over a Q sepharose FF column. The mobile phase is 50 mM Imidazole, pH 6.8, and the recombinant β-lactoglobulin protein is eluted on a 2M NaCl gradient. The gradient is run from 0-30% over 30 column volumes. Peak fractions are collected and analyzed on RP-HPLC. Peaks containing recombinant β-lactoglobulin protein with a purity of greater than 85% are pooled for final diaflltration into water. Example 2: Production of Polymer Comprising Recombinant β-lactoglobulin Protein Monomers [0214] A recombinant β-lactoglobulin protein of Example 1 is combined with a weak acid (e.g., 5% acetic acid) or base (e.g., 5% sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide) alone or in combination with an other ingredient (e.g., any of the other ingredients disclosed herein) to a final concentration of between about 2% and about 18% by mass of the recombinant β-lactoglobulin protein, a final pH of between about 4 and about 11, and a final conductivity of between about 10 ms/cm and about 300 mS/cm. The mixture is heated for 20 minutes at a temperature and a pH at which the recombinant β-lactoglobulin protein is mildly denatured (e.g.. at which less than 20% of the recombinant β-lactoglobulin protein is denatured), such that the free thiol group( s ) of the recombinant β-lactoglobulin protein are exposed but existing intra-molecular disulfide bonds are not broken (e.g., at between 10°C and 20°C below the Tm of the recombinant pdactoglobulin protein [as determined in Example 3]). The mixture is then cooled to 21°C or ambient temperature. The polymer is captured by centrifugation (e.g., at 4,000g for 20 min), filtration, solvent extractions, chromatography, or other method. The polymer is dried to a moisture content that still permits shaping. After shaping, the polymer is further dried to sei a final form.
Example 3: Production of Gel Comprising Recombinant B-Lactoglobulin Protein
[0215] A cylindrical (10-mm inner diameter, 40-mm length) stainless steel tube is filled with a solution comprising between about 2% and about 18% by mass of a recombinant β-lactoglobulin protein of Experiment 1 at a final pH of between about 4 and about 11 . The tube is closed with rubber stoppers, sealed with vinyl electrical tape, and placed vertically in a water bath. The solution is heated as described in Example 2 to obtain mildly denatured recombinant β-lactoglobulin protein. The gel is cooled to room temperature, and then left at 4 °C overnight, before being removed from the tube and cut into 10-mm diameter, 2-mm thick tablets, and dried in an enclosed desiccator until the tablets reach a constant mass (i.e., within ±0.001 g).
Example 4: Production of Film/Sheet Comprising Recombinant B-Lactoglobulin Protein
[0216] A solution comprising between about 20% and about 40% by weight of a recombinant β-lactoglobulin protein of Example 1 is heated as described in Example 2 to obtain mildly denatured recombinant β-lactoglobulin protein. The mildly denatured recombinant β-lactoglobulin protein is optionally combined with a synthetic co-polymer (e.g., polyvinyl acetate [PVAC], polyvinyl alcohol [PVA], polyvinyl pyrollidone [PVP]). The solution is then rapidly cooled in an ice water bath, The solution is kept overnight at 4°C to remove air bubbles. Finally, the solution is cast on the center of 27 x 27 cm2 glass plates then dried for 72 hours at ambient conditions (25°C).

Claims

1. A recombinant β-lactoglobulin protein comprising a modification compared to a native β- lactoglobulin protein, wherein the modification improves binding of a hydrophobic bioactive agent to the recombinant β-lactoglobulin protein.
2. The recombinant β-lactoglobulin protein of Claim 1, wherein the native β-lactoglobulin protein is native Bos taurus β-lactoglobulin protein (SEQ ID NO: 1), native Capra hircus β- lactoglobulin protein (SEQ ID NO: 3), or native Ovis aries β-lactoglobulin protein (SEQ ID NO: 5), and wherein the modification comprises: one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R, E62A, E62N. E62D. E62Q, K69L, K69I, K69F. K69W. K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71 W, I71 V, I84L, I84M, I84F, I84V, I84Y, I84W, A86G, A86H, A86Y, N90L N90K. N90R, N90Q. N90S. N90T, N90D, N90E. V92I, V92L, V92M, V92F, V92Y, F105I, F105V, M107I, M107V, L1221, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R, K138H. K138E, K138D. K138L, K138I, K141Q, K141L, K141M, K141R, K141F, K141H, A 142V. A142M, A1421, A142G, A142T, L143I, L143F, L143M, L143R, L143V, M145L, M145I, M145V, M145G, H146R, H146K, H146D, I147L, I147V, I147M, I147K, I147F. R148Q, R148W, R148H, R148K, and R148E.
3. The recombinant β-lactoglobulin protein of Claim 1 , wherein the native β-lactoglobulin protein is native Homo sapiens β-lactoglobulin protein (SEQ ID NO: 2), and wherein the modification comprises: one or more amino acid substitutions selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D. A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I84L, I84M, I84F, I84V, I84Y, I84W, N90I, N90K, N90R, N90Q, N90S, N90T, N90D, N90E, L122I, L122V, L122Y, L122F, L143I, L143F, L143M, L143R, L143V. H146R, H146K, and H146D.
4. The recombinant β-lactoglobulin protein of Claim 1, wherein the native β-lactoglobulin protein is native Equus asinus β-lactoglobulin protein (SEQ ID NO: 4), and wherein the modification comprises or consists of: one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37L A37M, A37V, A37Q. A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41I, V41 M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56 A. L58I, L58W, L58Y, L58M, I71L, I71F, I71 Y. I71W, I71V, F105I, F105V, L123L L123V, L123Y, L123F, L144I. L144F, L144M, L144R, L144V, H147R, H147K, and H147D.
5. The recombinant β-lactoglobulin protein of Claim 1, wherein the native β-lactoglobulin protein is native Equus caballus β-lactoglobulin protein (SEQ ID NO: 6), and wherein the modification comprises: one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q, K69L, K69I, K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, V92I, V92L, V92M. V92F, V92Y, M107I, M107V, L122I. L122V, L122Y, L122F. L143I, L143F, L143M, L143R, and L143V.
6. The recombinant β-lactoglobulin protein of Claim L wherein the native β-lactoglobulin protein is native Equus caballus β-lactoglobulin protein (SEQ ID NO: 7), and wherein the modification comprises: one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, L39I, L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43I, V43L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, I71L, I71F, I71Y, I71W, I71V, F105I, F105V, L123L L123V, L123Y, L123F, L144I, L144F, L144M, L144R, and L144V.
7. The recombinant β-lactoglobulin protein of Claim 1, wherein the native β-lactoglobulin protein is native Eqiius asinus β-lactoglobulin protein (SEQ ID NO: 8), and wherein the modification comprises or consists of: one or more amino acid substitutions selected from the group consisting of: A37L, A37I, A37M, A37V, A37Q, A37D, A37G, A37S. L39I, L39M, L39W, L39F, L39V, V41I, V41M. V41L, L46I, L46F, L46M, L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, E62A, E62N, E62D, E62Q. K69L, K69I, K69F, K69W. K69T, K69N, K69Q, K69R, K69H. I71L, I71F, I71Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, V92I, V92L, V92M, V92F, V92Y, M107I, M107V, L122I, L122V, L122Y, L122F. L143I, L143F, L143M. L143R, and L143V.
8, The recombinant β-lactoglobulin protein of Claim 1, wherein the the nati ve β-lactoglobulin protein is native Ovis aries β-lactoglobulin protein (SEQ ID NO: 9), and wherein the modification comprises: one or more amino acid substitutions selected from the group consisting of: S36T, S36D, S36E, S36N, S36Q, S36F, S36Y, A37L, A37L A37M, A37V, A37Q. A37D, A37G, A37S, L39L L39M, L39W, L39F, L39V, V41I, V41M, V41L, V43L V43L, 1,461, L.46F. L46M. L46V, L46Y, L46T, I56L, I56W, I56Y, I56A, L58I, L58W, L58Y, L58M, K60L, K60Y, K60F, K60H, K60E, K60T, K60R. E62A, E62N, E62D, E62Q, K69L. K69I. K69F, K69W, K69T, K69N, K69Q, K69R, K69H, I71L, I71F, I71 Y, I71W, I71V, I84L, I84M, I84F, I84V, I84Y, I84W, A86G, A86H, A86Y, N90I, N90K, N90R, N90Q, N90S, N90T. N90D, N90E, V92I, V92L, V92M, V92F, V92Y. F105I, F105V, M107I, M107 V, L122I, L122V, L122Y, L122F, D137N, D137Q, D137E, D137S, K138R, K138H, K138E, K138D, K138L, K138I, K141Q. K141L, K141M. K141R, K141F, K141H, A142V, A142M, A142I, A142G, A142T, L143I, L143F, L143M, L143R, L143V, M145L, M145I, M 145 V. M145G, H146R, H146K, H146D, I147L, I 147 V. I147M, I147K, I147F, R148Q, R148W, R148H, R148K, and R148E.
9, A composition comprising a recombinant β-lactoglobulin protein according to any of claims 1 to 8.
10. The composition of claim 9, wherein the composition further comprises a hydrophobic bioactive agent bound to the recombinant β-lactoglobulin protein.
11. A recombinant expression construct consisting of a polynucleotide comprising: a promoter sequence; an optional secretion signal sequence; a protein coding sequence encoding a recombinant β-lactoglobulin protein according to any of claims 1 to 8; and a termination sequence; wherein i) the promoter sequence is operably linked in sense orientation to the optional secretion signal sequence and the protein coding sequence, ii) the optional secretion signal sequence is operably linked in sense orientation to the protein coding sequence, and iii) the one or more terminator sequences are operably linked to the protein coding sequence.
12. A recombinant vector comprising a recombinant expression construct according to claim 11.
13. A recombinant host cell comprising the recombinant expression construct of Claim 11.
14. A method for obtaining the recombinant host cell of Claim 13, wherein the method comprises: obtaining i) a polynucleotide encoding a recombinant β-lactoglobulin protein according to any of claims 1 to 8, ii) a recombinant expression construct according to claim 11, or iii) a recombinant vector according to claim 12; and introducing the polynucleotide, the recombinant expression construct, or the recombinant vector into a host cell.
15. A method for producing a recombinant β-lactoglobulin protein according to any of claims 1 to 8, wherein the method comprises: fermenting the recombinant host cell according to claim 13 in a culture medium under conditions suitable for production of the recombinant β-lactoglobulin protein.
16. A method for producing a composition according to claim 9 or 10, wherein the method comprises obtaining a recombinant β-lactoglobulin protein according to any of claims 1 to 8.
17. The method of Claim 16, wherein the method further comprises binding a bioactive agent according to claim 10 to the recombinant β-iactoglobulin protein.
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