WO2019099427A1 - Compositions et procédés de stabilisation de protéines - Google Patents

Compositions et procédés de stabilisation de protéines Download PDF

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WO2019099427A1
WO2019099427A1 PCT/US2018/060879 US2018060879W WO2019099427A1 WO 2019099427 A1 WO2019099427 A1 WO 2019099427A1 US 2018060879 W US2018060879 W US 2018060879W WO 2019099427 A1 WO2019099427 A1 WO 2019099427A1
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polypeptide
identity
amino acid
seq
nos
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PCT/US2018/060879
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Samantha PISZKIEWICZ
Gary Joseph PIELAK
Thomas Christopher Clark BOOTHBY
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The University Of North Carolina At Chapel Hill
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)

Definitions

  • the invention relates to methods and compositions for stabilizing proteins using polypeptides such as ubiquitin, negative regulator of flagellin synthesis (Flg M), SRC Homology 3 (SH3) Domain, small ubiquitin-like modifier (SUMO), and/or serum albumin, and/or using sugar polymers.
  • polypeptides such as ubiquitin, negative regulator of flagellin synthesis (Flg M), SRC Homology 3 (SH3) Domain, small ubiquitin-like modifier (SUMO), and/or serum albumin, and/or using sugar polymers.
  • Recombumin® human serum albumin heterologously expressed in and purified from yeast
  • Recombumin® is also used as a stabilizer in drug formulations.
  • formulations containing Recombumin® still require refrigeration (AlbumedFix.
  • Some protein-based pharmaceuticals can be stored at room temperature if they are lyophilized (freeze dried); however, most protein-based pharmaceuticals denature as a result of either the freezing or drying process.
  • crowding agents can protect protein-based pharmaceuticals during lyophilization, but none of these crowding agents work universally.
  • the most effective additives for a given pharmaceutical is highly dependent on factors including the pi, b-sheet content, and melting temperature of the drug (Roughton et al. Comput. Chem. Eng. 58, 369-377 (2013)). Even with the addition of stabilizers, many protein-based pharmaceuticals are too unstable to survive lyophilization (Roughton et al. Comput. Chem. Eng. 58, 369-377 (2013)).
  • the present invention overcomes previous shortcomings in the art by providing new compositions and methods for stabilizing proteins and other biomedical material.
  • One aspect of the invention provides a liquid composition
  • a liquid composition comprising: at least one heterologous polypeptide and/or peptide of interest and (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin- like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin- like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer.
  • a second aspect provides a solid composition
  • a solid composition comprising: at least one heterologous polypeptide and/or peptide of interest and (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof.
  • a third aspect of the invention provides a method of stabilizing at least one heterologous polypeptide and/or peptide of interest, comprising, contacting the at least one heterologous polypeptide and/or peptide of interest with (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer to produce a liquid composition comprising the at least one heterologous polypeptide and/or peptide of interest and the at least one protective polypeptide and/or at least one sugar polymer, thereby stabilizing the at least one heterologous polypeptide and/or peptide of interest.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide,
  • the invention provides a method of stabilizing a heterologous cell, tissue or organ, comprising, contacting the heterologous cell, tissue or organ with a solution comprising: (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer, thereby stabilizing the heterologous cell, tissue or organ.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof.
  • the present invention provides a method of increasing drought or desiccation tolerance in an organism, comprising introducing into the organism at least one heterologous nucleotide sequence encoding a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; to produce a transgenic organism expressing the heterologous nucleotide sequence, thereby increasing the drought or desiccation tolerance of the transgenic organism.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptid
  • the present invention provides a method of producing a transgenic organism having increased tolerance to drought or desiccation, comprising introducing into the organism at least one heterologous nucleotide sequence encoding a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin- like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof, thereby producing a transgenic organism having increased drought or desiccation tolerance.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin- like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof, thereby producing a transgenic
  • the present invention provides a method of increasing drought or desiccation tolerance in a seed, comprising coating the seed with (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer, thereby increasing drought or desiccation tolerance of the seed.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof.
  • the invention provides a transgenic plant, fungus or bacterium having increased tolerance to drought or desiccation comprising in its genome at least one heterologous nucleotide sequence encoding (a) a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, and a serum albumin polypeptide, and any
  • the invention provides method of reactivating one or more desiccated and inactivated proteins, comprising resuspending the desiccated and inactivated protein in a solution comprising:(a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, a serum albumin polypeptide, and a tardigrade disordered polypeptide (TDP), and any
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, a serum albumin polypeptide, and a tardigrade disordered polypeptide (TDP),
  • transgenic organisms and/or transgenic cells comprising the heterologous nucleotide sequences or recombinant nucleic acid constructs of the invention.
  • Fig. I shows protection against desiccation-induced lactate dehydrogenase (LDH) inactivation at room temperature.
  • Fig. 2 shows protection of LDH activity against desiccation-plus-heat (95°C).
  • Fig. 3 shows protection against desiccation-induced LDH inactivation at room temperature.
  • Fig. 4 shows protection against desiccation-induced LDH inactivation at room temperature.
  • Fig. 5 shows protection against desiccation-induced LDH inactivation at room temperature.
  • Fig. 6 shows resurrection of desiccation-inactivated LDH DETAILED DESCRIPTION
  • the term“about,” as used herein when referring to a measurable value such as a dosage or time period and the like refers to variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
  • transitional phrase“consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term“consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to“comprising.”
  • nucleic acid molecule and/or a nucleotide sequence indicates that the nucleic acid molecule and/or a nucleotide sequence is transcribed and, optionally, translated.
  • a nucleic acid molecule and/or a nucleotide sequence may express a polypeptide of interest or a functional untranslated RNA.
  • contact refers to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., stabilizing the polypeptide, peptide, cell, tissue or organ).
  • the term "contact” may comprise any method in which a polypeptide, peptide, cell, organ and/or tissue is exposed to, provided with, or in which a TDP is applied.
  • the polypeptide and/or peptide of interest may be heterologous to the organism, to the genus or to the species from which the protective polypeptide (e.g., ubiquitin, Flg M, SH3, SUMO and serum albumin) and/or the sugar polymer is obtained.
  • A“heterologous cell, tissue or organ” as used herein, refers to a cell, tissue or organ that is heterologous to the organism, to the genus or to the species that naturally produces the polypeptide (e.g., ubiquitin, Flg M, SH3, SUMO and serum albumin) and/or sugar polymer used to protect the heterologous cell, tissue or organ.
  • polypeptide e.g., ubiquitin, Flg M, SH3, SUMO and serum albumin
  • “stabilizing” a heterologous polypeptide and/or peptide means maintaining the structure (1°, 2°, 3° and/or 4° structure) and the function of the polypeptide and/or peptide under either aqueous conditions or dried conditions, or after being frozen and/or dried and then thawed and/or rehydrated as compared to the heterologous polypeptide and/or peptide that has not been frozen and/or dried and then thawed and/or rehydrated.
  • the at least one heterologous polypeptide and/or peptide of interest may be stable at a temperature from about -80°C to about l00°C once the at least one heterologous polypeptide and/or peptide of interest (and/or cell, tissue, and/or organ) is contacted with the at least one protective polypeptide and/or sugar polymer.
  • at least about 10% to about 100% e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
  • the structure and function of the stabilized polypeptide and/or peptide (and/or cell, tissue and/or organ) is maintained.
  • the polypeptides and/or peptides when dried (e.g., solid compositions), may be stabilized over a range of temperature from about -80°C to. about l00°C.
  • the polypeptides and/or peptides (and/or the polypeptides and/or peptides in cells, tissues, and/or organs) in solution (liquid composition) may be stabilized over a range of temperatures from about -80°C to about 40°C.
  • “stabilizing” a cell, organ or tissue means maintaining the structure and function of a cell, organ or tissue under either aqueous conditions or dried conditions, or after being frozen and/or dried and then thawed and/or rehydrated.
  • “reactivating” or“reactivation” of a desiccated and inactivated protein means activating the desiccated and inactivated protein such that it has at least about 80% (e.g., about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, and any range or value therein) of its original level of activity (e.g., as compared to the same protein that has not been desiccated and inactivated).
  • 80% e.g., about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, and any range or value therein
  • a“cell, organ and/or tissue” refers to any cell, organ or tissue from an organism useful with this invention (e.g., a fungus, a bacterium, an archaeon, a protist, a plant, an animal).
  • an organ and/or tissue may include, but is not limited to, lung, liver, bladder, kidney, heart, brain, stomach, intestines (large and small), eye or any part thereof (e.g., lens, cornea), ear or any part thereof (e.g., earlobe, cochlea), gallbladder, esophagus, salivary gland, tongue, teeth, pancreas, ureter, urethra, ovary, uterus, vagina, fallopian tube, testes, vas deferens, penis, pituitary gland, thyroid gland, adrenal gland, lymph node, spleen, thymus, bone marrow, skin (including subcutaneous skin), connective tissue, muscle tissue, nervous tissue, epithelial tissue, mineralized tissue, meristematic tissue, petal, sepal, stamen, pistil, anther, pollen, flower, fruit, flower bud, ovule, seed, embryo, petiole, stem, root,
  • the terms“increase,”“increasing,”“increased,”“enhance,”“enhanced,” “enhancing,” and“enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
  • the activity of a desiccated and inactivated protein may be increased by contacting the desiccated/inactivated polypeptide with at least one protective polypeptide and/or sugar polymer of the invention (e.g., increase by at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control (e.g., the desiccated/inactivated polypeptide)).
  • at least one protective polypeptide and/or sugar polymer of the invention e.g., increase by at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control (e.g., the desiccated/inactivated polypeptide)).
  • an "increased tolerance to drought or desiccation” as used herein refers to the ability of an organism or part thereof that has been either contacted with at least one protective polypeptide (e.g., ubiquitin, Fig M, SH3, SUMO and serum albumin) and/or sugar polymer, or transformed with at least one heterologous nucleotide sequence encoding a protective polypeptide to withstand exposure to drought, or desiccation (e.g., water loss) better than a control organism or part thereof (e.g., an organism or part thereof that has been exposed to drought or desiccation but was not contacted with the at least one protective polypeptide and/or sugar polymer or transformed with at least one heterologous nucleotide sequence encoding a protective polypeptide as described herein).
  • at least one protective polypeptide e.g., ubiquitin, Fig M, SH3, SUMO and serum albumin
  • sugar polymer e.g., a heterologous nucleotide
  • Increased tolerance to drought or desiccation can be measured using a variety of parameters including, but not limited to, survival, metabolic capacity, reproductive capacity, ability to germinate, developmental potential, structural integrity, functional integrity, viability, morphological integrity, decreased necrosis/apoptosis, time required to recover to pre-desiccation/drought levels of metabolism, cell division, reproduction, germination, development, and/or function as compared to an organism or part thereof exposed to the same stress but not having been contacted with the protective polypeptide and/or sugar polymer.
  • A“part of an organism” refers to a multicellular organism and includes but is not limited to a cell, an organ, and other tissues from the organism.
  • A“part of an organism” may also include, but is not limited to, nucleic acids, proteins, lipids, carbohydrates, and the like, that are present in an organism.
  • an isolated cell refers to a cell that is separated from other components with which it is normally associated in its natural state.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier.
  • the terms“reduce,”“reduced,”“reducing,”“reduction,”“diminish,” and“decrease” describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control, ln particular embodiments, the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • the activity a polypeptide or peptide of interest may be reduced by about 10% to 100% when desiccated as compared to a non-desiccated control.
  • the activity of a polypeptide may be reduced by desiccation to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
  • A“native” or“wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence.
  • a“wild type mRNA” is an mRNA that is naturally occurring in or endogenous to the organism.
  • A“homologous” nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.
  • nucleic acid As used herein,“nucleic acid,”“nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single- stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or
  • the term "gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotide (AMO) and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions).
  • a gene may be "isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A.”
  • Complementarity between two single-stranded molecules may be "partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • isolated can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an“isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. “Isolated” does not mean that the preparation is technically pure
  • the recombinant nucleic acid molecules, nucleotide sequences and polypeptides of the invention are“isolated.”
  • An“isolated” nucleic acid molecule, an “isolated” nucleotide sequence or an“isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the isolated nucleic acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell.
  • the term“isolated” means that it is separated from the chromosome and/or cell in which it naturally occurs.
  • a polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences, and/or different position in the genome than as found in nature).
  • the recombinant nucleic acid molecules, nucleotide sequences and their encoded polypeptides are“isolated” in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.
  • nucleotide sequences and/or recombinant nucleic acid molecules of the invention can be operatively associated with a variety of promoters for expression in soybean plant cells.
  • a recombinant nucleic acid of this invention can further comprise one or more promoters operably linked to one or more nucleotide sequences.
  • operably linked or“operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • the term“operably linked” or“operably associated” as used herein refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence.
  • control sequences e.g., promoter
  • the control sequences need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered“operably linked” to the nucleotide sequence.
  • A“promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA.
  • a“promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription.
  • promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence.
  • the promoter region may comprise other elements that act as regulators of gene expression.
  • Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue- preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e.,“chimeric genes” or“chimeric polynucleotides.”
  • a “promoter” useful with the invention is a promoter capable of initiating transcription of a nucleotide sequence in a cell of interest.
  • a nucleic acid encoding a protective polypeptide may be operably linked to a promoter.
  • a nucleic acid encoding a protective polypeptide may be operably linked to a heterologous promoter.
  • the terms“coding region” and“coding sequence” are used interchangeably and refer to a polynucleotide region that encodes a polypeptide or functional RNA and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide or functional RNA.
  • a coding region can encode one or more polypeptides or functional RNAs.
  • a coding region can encode a polypeptide or functional RNA that is subsequently processed into two or more polypeptides or functional RNAs.
  • a regulatory sequence or regulatory region is a nucleotide sequence that regulates expression of a coding region to which it is operably linked.
  • Nonlimiting examples of regulatory sequences include promoters, transcription initiation sites, translation start sites, internal ribosome entry sites, translation stop sites, and terminators.“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence is“operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.
  • fragment as applied to a polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.
  • fragment as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence.
  • a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.
  • a“functional” polypeptide or“functional fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g., target protein binding).
  • the“functional” polypeptide or “functional fragment” substantially retains all of the activities possessed by the unmodified peptide.
  • substantially retains biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide).
  • A“non-functional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as protein binding can be measured using assays that are well known in the art and as described herein.
  • Polypeptides and fragments thereof of the invention may be modified for use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent.
  • a blocking agent can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered.
  • one or more non-naturally occurring amino acids such as D-alanine
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the peptide terminus can be modified, e.g., by acetylation of the N- terminus and/or amidation of the C-terminus.
  • the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable“carrier” proteins prior to use.
  • nucleic acids of the present invention may encode any suitable epitope tag, including, but not limited to, poly-Arg tags (e.g., RRRRR (SEQ ID NO:l) and RRRRRR (SEQ ID NO:2) and poly-His tags (e.g., HHHHHH (SEQ ID NO:3)).
  • poly-Arg tags e.g., RRRRR (SEQ ID NO:l) and RRRRRR (SEQ ID NO:2
  • poly-His tags e.g., HHHHHH (SEQ ID NO:3)
  • the nucleic acid may comprise a nucleotide sequence encoding a poly-Arg tag, a poly-His tag, a FLAG tag (i.e., DYKDDDDK (SEQ ID NO:4)), a Strep-tag IITM (GE Healthcare, Pittsburgh, PA, USA) (i.e., WSHPQFEK (SEQ ID NO:5)), and/or a c-myc tag (i.e., EQKLISEEDL (SEQ ID NO:6)).
  • proteins of the present invention may comprise any suitable epitope tag, including, but not limited to, poly-Arg tags (e.g., RRRRR (SEQ ID NO:l) and RRRRRR (SEQ ID NO:2) and poly-His tags (e.g., HHHHHH (SEQ ID NO:3)).
  • poly-Arg tags e.g., RRRRR (SEQ ID NO:l) and RRRRRR (SEQ ID NO:2
  • poly-His tags e.g., HHHHHH (SEQ ID NO:3
  • the polypeptide may comprise a poly-Arg tag, a poly-His tag, a FLAG tag (i.e., DYKDDDDK (SEQ ID NO:4)), a Strep-tag IITM (GE Healthcare, Pittsburgh, PA, USA) (i.e., WSHPQFEK (SEQ ID NO:5)), and/or a c-myc tag (i.e., EQKLISEEDL (SEQ ID NO:6)).
  • a poly-Arg tag i.e., DYKDDDDK (SEQ ID NO:4)
  • a Strep-tag IITM GE Healthcare, Pittsburgh, PA, USA
  • WSHPQFEK SEQ ID NO:5
  • a c-myc tag i.e., EQKLISEEDL (SEQ ID NO:6)
  • homologues include homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • Homology refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention.
  • Orthologous refers to homologous nucleotide sequences and/ or amino acid sequences in different species that arose from a common ancestral gene during speciation.
  • a homologue of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to said nucleotide sequence of the invention.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
  • “percent sequence identity” or“percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.
  • the phrase“substantially identical,” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, refers to two or more sequences or subsequences that have at least about 80%, least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • 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.
  • An“identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i. e. , the entire reference sequence or a smaller defined part of the reference sequence.
  • “percent sequence identity” or“percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison).
  • “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • An“identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • “percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • the percent of sequence identity can be determined using the“Best Fit” or“Gap” program of the Sequence Analysis Software PackageTM (Version 10; Genetics Computer Group, Inc., Madison, Wis.). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. “BestFit” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Smith et al. , Nucleic Acids Res. 77:2205 (1983)).
  • BLAST Basic Local Alignment Search Tool
  • BLAST programs allow the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.
  • Two nucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions.
  • two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.
  • Stringent hybridization conditions and“stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100
  • complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes.
  • a low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific
  • nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.
  • SDS sodium dodecyl sulfate
  • a recombinant nucleic acid molecule of the invention can be an “expression cassette” or can be comprised within an expression cassette.
  • expression cassette means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleotide sequences of the invention; e.g., a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to of any of SEQ ID NO:7- 209, a nucleotide sequence having at least about 70% identity to of any of SEQ ID NOs:210- 314, or a nucleotide sequence having at least about 80% identity to any of SEQ ID NOs:315- 419, and/or fragments thereof), wherein said nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter, a heterologous promoter).
  • a control sequence e.g., a promoter, a heterologous promoter
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in the cell in which the nucleotide sequence of interest is to be expressed.
  • a transcriptional and/or translational termination region i.e., termination region
  • a variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation.
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host organism, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the host organism, or any combination thereof).
  • a coding sequence's native transcription terminator can be used.
  • An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed organism and/or cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the transformed organism or cell expressing the marker and thus allows such transformed organisms or cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening.
  • a selective agent e.g., an antibiotic, herbicide, or the like
  • vector refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell.
  • a vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced.
  • Vectors for use in transformation of animals, plants and other organisms are well known in the art.
  • Non-limiting examples of general classes of vectors including but not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • a vector as defined herein can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., an autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from prokaryotic and eukaryotic organisms.
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or an animal or a plant cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • heterologous polypeptides and/or peptides may be stabilized in the presence of protective polypeptides, in both aqueous (liquid) and (solid) compositions.
  • a liquid composition comprising, consisting essentially of, or consisting of: at least one heterologous polypeptide and/or peptide of interest and (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof.
  • a solid composition comprising, consisting essentially of, or consisting of: at least one heterologous polypeptide and/or peptide of interest and (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer.
  • a solid composition may be produced by drying or partially drying a liquid composition of the invention.
  • a solid composition of the invention may comprise about 0% to about 5% by weight water (e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5% by weight water, or any range or value therein).
  • water e.g., about 0.001, 0.002, 0.003, 0.00
  • a solution comprising, consisting essentially of, or consisting of: (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer; and/or (c) any combination of (a) and (b).
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof.
  • a method of stabilizing at least one heterologous polypeptide and/or peptide of interest comprising: contacting the at least one heterologous polypeptide and/or peptide of interest with (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer to produce a liquid composition comprising the at least one heterologous polypeptide and/or peptide of interest and the at least one protective polypeptide and/or at least one sugar polymer, thereby stabilizing the at least one heterologous polypeptide and/or peptide of interest as compared to a control (e.g., the heterologous polypeptide and or peptide of interest that has not been
  • the invention provides a method of stabilizing a heterologous cell, tissue or organ, comprising: contacting the heterologous cell, tissue or organ with a solution or liquid composition of the invention comprising: (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide, and any combination thereof; and/or (b) at least one sugar polymer, thereby stabilizing the heterologous cell, tissue or organ as compared to a control (e.g., a heterologous cell, tissue or organ that has not been contacted with the liquid composition).
  • a control e.g., a heterologous cell, tissue or organ that has not been contacted with the liquid composition
  • Exemplary heterologous cells, tissues or organs may include, but are not limited to, those from a fungus, a bacterium, an archaeon, a plant, a protist, or an animal.
  • an animal may include, but is not limited to, a mammal, a bird, a fish, an insect, a reptile, a nematode, or an amphibian.
  • a mammal includes, but is not limited to, a human, a non-human primate, a dog, a cat, a goat, a horse, a pig, a cow, a sheep, a rat, a guinea pig, a mouse, a gerbil, or a hamster.
  • TDPs tardigrade disordered polypeptides
  • the invention provides a method of reactivating one or more desiccated and inactivated proteins, comprising resuspending the one or more desiccated and inactivated proteins in a solution comprising: (a) at least one protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, a serum albumin polypeptide, and a TDP, and/or any combination thereof.
  • a protective polypeptide selected from the group consisting of a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, a serum albumin polypeptide, and a TDP, and/or any combination thereof.
  • a method of increasing drought or desiccation tolerance in a seed comprising: coating the seed with a solution of the present invention (e.g., a solution comprising at least one ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, and a serum albumin polypeptide; and/or (b) at least one sugar polymer) to produce a coated seed, thereby increasing drought or desiccation tolerance of the seed.
  • the coating may be dried or partially dried on the seed.
  • Drying of a liquid composition or solution of the invention may commence any time following the contacting or coating with the liquid composition or solution. Any method of drying a liquid composition or solution may be used including, but not limited to, freeze-drying, air-drying, spray-drying, spray-freeze-drying, vacuum drying, and/or foam drying.
  • “partially drying” refers to drying a composition or solution such that it comprises less water than when the drying process began.
  • “partially drying” can mean removing about 0.001 % to about 100% of the water that was present in the composition or solution prior to initiating the drying process. (e.g., about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,
  • the amount of water removed when a composition or solution is partially dried can be from about 0.001 to about 0.01%, about 0.001 to about 0.1%, about 0.001 to about 1%, about 0.01 to about 1%, about 0.1 to about 1%, about 0.001 to about 10%, about 0.01 to about 10%, about 0.1 to about 10%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 10%
  • a solid composition of the invention may comprise a hydration level of about 0 to about 10 g water per gram of dried protein (e.g., up to about 10 g water per gram of dried protein; e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, and any range or value therein).
  • a solid composition of the invention may comprise a hydration level of about Og to about 0.005g, about Og to about O.Olg, about 0.005g to about O.Olg, about 0.005g to about O. lg, about O.Olg to about O.lgwater per gram of dried protein, and any value or range therein.
  • the amount of a protective polypeptide (e.g., ubiquitin, FlgM, SH3 domain, SUMO serum albumin) and/or a sugar polymer in a liquid composition, solid composition, and/or solution of the invention can vary depending on the heterologous polypeptide and/or peptide of interest, whether it is a liquid or a solid, and/or whether the composition is a liquid composition or solution that will be dried.
  • a protective polypeptide e.g., ubiquitin, FlgM, SH3 domain, SUMO serum albumin
  • the concentration of a protective polypeptide and/or a sugar polymer in a liquid composition and/or solution of the invention may be about 1 g/L to about 300 g/L or any range or value therein (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
  • the protective polypeptide and/or sugar polymer concentration in a liquid composition or solution of the invention may be about 1 g/L to about 250 g/L, about 1 g/L to about 200 g/L, about 1 g/L to about 100 g/L, about 1 g/L to about 50 g/L, about 1 g/L to about 20 g/L, about 1 g/L to about 10 g/L, about 5 g/L to about 300 g/L, about 5 g/L to about 250 g/L, about 5 g/L to about 200 g/L, about 5 g/L to about 100 g/L, about 5 g/L to about 50 g/L, about 5 g/L to about 20 g/L, about 5 g/L to about 10 g/L, 10 g//L
  • a liquid composition, solid composition, and/or solution may comprise about 50% to about 99.9% of protective polypeptide and/or sugar polymer (total weight) (e.g., about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
  • protective polypeptide and/or sugar polymer total weight
  • a liquid composition, solid composition, and/or solution may comprise about 90% to 99.99% of protective polypeptide and/or sugar polymer (total weight) (e.g., about 90, 91, 92, 93, 94, 95, 96,
  • the mass ratio of the at least one heterologous polypeptide and/or peptide of interest, or the one or more desiccated and inactivated proteins to the at least one protective polypeptide and/or sugar polymer in a liquid or a solid composition may be about 1 :1000 to about 1:1 (e.g., about 1:1000, 1:950, 1:900, 1:850, 1:800, 1 :750, 1:700, 1:650, 1:600, 1 :550, 1 :500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:150, 1 :125, 1 :100, 1:95, 1:90, 1 :85, 1 :80, 1:75, 1 :70, 1:65, 1:60, 1:55, 1 :50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:15, 1 :10 1:9, 1:8,
  • the at least one heterologous polypeptide and/or peptide of interest to the at least one protective polypeptide and/or sugar polymer in a liquid or a solid composition may be about 1 : 100 to about 1 :10, about 1 :20 to about 1 : 10, or about 1 :20 to about 1:1.
  • liquid compositions, solid compositions, and/or solutions of this invention may comprise any number or combination of protective polypeptides and/or sugar polymers.
  • the liquid compositions, solid compositions, and/or solutions can comprise, consist essentially of, or consist of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more different protective polypeptides and/or sugar polymers (e.g., from about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 2 to about 10, about 2 to about 5, about 4 to about 10, about 6 to about 10 different protective polypeptides and/or sugar polymers and the like).
  • ubiquitin polypeptide may be used as protective polypeptide as described herein.
  • exemplary ubiquitin polypeptides include, but are not limited to, those from a mammal, a fish, an insect, a bird, a plant, a bacterium, a protist, a fungus, a plant, a nematode, or an archaeon.
  • Exemplary mammals include, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), primates such as a human or a non-human primate (e.g., a chimpanzee, baboon, monkey, orangutan, gorilla, etc.).
  • a laboratory animal e.g., a rat, mouse, guinea pig, rabbit, etc.
  • a farm or commercial animal e.g., a bovine, pig, horse, goat, donkey, sheep, etc.
  • a domestic animal e.g., cat, dog, ferret, etc.
  • primates e.g., a chimpanzee, baboon, monkey, orangutan, gorilla, etc
  • the protective polypeptide may be a ubiquitin polypeptide having at least 70% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9, 100%) to an amino acid sequence of any of SEQ ID NOs:7-30; and/or may be a ubiquitin polypeptide encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:7-30, or any combination thereof.
  • 70% identity e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
  • SRC Homology 3 (SH3) Domain polypeptide Any SRC Homology 3 (SH3) Domain polypeptide may be used as protective
  • SH3 domain polypeptides include, but are not limited to, those obtained from a eukaryote polypeptide or a prokaryote polypeptide comprising an SH3 domain.
  • a SH3 domain polypeptide may be obtained from, for example, a polypeptide from a primate such as a human or a non-human primate (e.g., a chimpanzee, baboon, monkey, orangutan, gorilla, etc.), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a reptile (e.g., a lizard, turtle, snake, etc.), a bacterium (e.g., Escherich
  • the protective polypeptide may be a SH3 domain polypeptide having at least 70% identity to an amino acid sequence of any of SEQ ID NOs:31-53; and/or may be a SH3 domain polypeptide encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:31-53, or any combination thereof.
  • Fig M flagellin synthesis polypeptide
  • Fig M polypeptides include, but are not limited to, those obtained from a monotrichous bacterium or a lophotrichous bacterium.
  • a Flg M polypeptide may be obtained from Escherichia, Pseudomonas, Bacillus, Vibrio, Helicobacter, Klebsiella, Burkholderia, Salmonella, Campylobacter, Enterobacter, Kluyvera, Proteobacteria, Shigella, Citrobacter, Diaporthe, Beauveria and/or Caulobacter.
  • the protective polypeptide may be a Flg M polypeptide having at least 70% identity to an amino acid sequence of any of SEQ ID NOs:54-64; and/or may be a Flg M polypeptide encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:54-64, or any combination thereof.
  • SUMO polypeptide Any small ubiquitin-like modifier (SUMO) polypeptide may be used as a protective polypeptide as described herein.
  • exemplary SUMO polypeptides include those from a eukaryote or a prokaryote.
  • a SUMO polypeptide may include, but is not limited to, those from a human or a non-human primate (e.g., monkey, gorilla, orangutan, chimpanzee), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a reptile (e.g., a lizard, turtle, snake, etc.), a bacterium (e.g., Escherichia), a plant, e.
  • the protective polypeptide may be a SUMO polypeptide having at least 70% identity to an amino acid sequence of any of SEQ ID NOs:65- 86; and/or may be a SUMO polypeptide encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:65-86, or any combination thereof.
  • a serum albumin polypeptide useful as a protective polypeptide may be any serum albumin polypeptide.
  • Exemplary serum albumin polypeptides include those from any animal including but not limited to a human, a non-human primate (e.g., monkey, gorilla, orangutan, chimpanzee), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a fish (e.g., salmon, danio), an amphibian (e.g., frog), reptile (e.g., turtle, lizard, snake) and/or an avian (e.g., chicken, parakeet, parrot).
  • a human e.g., monkey, gorilla, orangutan, chim
  • the protective polypeptide may be a serum albumin polypeptide having at least 70% identity to an amino acid sequence of any of SEQ ID NOs:87-104; and/or may be a serum albumin polypeptide encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:87-104, or any combination thereof.
  • a sugar polymer may be used to protect a heterologous polypeptide of interest.
  • exemplary sugar polymers include, but are not limited to, neutral, highly branched, hydrophilic polysaccharides (e.g., FICOLL ® ), branched glucose polymers with reducing linkages (e.g., dextran), branched glucose polymers with non-reducing linkages (glycogen), branched or linear glucose polymers with non-reducing linkages (alpha glycosidic linkages, e.g., starch (e.g., amylose, amylopectin)), and/or glucose polymers with beta glycosidic linkages (e.g., cellulose).
  • neutral, highly branched, hydrophilic polysaccharides e.g., FICOLL ®
  • branched glucose polymers with reducing linkages e.g., dextran
  • branched glucose polymers with non-reducing linkages glycogen
  • TDPs tardigrade polypeptides
  • Any number or combination of tardigrade polypeptides (TDPs) obtained from any tardigrade genus or species may be used as a protective polypeptide alone or in combination with any other protective polypeptides as described herein for reactivating a desiccated and inactivated protein.
  • Tardigrades are a phylum of microscopic animals renowned for their ability to survive a vast array of environmental extremes, including essentially complete desiccation for up to a decade (Goldstein and Blaxter, Curr. Biol. 12, R475 (2002)).
  • Exemplary tardigrade genera from which a TDP may be obtained include Macrobiotus spp., Isohypsibius spp., Diphascon spp., Echiniscus spp., Minibiotus spp., Doryphoribius spp., Paramacrobiotus spp., Hypsibius spp., Milnesium spp., Pseudechiniscus spp., Ramazzottius spp., Batillipes spp., Bryodelphax spp., Dactylobiotus spp., Echiniscoides spp., Calcarobiotus spp., Tenuibiotus spp., Itaquascon spp., Cornechiniscus spp., and/or Halechiniscus spp.
  • the at least one TDP may be obtained from the tardigrade genera of Hypsibius spp., Paramacrobiotus spp., Milnesium spp. and/or Ramazzottius spp.
  • the TDP may be obtained from one or more of the exemplary tardigrade species provided in Table 1.
  • a TDP may be obtained from Hypsibius dujardini, Paramacrobiotus richters, Milnesium tardigradum and/or Ramazzottius varieornatus.
  • a solution may comprise, consist essentially of, or consist of a TDP comprising an amino acid sequence having at least about 70% identity to any of SEQ ID N0s:104-208; an amino acid sequence encoded by a nucleotide sequence having at least about 80% identity to any one of SEQ ID NOs:210-314; or an amino acid sequence encoded by a nucleotide sequence having at least about 80% identity to any one of SEQ ID NOs:315-419; or any combination thereof.
  • a solution may comprise, consist essentially, or consist of a TDP comprising an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:121, 123, 136, 139, and/or 148; an amino acid sequence encoded by a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs:226, 228, 241, 244, and/or 247; or an amino acid sequence encoded by a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs:331, 333, 346, 349 and/or 352; or any combination thereof.
  • a liquid composition or a solid composition of the invention may further comprise one or more excipients.
  • an excipient includes, but is not limited to, trehalose, sucrose, maltose, mannitol, sorbitol, polysorbate, a buffer, a salt, an antioxidant, a preservative, a colorant, and/or a flavorant.
  • the at least one heterologous polypeptide and/or peptide of interest may be a therapeutic agent or it may be part of a protein-based food.
  • the at least one heterologous polypeptide and/or peptide of interest may be in purified form or it may be in a mixture (unpurified or partially purified).
  • the at least one heterologous polypeptide and/or peptide of interest may be obtained from, for example, an organism (bacteria, fungi, animals, plants, archaea, protists, etc.), the cells of an organism (either isolated or cultured), from serum and/or from in vitro expression systems.
  • heterologous polypeptides and/or peptides so produced may then be protected (stabilized) by contacting them with at least one protective polypeptide (e.g., ubiquitin polypeptide, Flg M polypeptide, SH3 Domain polypeptide, SUMO polypeptide, and/or a serum albumin polypeptide) immediately without any further isolation or purification or they may be contacted with the at least one protective polypeptide (e.g., ubiquitin polypeptide, Flg M polypeptide, SH3 Domain polypeptide, SUMO polypeptide, and/or a serum albumin polypeptide) after they are purified or partially purified.
  • at least one protective polypeptide e.g., ubiquitin polypeptide, Flg M polypeptide, SH3 Domain polypeptide, SUMO polypeptide, and/or a serum albumin polypeptide
  • a mixture may include, for example, serum, cell culture, and/or one or more constituents of an organism or cell thereof, and/or of an in vitro expression system, and the like.
  • a protein based-food may have multiple additional components (e.g., a mixture), which additional components may or may not be proteinaceous.
  • a therapeutic protein may be any protein based molecule (e.g., a biologic) including, but not limited to, a vaccine, an antibody, an enzyme, hormone, and/or a globular protein.
  • a protein based molecule e.g., a biologic
  • a vaccine e.g., an antibody, an enzyme, hormone, and/or a globular protein.
  • antibody or “antibodies” as used herein refers to all types of
  • the antibody can be monoclonal or polyclonal and can be of any species of origin, including (for example) mouse, rat, rabbit, horse, goat, sheep, camel, or human, or can be a chimeric antibody. See, e.g. , Walker et al. , Molec. Immunol. 26:403 (1989).
  • the antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Patent No. 4,474,893 or U.S. Patent No. 4,816,567.
  • the antibodies can also be chemically constructed according to the method disclosed in U.S. Patent No. 4,676,980.
  • antibody also refers to antibody fragments, for example, Fab, Fab', F(ab') 2 , and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Also included within the scope of the present invention are antibodies, which are altered or mutated for compatibility with species other than the species in which the antibody was produced. For example, antibodies may be humanized or camelized.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • A“protein-based food” is any food that comprises protein including, but not limited to, meat, seafood, a food comprised of plant based proteins (tofu, tempeh), and/or fungal based proteins (tempeh, meat-substitutes) and the like.
  • a protective polypeptide e.g., ubiquitin polypeptide, Flg M polypeptide, SH3 Domain polypeptide, SUMO polypeptide, and/or a serum albumin polypeptide
  • ubiquitin polypeptide e.g., Flg M polypeptide, SH3 Domain polypeptide, SUMO polypeptide, and/or a serum albumin polypeptide
  • the concentration of the salt can be about 0.01 mM to about 100 mM or any range or value therein (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM and any range or value therein).
  • the salt concentration can be about 0.1 mM to 50 mM and any value or range therein).
  • Any appropriate physiologically compatible salt may be used, for example, NaCl.
  • the pH of a liquid composition, solid composition and/or solution of the invention may be about 5 to about 9, or any range or value therein (e.g., about 5, 5.1, 5.2, 5.3, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.5, 8.6, 8.7, 8.8, 8.9, 9, and the like).
  • the pH of a liquid composition, solid composition and/or solution of the invention may be, for example, about pH 6 to about pH 8, about pH 6.5 to about pH7.5, optionally about pH 7.
  • the liquid compositions, solid compositions and/or solutions of the invention may comprise a buffer. Any buffer may be used provided the buffer is within the pH range of about pH 5 to about pH 9, and within the salt concentration of about 0 to 100 mM.
  • the present invention further provides methods of producing transgenic cells or transgenic organism having increased tolerance to drought or desiccation.
  • a method of producing a transgenic cell having increased tolerance to drought or desiccation comprising: introducing into a cell at least one heterologous
  • polynucleotide encoding a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, a serum albumin polypeptide, or any combination thereof; to produce a transgenic organism expressing the heterologous polynucleotide, thereby producing a transgenic cell having increased tolerance to drought or desiccation as compared to a control (e.g., the cell in which the at least one heterologous polynucleotide encoding a ubiquitin polypeptide, a Flg M polypeptide, a SH3 Domain polypeptide, a SUMO polypeptide, or a serum albumin polypeptide is not introduced).
  • a control e.g., the cell in which the at least one heterologous polynucleotide encoding a ubiquitin polypeptide, a Fl
  • a cell may be from an animal, a fungus, a bacterium, an archaeon, a protist, or a plant.
  • animals include a mammal, a bird, a fish, an insect, a reptile, a nematode, or an amphibian.
  • Exemplary mammals include a human, a non- human primate (e.g., a monkey, a gorilla, a chimpanzee, a baboon, and the like), a cow (cattle), a dog, a cat, a ferret, a horse, a donkey, a goat, a sheep, a rabbit, a guinea pig, a hamster, a mouse, and/or a rat.
  • the transformation may be performed in vitro (e.g., single cells, cell masses, or tissues) or in vivo (e.g., one or more cells in an organism).
  • a transformed cell may be used to regenerate a transgenic multicellular organism.
  • the method further comprising regenerating a transgenic plant from the transgenic cell, the regenerated transgenic plant comprising the heterologous nucleotide sequence encoding a protective polypeptide (e.g., a ubiquitin polypeptide, a Flg M polypeptide, a SH3 Domain polypeptide, a SUMO polypeptide, a serum albumin polypeptide, or any combination thereof) in its genome.
  • a transgenic plant of the invention may produce transgenic seed, the genome of the seed comprising a heterologous nucleotide sequence encoding a protective polypeptide.
  • a method of producing a transgenic organism having increased tolerance to drought or desiccation comprising: introducing into the organism at least one heterologous polynucleotide encoding a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin-like modifier (SUMO) polypeptide, a serum albumin polypeptide, or any combination thereof, thereby producing a transgenic organism having increased drought or desiccation tolerance as compared to a control (e.g., the organism in which the at least one heterologous polynucleotide encoding a ubiquitin polypeptide, a Flg M polypeptide, a SH3 Domain polypeptide, a SUMO polypeptide, or a serum albumin polypeptide is not introduced).
  • a control e.g., the organism in which the at least one heterologous polynucleotide encoding
  • a method of increasing drought or desiccation tolerance in an organism comprising: introducing into the organism at least one heterologous polynucleotide encoding a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin- like modifier (SUMO) polypeptide, a serum albumin polypeptide, or any combination thereof, to produce a transgenic organism expressing the heterologous polynucleotide, thereby increasing the drought or desiccation tolerance of the transgenic organism.
  • Flg M negative regulator of flagellin synthesis
  • SH3 Domain polypeptide SRC Homology 3 (SH3) Domain polypeptide
  • SUMO small ubiquitin- like modifier
  • An organism useful with this invention may be any organism that can be transformed and includes, but is not limited to, an animal, a fungus, a bacterium, an archaeon, a protist, or a plant.
  • Non-limiting examples of an animals includes a mammal, a bird, a fish, an insect, a reptile, a nematode, or an amphibian.
  • Exemplary mammals include a human, a non-human primate (e.g., a monkey, a gorilla, a chimpanzee, a baboon, and the like) a bovine, a dog, a cat, a ferret, a horse, a donkey, a goat, a sheep, a rabbit, a guinea pig, a hamster, a mouse, and/or a rat.
  • the organism that can be transformed is not a human.
  • Any polynucleotide encoding a ubiquitin polypeptide may be introduced into a cell or organism as described herein.
  • Exemplary ubiquitin encoding polynucleotides include, but are not limited to, those from a mammal, a fish, an insect, a bird, a plant, a bacterium, a protist, a fungus, a plant, a nematode, or an archaeon.
  • Exemplary mammals include, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), primates such as a human or a non-human primate (e.g., a chimpanzee, baboon, monkey, orangutan, gorilla, etc.).
  • a laboratory animal e.g., a rat, mouse, guinea pig, rabbit, etc.
  • a farm or commercial animal e.g., a bovine, pig, horse, goat, donkey, sheep, etc.
  • a domestic animal e.g., cat, dog, ferret, etc.
  • primates e.g., a chimpanzee, baboon, monkey, orangutan, gorilla, etc
  • a ubiquitin polypeptide may be encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9, 100%) to any of SEQ ID NOs:7-30; and/or any combination thereof.
  • 70% identity e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9, 100%
  • Any polynucleotide encoding a SRC Homology 3 (SH3) Domain may be introduced into a cell or organism as described herein.
  • Exemplary SH3 domain encoding polynucleotides include, but are not limited to, those obtained from a eukaryote polypeptide or a prokaryote polynucleotide comprising an SH3 domain.
  • a SH3 domain encoding polynucleotide may be obtained from, for example, from a primate such as a human or a non- human primate (e.g., a chimpanzee, baboon, monkey, orangutan, gorilla, etc.), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a reptile (e.g., a lizard, turtle, snake, etc.), a bacterium (e.g., Escherichia ), a plant, e.g.,
  • a SH3 domain may be encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:31-53; and/or any combination thereof.
  • any polynucleotide encoding a negative regulator of flagellin synthesis (Flg M) polypeptide may be introduced into a cell or organism as described herein.
  • Exemplary Flg M encoding polynucleotides include, but are not limited to, those obtained from a monotrichous bacterium or a lophotrichous bacterium.
  • a Flg M polypeptide may be obtained from Escherichia, Pseudomonas, Bacillus, Vibrio, Helicobacter, Klebsiella,
  • a Flg M polypeptide may be encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:54-64; and/or any combination thereof.
  • Any polynucleotide encoding a small ubiquitin-like modifier (SUMO) polypeptide may be introduced into a cell or organism as described herein.
  • SUMO encoding polynucleotides include those from a eukaryote or a prokaryote.
  • a SUMO encoding polynucleotide may be obtained from a polypeptide from, for example, a human or a non-human primate (e.g., monkey, gorilla, orangutan, chimpanzee), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a reptile (e.g., a lizard, turtle, snake, etc.), a bacterium (e.g., Escherichia ), a plant, e.g., Arabidopsis), a fungus (e.g., yeast), protist (e.g., Monosiga), insect (e.g., Drosophila), fish (e.g., salmon,
  • a SUMO polypeptide may be encoded by a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to any of SEQ ID NOs:65- 86; and/or any combination thereof.
  • any polynucleotide encoding a serum albumin polypeptide useful as a protective polypeptide may be introduced into a cell or organism as described herein.
  • Exemplary serum albumin encoding polynucleotides include those from any animal including, but not limited to, a human, a non-human primate (e.g., monkey, gorilla, orangutan, chimpanzee), a farm or commercial animal (e.g., a bovine, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.), a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, etc.), a fish (e.g., salmon, danio), an amphibian (e.g., frog), reptile (e.g., turtle, lizard, snake) and/or an avian (e.g., chicken, parakeet, parrot
  • the at least one heterologous polynucleotide may be operably linked to a promoter.
  • the promoter may be heterologous to the polynucleotide and/or heterologous to the organism or cell into which the polynucleotide is introduced.
  • the promoter may be endogenous to the polynucleotide that is introduced but heterologous to the organism or cell into which the polynucleotide is introduced.
  • the heterologous polynucleotide may be heterologous to the organism or cell into which the heterologous polynucleotide is being introduced.
  • the heterologous polynucleotide may be endogenous to the organism or cell into which the heterologous polynucleotide is being introduced but may be heterologous to the promoter that is operably linked to the polynucleotide.
  • the present invention further provides transgenic cells produced by the methods of the invention comprising at least one heterologous polynucleotide encoding a protective polypeptide (e.g., ubiquitin, SH3 Domain, Flg M, SUMO, serum albumin) and having increased tolerance to drought or desiccation as compared to a control (e.g., a cell that does not comprise a protective polypeptide (e.g., ubiquitin, SH3 Domain, Flg M, SUMO, serum albumin) and having increased tolerance to drought or desiccation as compared to a control (e.g., a cell that does not comprise a control (e.g., a cell that does not comprise a control (e.g., a cell that does not comprise a control).
  • a protective polypeptide e.g., ubiquitin, SH3 Domain, Flg M, SUMO, serum albumin
  • heterologous polynucleotide encoding a ubiquitin polypeptide, a negative regulator of flagellin synthesis (Flg M) polypeptide, a SRC Homology 3 (SH3) Domain polypeptide, small ubiquitin- like modifier (SUMO) polypeptide, and/or a serum albumin polypeptide.
  • Flg M negative regulator of flagellin synthesis
  • SH3 SRC Homology 3
  • SUMO small ubiquitin- like modifier
  • the cell can be, but is not limited to, an animal cell (e.g., a mammalian cell, an avian cell, a reptile cell, an amphibian cell, an insect cell, or a fish cell, a sperm cell, an egg cell, a stem cell, a red blood cell, muscle cell, and the like), a fungal cell, a bacterial cell, or a plant cell.
  • an animal cell e.g., a mammalian cell, an avian cell, a reptile cell, an amphibian cell, an insect cell, or a fish cell, a sperm cell, an egg cell, a stem cell, a red blood cell, muscle cell, and the like
  • a fungal cell e.g., a mammalian cell, an avian cell, a reptile cell, an amphibian cell, an insect cell, or a fish cell, a sperm cell, an egg cell, a stem cell, a red blood cell, muscle cell, and the
  • a transgenic organism e.g., a transgenic animal, plant, fungus, protist, archaeon, or bacterium
  • the transgenic organism comprises in its genome at least one heterologous polynucleotide encoding a protective polypeptide.
  • the invention provides a seed of a transgenic plant produced by the methods of the invention, wherein the seed comprises in its genome at least one heterologous polynucleotide encoding a protective polypeptide.
  • the invention provides a crop comprising a plurality of transgenic plants of the invention, planted together in an agricultural field, a golf course, a residential lawn, a road side, an athletic field, and /or a recreational field.
  • “Introducing,” in the context of a heterologous polynucleotide encoding a protective polypeptide means presenting the polynucleotide to the cell of an organism in such a manner that the polynucleotide gains access to the interior of the cell.
  • the methods of the invention do not depend on a particular method for introducing one or more polynucleotides into an organism, only that they gain access to the interior of at least one cell of the organism.
  • these polynucleotides can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotides or nucleic acid constructs, and can be located on the same or different expression constructs or transformation vectors. Accordingly, these polynucleotides may be introduced into cells in a single transformation event, in separate transformation events, or, for example, they may be incorporated into an organism as part of a breeding protocol.
  • transformation refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, in some embodiments, a cell of the invention may be stably transformed with a nucleotide sequence of the invention. In other embodiments, a cell may be transiently transformed with a nucleotide sequence of the invention.
  • Transient transformation in the context of a polynucleotide means that a
  • polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • stably introducing or“stably introduced” in the context of a polynucleotide introduced into a cell is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.
  • Gene as used herein also includes the nuclear, mitochondrial, and plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast or mitochondrial genome.
  • Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.
  • Transient transformation may be detected by, for example, an enzyme-linked
  • ELISA immunosorbent assay
  • Western blot which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism.
  • transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant).
  • Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences, which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism.
  • Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
  • PCR polymerase chain reaction
  • Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
  • a polynucleotide of the invention e.g., a nucleotide sequence encoding an amino acid sequence having at least about 70% identity to of any of SEQ ID NO: 7-209, a nucleotide sequence having at least about 70% identity to of any of SEQ ID NOs:210-314, or a nucleotide sequence having at least about 80% identity to any of SEQ ID NOs:315-419, and/or fragments thereof
  • transformation of a cell comprises nuclear transformation.
  • transformation of a cell comprises mitochondrial or chloroplast
  • Polynucleotides encoding protective polypeptides can be delivered directly into a cell by any method known in the art, e.g., by transfection or microinjection. Those skilled in the art will appreciate that the isolated polynucleotides encoding the protective polypeptides will typically be associated with appropriate expression control sequences, e.g.,
  • promoter/enhancer elements can be used depending on the level and tissue-specific expression desired.
  • the promoter can be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • the promoter is chosen so that it will function in the target cell(s) of interest.
  • a promoter or other regulatory element may be heterologous to the organism into which it is being introduced and/or it may be heterologous to the polynucleotide to which it is operably linked.
  • polynucleotides encoding protective polypeptides can be incorporated into an expression vector.
  • Expression vectors compatible with various host cells are well known in the art and contain suitable elements for transcription and translation of nucleic acids.
  • an expression vector contains an“expression cassette,” which includes, in the 5’ to 3’ direction, a promoter, a coding sequence encoding a double stranded RNA operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.
  • Non-limiting examples of animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the El A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, a- actin, tubulin and the like), the promoters
  • Non-limiting examples of plant promoters include the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal).
  • PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters. Pnr is induced by nitrate and repressed by ammonium and Pdcal is induced by salt.
  • Other constitutive plant promoters include but are not limited to oestrum virus promoter (cmp) (U.S. Patent No.
  • tissue-specific promoters for plants include those associated with genes encoding the seed storage proteins (such as b-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4).
  • seed storage proteins such as b-conglycinin, cruciferin, napin and phaseolin
  • zein or oil body proteins such as oleosin
  • proteins involved in fatty acid biosynthesis including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)
  • other nucleic acids expressed during embryo development such as Bce4
  • Non-limiting examples of promoters functional in chloroplasts include the bacteriophage T3 gene 9 5' UTR, the S-E9 small subunit RuBP carboxylase promoter, the Kunitz trypsin inhibitor gene promoter (Kti3).arid other promoters disclosed in U.S. Patent No. 7,579,516.
  • kits for use in the methods of the invention can comprise one or more protective polypeptides of the invention in a form suitable for stabilizing vaccines, antibodies, a heterologous cell, tissue, organ and/or other biologies or in a form suitable for introducing into an organism.
  • the kit can further comprise other components, such as therapeutic agents, carriers, buffers, containers, devices for administration/contacting, compositions for transformation, and the like.
  • the kit can be designed for therapeutic use, diagnostic use, and/or research use and the additional components can be those suitable for the intended use.
  • the kit can further comprise labels and/or instructions, e.g., for stabilizing a heterologous polypeptide, cell, tissue, or organ, or for, e.g., imparting drought or desiccation resistance/tolerance to an organism.
  • labeling and/or instructions can include, for example, information concerning the amount, frequency and method of administration of the one or more TDPs.
  • LDH lactate dehydrogenase
  • BSA bovine serum albumin
  • TDP tardigrade polypeptides
  • CAHS Cytosolic Abundant Heat Soluble
  • ubiquitin was used at concentrations of between 0.01 g/L to 200 g/L.
  • Trehalose is a positive control. The results are provided in Fig. 1 and show that at room temperature, ubiquitin protects against desiccation-induced inactivation as well as the CAHS protein.
  • LDH lactate dehydrogenase
  • LDH lactate dehydrogenase
  • LDH lactate dehydrogenase
  • LDH lactate dehydrogenase
  • the protective polypeptides may function by facilitating proper refolding upon rehydration rather than protecting against misfolding during desiccation.

Abstract

La présente invention concerne des procédés et des compositions pour stabiliser des protéines. L'invention concerne des compositions comprenant au moins un polypeptide protecteur et/ou un polymère de sucre, et au moins un polypeptide hétérologue et/ou un peptide d'intérêt. L'invention concerne en outre des procédés de stabilisation de protéines, des procédés de stabilisation de cellules, de tissus et d'organes à l'aide des compositions et des solutions de l'invention. L'invention concerne également des procédés de production d'une cellule et/ou d'un organisme présentant une tolérance accrue à la dessiccation et/ou à la sécheresse, comprenant l'introduction dans l'organisme et/ou la cellule d'au moins un polynucléotide codant un polypeptide protecteur.
PCT/US2018/060879 2017-11-14 2018-11-14 Compositions et procédés de stabilisation de protéines WO2019099427A1 (fr)

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