Patent Application
20200236971
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 7, 2020, is named 45460US_CRF_sequencelisting.txt and is 1,704,433 bytes in size.
The invention relates to methods and compositions for the recombinant production of animal proteins, and also relates to the use of recombinantly produced animal protein in animal food.
Pets need essential amino acids in the form of protein and these amino acids are often provided by meat from different animals, most often domesticated animals like chicken, cows, and pigs. Another common protein source is fish. Animal meat also contains other nutrients like vitamins, minerals, and fatty acids that are important for the health of pets. The use of meat for feeding pets presents some problems. For instance, animal agriculture is a climate change driver releasing 14.5% of anthropogenic greenhouse gases globally [10]. It also causes biodiversity loss; only 15 mammals and bird species make up over 90% of the livestock production and 60% of mammals are made up by domesticated livestock (on a weight basis), mainly cows and pig, whereas humans account for 36% and wild mammals only 4% [11]. The biomass of poultry is about three times higher than that of wild birds [11]. Populations of vertebrate animals, such as mammals, birds, and fish, have declined by 58% between 1970 and 2012 [12]. Meat also frequently contains pathogens like Salmonella, Shigella, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus. These pose a risk to both the companion animal and the pet parent [13]. Antibiotic resistance is an immeasurable challenge for humanity and animal agriculture is one of the contributors to the trend [14]. Numerous studies have demonstrated a link between antibiotic use in livestock and increased prevalence of antibiotic-resistant organisms associated with those animals [15] [16]. Of all antibiotics sold in the United States, approximately 80% are sold for use in animal agriculture; about 70% of these are from classes important to human medicine [17].
Although plant sources, e.g., different types of legumes, contain a fair amount of protein, they also contain anti-nutritional factors like fiber, phytate, and protease inhibitors, that limit digestion and absorption [1] [2]. Soybean, a commonly used protein source, decreases the digestibility in canine foods when present in concentrations over 15% [3]. Many plant sources also lack in one or more essential amino acids [4] and thus vegetarian pet foods often contain an inadequate concentration of essential amino acids [5]. In fact, tryptophan and lysine are scarce in corn, lysine in wheat and other cereals, and methionine in soybeans and other legumes [6]. Numerous studies have shown that vegetarian commercial diets for cats and dogs are frequently nutritionally inadequate [7], [8]. Some commercial legume-rich diets may lead to diluted cardiomyopathy in dogs [9]. For all the forgoing reasons, a need exists for improved food for animals that require protein in their diet.
Disclosed herein are improved methods and compositions for manufacturing food for animals, particularly companion animals. In certain embodiments, the methods entail producing animal proteins recombinantly in a microbial host, as described herein. The recombinant proteins produced by the method can provide equivalent or better nutrition than conventionally harvested animal proteins or plant-derived proteins, without the associated problems described above. In certain embodiments, the recombinant animal proteins described herein can be incorporated into or serve as food for humans, wild animals and/or zoo animals. In preferred embodiments, the food composition is substantially free of antibiotics, animal growth hormones, and/or meat from farmed, caught or slaughtered animals.
One or a plurality of recombinant proteins can be produced in one organism, or one strain, thereby allowing the amino acid profile to be tailored to the particular nutritional needs of targeted pets and other animals, including humans. Alternatively, a single recombinant animal protein can be produced in one strain (or organism) and mixed with a protein or proteins produced in a different strain (or organism) to yield a final product with the desired proportions of amino acids and other nutrients. Thus, the amino acid profile (and/or the profile of other nutrients) can be customized for the targeted animal, including pets and humans.
Terms used in the claims and specification are defined as set forth below unless otherwise specified.
The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a nutritional deficiency disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.
The term “in situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.
The term “in vivo” refers to processes that occur in a living organism.
The term “mammal” as used herein includes both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences having a specified percentage of nucleotides or amino acid residues that are identical, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer-based program, sub-sequence coordinates are designated, if necessary, and sequence comparison program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.
The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Proteins
The recombinant proteins produced are animal proteins, e.g., proteins from any organ of an animal, including, but not limited to proteins expressed in the brain, skin, scales, feathers, eyes, shells, hair, horns, ears, liver, heart, kidney, stomach, intestines, and muscle tissue. In certain embodiments, the recombinant proteins are animal muscle proteins. In related embodiments, the animal protein is an animal cytoskeletal protein. In other related embodiments, the animal protein is an animal myosin protein or an animal actin protein.
The animal muscle proteins include those proteins normally found in animal muscle tissue (or relatives of those proteins). In addition to myosin and actin, these proteins include troponin, tropomyosin, actinin, titin, connectin, skeletal receptor, myosin-binding protein, desmin, leiomodin, tubulin, myotubularin, myozenin, telethonin, calsarcin, myotilin, nebulin, nebulin-related anchoring protein, myomesin, vinculin, paxillin, beta-enolase, myotubularin, calponin, caldesmon, transgelin, tropomodulin, supervillin, gelsolin, twinfilin, profilin, caveolin, catenin, cofilin, capping protein, leiomodin, tensin, M-protein, radixin, filamin, keratin, myopalladin, calsequestrin, caveolae-associated protein, nebulette, coronin, talin, dystrophin, dystroglycan, integrin, ankyrin, syncoilin, smoothelin-like-1, spectrin, synemin, paranemin, ponsin, plectin, skelemin, sarcoglycan, LIM protein, myoblast determination protein, myocyte-specific enhancer, and myocilin.
The recombinant animal proteins may be full-length proteins, truncated proteins, or fragments of proteins. The sequences of the recombinantly expressed proteins may be modified by replacing one or more amino acids with a different amino acid (e.g., by changing the nucleotide sequence of the recombinant gene encoding the protein). The modifications may improve the yield of protein produced by the organism that has been engineered to express the protein, e.g., by improving the efficiency of transcription and/or translation of the protein, by improving the stability of the protein, by altering the rate at which the protein is secreted by the organism, or by changing the activity of the protein so any deleterious effects of expression of the protein on the recombinant host cell are minimized.
Protein Origin
The origin of the recombinantly expressed protein sequence (i.e., the species of animal from which the sequence to be recombinantly expressed is found in nature) can be any species within the biological kingdom of Animalia. Preferably, the origin is a vertebrate animal, which can be a fish, bird, mammal, amphibian, or reptile. The origin may be a placental mammal, monotreme mammal, or marsupial mammal (metatheria). The origin may furthermore be a bird or another vertebrate from the reptile Glade.
In some embodiments the gene origin is a placental mammal, including but not limited to carnivores (including lion, bear, weasel, seal, wolf, coyote, fox), equidae (including horse and donkey), even-toed ungulates (including pig, camel, cattle, and deer), Afrotheria (including elephants, golden moles, and manatees), and Boreoeutheria (including primates, rabbits, hares, pikas, rodents, moles, whales, bats, dogs, cats, seals, hoofed mammals). In some embodiments the origin is a monotreme mammal, including but not limited to platypus and echidna. In some embodiments the origin is a marsupial mammal, including but not limited to koala, possums, tapirs, kangaroos, wallabies, and marsupial lions. In some embodiments the origin is a hoofed mammal, including but not limited to cattle, antelope, deer, reindeer, elk, sheep, goat, camels, carabao, yak, bison, buffalo, caribou, water buffalo, pig, horse, and donkey. In some embodiments the origin is an endothermic vertebrate, classified as Ayes, including but not limited to chicken, turkey, duck, pigeon, penguin, ostrich, goose, pheasant, and quail. In some embodiments the gene origin is a reptile, including but not limited to alligators and crocodiles. In some embodiments the gene origin is an aquatic animal, including but not limited to shark, tuna, trout, salmon, jacks, carp, catfish, cod, flounder, bass, tilapia, sturgeon, crab, lobster, shrimp, prawns, oysters, mussels, eels, shellfish, cuttlefish, starfish, crayfish, and jellyfish. In some embodiments the gene origin is an amphibian, including but not limited to frogs, salamanders, and toads. In some embodiments, the gene origin is an insect.
Truncated/Mutated Proteins
It is well known that expression of mammalian and avian cytoskeletal proteins, such as actin and tubulin, in microbial hosts can be toxic to those hosts, thus limiting expression levels. This is due to the biological activity of these proteins interfering with the metabolism of the host cell. The nutritional properties of these animal derived proteins will remain essentially unchanged regardless of the biological activity of the protein. Expression levels of toxic recombinant animal proteins can be increased by decreasing their host toxicity via targeted mutagenesis (to decrease the biological activity responsible for the protein's toxicity, for example). Examples are provided herein for improving expression of actin, but these examples should not be construed as limiting. Similar methodology can be used for other proteins.
Preferred protein sequences are listed in Table 1, below. They are grouped according to the tissue in which they are highly expressed (when known). If it is not known in what tissue a protein is expressed, the protein is grouped according to the tissue for which its expression is required (e.g., for normal development of the tissue). For example, it is known that myotubularin is required for normal skeletal muscle growth. Thus, it is grouped with the skeletal muscle proteins. In certain embodiments, the food compositions described herein comprise one or more of the recombinantly produced proteins set forth in Table 1. In related embodiments, the food compositions described herein additionally, or alternatively, comprise one more recombinantly expressed homologs of the recombinantly produced proteins set forth in Table 1. In other related embodiments, the food compositions described herein comprise one more recombinantly expressed proteins that are at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% identical, but less than 100% identical, to the proteins set forth in Table 1 (i.e., the protein sequences are modified to alter their amino acid content, e.g., to improve nutrition, to improve digestibility, to optimize expression or to optimize secretion). In other related embodiments, the food compositions described herein comprise one or more of the recombinantly expressed skeletal muscle tissue proteins of Table 1, or one or more of the recombinantly expressed cardiac muscle tissue proteins of Table 1, or one or more of the recombinantly expressed smooth muscle tissue proteins of Table 1, or one or more of the skeletal/cardiac muscle tissue proteins of Table 1, or one or more of the skeletal/smooth muscle tissue proteins of Table 1, or one or more of the cardiac/smooth muscle tissue proteins of Table 1, or one or more of the skeletal/cardiac/smooth muscle tissue proteins of Table 1. In yet other related embodiments, the food compositions described herein comprise proteins from two or more of the above-mentioned categories of proteins described in Table 1.
Recombinant Genes Encoding Animal Proteins
Genes encoding recombinant animal proteins can be isolated by taking a sample from an animal and extracting nucleic acids, such as mRNA, from that sample and then amplifying the gene by reverse transcription followed by PCR. The cell sample could be a tissue sample, blood sample, mucus, skin, saliva, or hair. Another option is to have the gene synthesized on demand by a company which performs such work.
The gene sequences (DNA/nucleotide sequences) or protein sequences can be obtained by searching appropriate databases (e.g., UniProtKB and NCBI). A polynucleotide can be obtained using chemical synthesis, molecular cloning or recombinant methods, DNA or gene assembly methods, artificial gene synthesis, PCR, or any combination of those.
The gene sequences are those encoding the types of protein described above. The gene sequences may have non-coding introns.
In the case that there are not sequences available, conserved regions can be used to amplify segments of the genes and the flanking regions can be sequenced in order to obtain the full-length sequence. Multiple sequence alignments of a specific protein in several different organisms will show where the conserved regions lie, and which are the most suitable stretches to use for primer design. Primers with alternative nucleotides can be used when needed.
Expression Constructs
A genetic element is any coding or non-coding nucleic acid sequence. A genetic element can be a nucleic acid that codes for an amino acid, a peptide or a protein. Genetic elements may be operons, genes, gene fragments, promoters, exons, introns, regulatory sequences, or any combination of those. A genetic element includes an entire open reading frame of a protein, or the entire open reading frame and one or more (or all) regulatory sequences associated therewith. The genes may be codon optimized for different hosts.
The term “engineered” or “recombinant” refers to a cell into which a recombinant gene, such as a gene encoding a muscle protein, or part of a muscle protein, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene that is introduced by transfection or transformation techniques. Recombinantly introduced genes will either be in the form of a cDNA (i.e., they will not contain introns), a copy of a cDNA gene, genomic DNA (with or without introns; for expression in prokaryotic hosts, the DNA should be without introns), or will include DNA sequences positioned next to a promoter not naturally associated with the particularly introduced gene.
Disclosed herein are expression vectors comprising a genetic element encoding an animal protein or part of an animal protein and the use thereof for the recombinant expression of the animal protein. The expression vector may further comprise a promoter. The promoter may be a constitutive promoter, an inducible promoter, or a hybrid promoter. In the expression vector, the gene construct and the method, the promoter may be a viral promoter, a prokaryotic promoter or a eukaryotic promoter. The promoter may be a synthetic promoter from a promoter library. The promoter may be any scientifically known promoter or a novel promoter. The promoter may be an engineered form of a known promoter or a hybrid promoter.
The eukaryotic promoter may be a fungi promoter, a plant promoter, or an animal promoter. The fungi promoter may be the promoter of the genes phosphoglycerate kinase (PGK, PGK1, PGK3), enolase (ENO, ENOl), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase (pki1), triosephosphate isomerase, phosphoglucose isomerase, glucokinase, alcohol dehydrogenase promoter (ADH1, ADH2, ADH4), isocytochrome C, acidic phosphatase, galactose metabolism enzymesGAL (GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10), alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), CUP1, AHSB4m, adhl+, AINV, alcA, AXDH, cellobiohydrolase I (cbhl), ccg-1, cDNA1, cellular filament polypeptide (cfp), cpc-2, ctr4+, dihydroxyacetone synthase (DAS), FMD, formate dehydrogenase (FMDH), formaldehyde dehydrogenase (FLD1), GAA, GCW14, glucoamylase (glaA, gla-1), inv1, isocitrate lyase (ICL1), glycerol kinase (GUT1), acetohydroxy acid isomeroreductase (ILV5), β-galactosidase (lac4), LEU2, melO, MET3, MET25, KAR2, KEX2, methanol oxidase (MOX), nmtl, peroxin 8 (PEX8), pcbC, PET9, PH05, PH089, PYK1, phosphatidylinositol synthase (PIS1), RPS7, TEF, translation elongation factor 1 alpha (TEF1), sorbitol dehydrogenase (SDH), SSA4, THI11, homoserine kinase, XRP2, TPI, and YPT1, PHO5, CYC1, HISS, ADC1, TAP1, URA3, LEU2, TP1, TDH1, TDH3, FBA1, ADR1, TPI1, or any combination of those.
The plant promoter may be the promoter of the gene phol, TPI, TPS1, and any combination of these.
The animal promoter may be a heat-shock protein promoter, proactin promoter, immunoglobulin promoter, or the promoter of the gene B2, HSP82, Ser1, triose phosphate isomerase (TPI1), or any combination of those. However, any promoters can be used if they drive the expression of recombinant proteins in a particular host cell.
The expression vector may include an auxotrophic marker. The auxotrophic marker may be selected from the group consisting of adel, arg4, his4, ura3, and met2. The expression vector may comprise a selectable marker, which may be a resistance gene. The resistance gene may confer resistance to drugs including, but not limited to, zeocin, ampicillin, blasticidin, kanamycin, nurseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir.
The prokaryotic promoter may be the T7, heat-shock protein (HSP) 70, beta-lactamase, a-amylase, alkaline phosphatase, trp operon, lac operon, tac, trc, gdhA, fic, bgl, xylA, xylS, AraC, lacSynth, P11, P43, trnQ, sigX, groES, ftsH, and Pm.
In certain embodiments, the compositions of the invention include a host cell transformed with a vector to express one or more recombinant animal muscle proteins. The vector or required genetic segments along with genes of interest may be genomically integrated. Alternatively, or in addition, the host cell may comprise multiple copies of an expression vector. The expression vector may comprise a plasmid. Cells may be transformed by transfection, endocytosis, F-mating, PEG-mediated protoplast fusion, Agrobacterium tumefaciens-mediated transformation, chemical transformation, electroporation, or biolistic transformation.
The construct may encode a signal peptide. A signal peptide, also identified as a signal sequence, targeting signal, localization signal, localization sequence, secretion signal, transit peptide, leader sequence, or leader peptide, may cause secretion of a protein. Extracellular secretion of a recombinant or heterologously expressed animal protein from a host cell simplifies protein purification. Recovery of a recombinant animal protein from a cell culture supernatant may be preferable to lysing host cells to release a complex mixture of proteins including intracellular proteins of the host cell. Secretion may reduce harmful effects that intracellular overexpression of a recombinant or heterologous animal protein may have on a host cell such as toxicity or reduced growth rate. Secretion may produce higher amounts of heterologous animal protein compared to intracellular expression. Secretion of a protein may also enable post-translational modification (e.g., glycosylations) or aid in folding the protein correctly and allow for the formation of disulfide bonds.
Food and Feed Compositions
The recombinant protein composition may be incorporated into food or feed at percentages (based on dry mass) of 0.1-95%, typically between 10% and 90%, more typically between 5% and 50%, including ranges of 5%-10%, 10-20%, 20-30%, 30-40%, 40-50%, but also including 60-70%, 70-80% and 80%-90% and combinations of these ranges (e.g., 30%-70%). The recombinant protein compositions can be used in dry or wet food. In preferred embodiments, the end users are companion animals such as dogs and cats. Other users include the animals described herein, including humans.
The recombinant animal protein is typically mixed with other ingredients. The ingredients are any ingredient suitable for consumption by the pet or person. Typical food ingredients include but are not limited to carbohydrates, fats, proteins, fibers, and nutrients such as vitamins, minerals, and trace elements. People skilled in the art can select the amount and type of food ingredients for a typical food based upon the dietary requirements of the pet or person, which can depend on the species, age, size, weight, health, and function.
The food composition can comprise 100% of any particular food ingredient or can comprise a mixture of food ingredients in various proportions. Preferably, the food composition will include, by mass, 5-50% protein, 0.01-1.5% sodium, 0.01-1.5% potassium, 0-50% fat, 0-75% carbohydrate, 0-40% dietary fiber, and 0-15% of other nutrients.
Fat and carbohydrates are obtained from a variety of sources including but not limited to animal fat, fish oil, vegetable oil, meat, meat by-products, grains, other animal or plant sources, and combinations of those. Grains include but are not limited to rice, wheat, corn, barley, buckwheat, sorghum, oats, and quinoa. Other plant sources include but are not limited to pulses (chickpeas and different beans) and edible roots (e.g. potato, sweet potato, carrot, cassava, and turnips).
The supplemental protein food ingredient can be obtained from a variety of sources including plants, animals, or microbes (unicellular and multicellular). Animal protein includes meat, meat by-products, dairy, and eggs. Meats include the flesh from poultry, fish, and animals such as cattle, swine, sheep, goats, and the like. Meat by-products include but are not limited to kidneys, lungs, livers, stomachs, and intestines. The supplemental protein food ingredient may also be free amino acids and/or peptides.
The fiber food ingredient is obtained from a variety of sources such as vegetable fiber sources, including but not limited to beans, cellulose, beet pulp, parsnips, broccoli, peanut hulls, carrots, spinach, and soy fiber.
The nutrients are obtained from a variety of sources known to people skilled in the art, e.g., vitamin and mineral supplements and food ingredients. Vitamins and minerals can be included in amounts required to avoid deficiency and maintain health. These amounts are readily available in the art. The National Research Council (NRC) provides recommended amounts of such nutrients for farm animals. See, e.g., Nutrient Requirements of Swine (11th Rev. Ed., National Academy Press, Wash. D.C., 2012), Nutrient Requirements of Poultry (9th Rev. Ed., National Academy Press, Wash. D.C., 1994), Nutrient Requirements of Horses (6th Rev. Ed., National Academy Press, Wash. D.C., 2007). The American Feed Control Officials (AAFCO) provides recommended amounts of such nutrients for dogs and cats. See American Feed Control Officials, Inc. (Official publication, 2018). Vitamins generally useful as food additives include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin D, biotin, vitamin K, folic acid, inositol, niacin, and pantothenic acid. Minerals and trace elements useful as food additives include calcium, phosphorus, sodium, chloride, potassium, magnesium, iron, copper, zinc, selenium, and iodine. In certain embodiments, the food compositions comprise taurine.
In certain embodiments, the recombinant animal protein is not isolated from the host organism recombinantly expressing the protein prior to mixing with the food composition. The host organism itself, and the recombinant protein expressed within or on its surface, can be added to the food composition.
The food compositions can be in any form useful for feeding the composition to an animal, e.g., kibbles, treats, and toys for animal food. The food compositions can be freeze-dried, dehydrated, air dried, or used as coatings. Kibbles are generally formed using an extrusion process in which the mixture of dry and wet ingredients is mechanically worked at high temperature and pressure and pushed through small openings and cut off into kibble by a rotating knife. The wet kibble is dried and can be coated with one or more topical coatings such as flavors, fats, oils, powders, etc. Kibble also can be made using a baking process when the mix is placed into a mold before dry-heat treatment. Treats may be nutritional and may contain one or more nutrients and may have a food-like composition. Non-nutritional treats are any other treats that are non-toxic. The composition or components are coated onto the treat, incorporated into the treat, or both. Treats of the present invention can be prepared by an extrusion or baking process similar to those used for dry food. Other processes may also be used to either coat the composition on the exterior or inject the composition into an existing treat form. Toys include chewable toys such as artificial bones and food compositions shaped to look like natural foods that are appealing to the animal. The food compositions can be packaged in cans, trays, tubs, pouches, bags, or any other suitable container.
Methods for treatment of diseases are also encompassed by the present invention. Said methods of the invention include administering a therapeutically effective amount of the recombinant animal protein or a food composition comprising the recombinant animal protein. The recombinant animal proteins of the invention can be formulated in pharmaceutical compositions or therapeutic food compositions. These compositions can comprise, in addition to one or more of the recombinant animal proteins, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.
For treatment purposes, administration of the food composition comprising the recombinantly produced animal protein is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the animal or individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g., decisions on dosage, etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
A food composition of the invention described herein can be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
In certain embodiments, the invention includes cultivating the host cell and expressing the recombinant animal proteins intracellularly or secreted into the culture broth.
Harvesting and Processing
Cells expressing recombinant animal protein may be harvested by batch centrifugation, continuous flow centrifugation, filter press, flocculation, rotary drum vacuum filtration, tangential flow filtration, or ultrafiltration.
Cells may be lysed by raising temperature, autolysis, by high pressure homogenization (e.g., French press), ultrasonic cavitation, bead beating, rotor-stator processors, freeze-thaw cycles, enzymatic lysis (e.g., lysozyme, lysostaphin, zymolase, cellulose, protease or glycanase), osmotic shock methods, chemical lysis (by alkaline, detergent or organic solvent) or a combination of these methods. Secreted recombinant animal proteins can be isolated from growth media using well-known techniques.
The whole cell fraction or proteins may be dried by drum drying, spray drying, lyophilization, fluidized bed drying, or other commonly used methods.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992). Also referred to below are the following references: (1) M. R. Green and J. Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2012, pp. 1009-1011. (2) G. C. U. F. T. Tool, GenScript, [Online]. Available: https://www.genscript.com/tools/codon-frequency-table. [Accessed 18 12 2018]. (3) S. Wu and L. J. Geoffrey, “High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol,” Drug Discovery and genomic technologies, vol. 36, no. 1, pp. 152-154, 2004. (4) S. Kawai, W. Hashimoto and K. Murata, “Transformation of Saccharomyces cerevisiae and other fungi,” Bioengineered Bugs, vol. 1, no. 6, pp. 395-403, 2010. (5) P. Manivasakam and R. H. Schiestl, “High efficiency transformation of Saccharomyces cerevisiae by electroporation,” Nucleic Acids Research, vol. 21, no. 18, pp. 4414-4415, 1993.
Actin is the major component of the cytoskeleton. It exists in two different forms, a monomeric form (G-actin) and a filamentous form (F-actin). G-actin polymerizes to form F-actin, and it is primarily these filaments that participate in processes such as cell motility, transport, and cytokinesis [20]. The actin-binding domain is highly conserved amongst species. Actin-binding proteins share a common binding area on the actin surface, consistent of the cleft between actin sub domains 1 and 3 [21]. There is also a nucleotide-binding site, which is a cleft between subdomains 2 and 4. The binding of adenosine 5′-triphosphate or ATP and subsequent hydrolysis into adenosine 5′-diphosphate or ADP is known to be a critical element in controlling the association of actin with itself and with other proteins. When ATP is bound to actin it polymerizes faster and dissociates slower than ADP-actin [22].
Single and double mutants of the ATP-binding site of actin will ablate its toxicity in prokaryotic expression hosts and thus increase expression levels. The residues targeted by mutagenesis are P-72, E-74, 1-77, and T-79 (numbering for pig (SEQ P68137), chicken (SEQ P68139), and cow (SEQ P68138)). Recombinant actin protein mutated at these sites will be over-expressed in a prokaryotic host organism, isolated, and incorporated into a companion animal food product.
In one embodiment, then, the invention provides a food composition comprising a recombinant actin protein, wherein said recombinant actin protein comprises one or more mutations from the group consisting of P-72, E-74, 1-77 and T-79. In certain related embodiments, the recombinant actin protein is a fragment of actin protein comprising the aforementioned residues.
Actin is highly conserved between widely divergent species. For instance, there is 87% sequence identity (325 of 374 amino acids) between yeast and human actin. Comparing chicken, cow, pig, human, and Saccharomyces cerevisiae, there are 319 conserved residues. A library of point mutations is made at each of these conserved positions and those mutations that are permissive of high levels of expression of mutant actin are identified.
Error-prone PCR with/without shuffling will be used across the DNA coding sequence (cDNA) to create mutated DNAs encoding animal protein sequences. Prokaryotic hosts recombinantly expressing the mutant sequences will be screened for high growth and high expression of the target protein.
The genes and the proteins encoded by the genes may also be truncated in order to yield a high expression and fast cell growth. Modifications of the gene sequence (e.g., the addition or removal of certain amino acids) will, in some cases, increase cell viability and increase the rate of cell division. Proteins that are too large to overexpress efficiently will be truncated in order to increase the expression level.
The prokaryotic promoter may be the T7, heat-shock protein (HSP) 70, beta-lactamase, a-amylase, alkaline phosphatase, trp operon, lac operon, tac, trc, gdhA, fic, bgl, xylA, xylS, AraC, lacSynth, P11, P43, trnQ, sigX, groES, ftsH, and Pm.
The expression vector pNZ8152 (MoBiTec GmbH, Gottingen, Germany) is a Gram-positive broad host range vector. Taking advantage of the nisA promoter, it allows intracellular recombinant protein expression via induction with nisin. The vector pNZ8152 also contains the selectable marker alanine racemase gene alr, which restores auxotrophy to D-alanine caused by a deletion of the host cell copy of alr. The vector is linearized using HindIIII restriction enzyme (New England Biolabs, Ipswich, Mass.) and dephosphorylated using established molecular cloning methods [1]. Linearized vector is separated using agarose gel electrophoresis. An agarose gel section containing linearized vector is collected and the linearized plasmid is purified from the agarose using a commercially available DNA purification kit, e.g. the QIAquick Gel Extraction Kit (Qiagen, Germantown, Md.).
The gene sequence for chicken myosin regulatory light chain 2, skeletal muscle isoform, (MYLPF) can be obtained from UniProt.org under accession number P02609. The double-stranded DNA is constructed through chemical gene synthesis from either ATUM (Newark, Calif.), Genscript (Piscataway, N.J.), or IDT (Coralville, Iowa). It is supplied in a vector of choice. The DNA sequence can also be obtained via amplification of cDNA generated directly from a mRNA of a biological sample, such as a tissue or a blood sample from a chicken donor. The gene sequence is modified to aid in cloning, gene expression, or enhance production. It is “codon optimized”, i.e. triplet DNA sequences that are not commonly used in the expression host are changed to those that are commonly used.
The codon optimized myosin regulatory light chain 2, skeletal muscle isoform, gene (MYLPF), containing exons, but no introns, is ligated to the linearized and purified pNZ8152 vector via enzymatic ligation to generate a vector capable of being inserted into a host organism. The method used is known in the art and the protocol can be obtained from a molecular cloning manual [1].
The host organism in this example is Lactococcus lactis strain NZ9130 (MoBiTech). This strain contains genes nisR and nisK, which are needed for nisin-regulated gene expression, as well as a deletion of the alr gene encoding an alanine racemase, causing auxotrophy to D-alanine.
The pNZ8152 vector construct containing the codon optimized myozenin gene is transformed into host strain Lactococcus lactis NZ9130 via electroporation using instrument settings of 2.0 kV, 25 μf, and 200Ω. Electrocompetent cells are obtained via methods known in the art [3]. Chemical transformation or another method can also be used. Successful clones are selected for on agar plates prepared with M17 medium containing 0.5% (w/v) glucose grown at 30° C. As this medium does not contain D-alanine, only host cells that contain a copy of the plasmid pNZ8152 will be able to proliferate. Colonies are picked and cultured at 30° C. in M17 medium with 0.5% (w/v) glucose for 48 hours in a non-aerated environment. A successful clone containing a copy of the chicken myosin regulatory light chain 2 gene, skeletal muscle isoform, is confirmed by sequencing for insert identity and orientation using established methods such as PCR [1]. Protein expression using the successful clone is induced by culturing in M17 medium with 0.5% (w/v) glucose at 30° C. in a non-aerated environment for 4 hours after cells have reached stationary phase by supplementing 1 ng/mL of nisin. The cells are pelleted by centrifugation and the cell pellets are lysed by methods known in the art, e.g. by sonication [1] and analyzed for protein expression by SDS-PAGE.
Other variants of Lactococcus or Lactobacillus can also be used. The codon usage table is obtained from Kazusa DNA Research Institute.
The expression vector pNZ8121 (MoBiTec GmbH, Gottingen, Germany) is a Gram-positive broad host range vector. Taking advantage of the nisA promoter, it allows intracellular recombinant protein expression via induction with nisin. It also allows for recombinant protein secretion using the signal sequence PrtP. The vector pNZ8121 also contains the selectable marker conferring resistance to chloramphenicol. The vector is linearized using EcoRV restriction enzyme (New England Biolabs, Ipswich, Mass.) and dephosphorylated using established molecular scolong methods [1]. Linearized vector is separated using agarose gel electrophoresis. An agarose gel section containing linearized vector is collected and the linearized plasmid is purified from the agarose using a commercially available DNA purification kit, e.g. the QIAquick Gel Extraction Kit (Qiagen, Germantown, Md.).
The gene sequence for chicken myosin regulatory light chain 2, skeletal muscle isoform, (MYLPF) can be obtained from UniProt.org under accession number P02609. The double-stranded DNA is constructed through chemical gene synthesis from either ATUM (Newark, Calif.), Genscript (Piscataway, N.J.), or IDT (Coralville, Iowa). It is supplied in a vector of choice. The DNA sequence can also be obtained via amplification of cDNA generated directly from a mRNA of a biological sample, such as a tissue or a blood sample from a chicken donor. The gene sequence is modified to aid in cloning, gene expression, or enhance production. It is “codon optimized”, i.e. triplet DNA sequences that are not commonly used in the expression host are changed to those that are commonly used.
The codon optimized myosin regulatory light chain 2, skeletal muscle isoform, gene (MYLPF), containing exons, but no introns, is ligated to the linearized and purified pNZ8152 vector via enzymatic ligation to generate a vector capable of being inserted into a host organism. The method used is known in the art and the protocol can be obtained from a molecular cloning manual [1].
The host organism in this example is Lactococcus lactis strain NZ9100 (MoBiTech GmbH). This strain contains genes nisR and nisK, which are needed for nisin-regulated gene expression.
The pNZ8121 vector construct containing the codon optimized myozenin gene is transformed into host strain Lactococcus lactis NZ9100 via electroporation using instrument settings of 2.0 kV, 25 μf, and 200Ω. Electrocompetent cells are obtained via methods known in the art [3]. Chemical transformation or another method can also be used. Successful clones are selected for on agar plates prepared with M17 medium containing 0.5% (w/v) glucose and 10 micrograms/mL chloramphenicol grown at 30° C. Colonies are picked and cultured at 30° C. in M17 medium with 0.5% (w/v) glucose and 10 micrograms/mL chloramphenicol for 48 hours in a non-aerated environment. A successful clone containing a copy of the myosin regulatory light chain 2 gene, skeletal muscle isoform, is confirmed by sequencing for insert identity and orientation using established methods such as PCR [1]. Protein expression using the successful clone is induced by culturing in M17 medium with 0.5% (w/v) glucose at 30° C. in a non-aerated environment until stationary phase at which 1 ng/mL of nisin is supplemented for induction. The induction phase is approximately 4 hours. Cultures are clarified via centrifugation and supernatant is analyzed for protein expression by SDS-PAGE.
Other variants of Lactococcus or Lactobacillus can also be used. The codon usage table is obtained from Kazusa DNA Research Institute.
While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.
[5] K. Kanakubo, A. J. Fascetti and J. A. Larsen, “Assessment of protein and amino acid concentrations and labeling adequacy of commercial vegetarian diets formulated for dogs and cats,” Journal of the American Veterinary Medical Association, vol. 247, no. 4, pp. 385-392, 2015.
salar OX = 8030 GN = LOC106593168 PE = 3 SV = 1
gallus OX = 9031 GN = CAPZA2 PE = 3 SV = 1
Sus scrofa (Pig)
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/798,449, filed on Jan. 29, 2019, which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62798449 | Jan 2019 | US |
