Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4717—Plasma globulins, lactoglobulin
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
  • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
  • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
  • A23C11/06—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing non-milk proteins
  • A23C11/065—Microbial proteins, inactivated yeast or animal proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/18—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from yeasts
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/04—Animal proteins
  • A23J3/08—Dairy proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/04—Animal proteins
  • A23J3/08—Dairy proteins
  • A23J3/10—Casein
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/20—Proteins from microorganisms or unicellular algae
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/30—Artificial sweetening agents
  • A23L27/31—Artificial sweetening agents containing amino acids, nucleotides, peptides or derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
  • C07K14/43—Sweetening agents, e.g. thaumatin, monellin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4732—Casein
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/77—Ovalbumin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
  • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention generally relates to a novel protein composition which can be used in formulating dairy products.
• the present invention relates to a recombinant fusion protein, and a method for producing such recombinant protein, which has a sweet taste, is palatable and can be used as an animal-free dairy substitute/s.
• Milk is considered as a rich source of nutrients and is usually desired to be consumed along with sugars in many of the milk-based beverages and other food items.
• the global population is estimated to exceed 9 billion consumers by 2050 (United countries, 20/3) and so would be the increase in demand for nutrition of which dairy constitutes a major component.
• sugars are added to several milk, milk-based beverages or other food items usually to impart sweet taste and desired aroma. While milk is considered a rich source of nutrients, there is a growing concern worldwide about co-consuming carbohydrate sugars that are concerned to cause obesity, diabetes, caries and/or hyperlipemia. Some of the naturally occurring sugars in natural dairy such as lactose are responsible for intolerances such as lactose intolerance.
• Taste receptors on the tongue, identify perfectly fitting molecules with a particular structure (which is common to carbohydrate sugars, artificial low-calorie sweeteners and sweet-tasting proteins) and send signals to the brain indicating it to identify it as a ‘Sweet’ tasting compound.
• fusion protein in which one or more protein is fused along with one or more sweet-tasting protein using one or more linkers in a chain or in tandem.
• the fusion protein would serve not only as a rich source of protein but also as low-calorie non-carbohydrate sweeteners overcoming the otherwise associated health concerns.
• the present invention is based on the finding that constructing fusion genes with components encoding one or more proteins present in milk along with one or more sweet-tasting proteins that have naturally originated from plants would result in fusion protein molecules that would serve not only as rich source of proteins but also as low- calorie sweet tasting protein thereby overcoming the otherwise associated health concerns posed by alternative sweeteners being used currently.
• the resulting composition thereby not only preserves native nutritive properties of targeted dairybased beverages and food items but also imparts desired taste and/or aroma, stability and solubility, while ensuring to avoid possible health concerns that currently prevail worldwide.
• the milk protein part of the resultant fused protein would serve the same nutritive and functional properties as served by the animal-derived milk protein and also act as a substitute for all of its applications and products derived thereupon. Furthermore, the sweet-tasting protein part of the resultant fused protein would serve to replace/reduce lactose or other sugars in dairy applications and products derived thereupon while increasing the overall protein content of such products.
• the present invention provides a recombinant fusion protein comprising: a) at least a first protein; b) at least a second protein; and c) at least a linker comprising an amino acid sequence coded by the nucleotide sequence which is 80% similar to sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:9, and SEQ ID NO.: 10
• the present invention provides a recombinant fusion protein, comprising: at least a first protein comprising an amino acid sequence having at least 80% sequence identity to a polypeptide as encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3.
• SEQ ID NO: 4 SEQ ID NO:5 SEQ ID NO:6, and SEQ ID NO:7; at least a second protein comprising an amino acid sequence having at least 80% sequence identity to a polypeptide as encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, and SEQ ID NO.: 13; and at least a linker comprising an amino acid sequence coded by the nucleotide sequences which is 80% similar to sequences selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO :16, SEQ ID NO :17, SEQ ID NO : 18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO :21.
• the present invention provides a recombinant fusion protein, comprising: at least a first protein comprising an amino acid sequence having at least 80% sequence identity to a polypeptide as encoded by the nucleotide sequences set forth in SEQ ID NO:1; at least a second protein comprising an amino acid sequence having at least 80% sequence identity to the polypeptide as encoded by the nucleotide sequences set forth in SEQ ID NO:2; and at least a linker comprising an amino acid sequence coded by the nucleotide sequences which is 80% similar to sequences selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO : 16, SEQ ID NO :17, SEQ ID NO :18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO :21.
• the linker is an inert peptide linker.
• the present invention provides a recombinant fusion protein, comprising: a first protein comprising an amino acid sequence having at least 95% sequence identity to the polypeptide as encoded by the nucleotide sequences set forth in SEQ ID NO:1; a second protein comprising an amino acid sequence having at least 95% sequence identity to the polypeptide as encoded by the nucleotide sequences as set forth in SEQ ID NO:2; and a linker comprising an amino acid sequence coded by nucleotide sequence which is at least 80% similar to sequences selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO :16, SEQ ID NO :17, SEQ ID NO : 18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO :21.
• the linker is an inert peptide linker and consists of repetitive sequences of amino acids.
• the first protein is selected from the group comprising of a milk protein, a non-milk bovine protein, and a non-bovine protein.
• the milk protein is a recombinantly expressed protein selected from the group comprising of alpha-lactalbumin, beta-lactoglobulin, kappa-casein, alpha-casein, beta- casein, lactoferrin and the like.
• the milk protein is a recombinant beta-lactoglobulin.
• the second protein is selected from the group comprising of a low-calorie non-carbohydrate sweetener or a non-milk bovine protein or a non-bovine protein of microbial, animal, plant, or recombinant origin.
• the low-calorie sweetener is Brazzein.
• the milk proteins and low-calorie non-carbohydrate sweetener are produced in microbial systems.
• the recombinant fusion protein comprises any combination of the listed one or more first protein and one or more second protein from any origin using one or more linkers.
• the fusion protein comprises any combination of the listed one or more first protein and one or more second protein from any origin, in both orientation, using one or more linkers.
• the fusion protein comprises at least one milk protein; at least one low-calorie sweet tasting protein; and at least one inert peptide linker. In another embodiment, at least one milk protein is fused with the low-calorie non- carbohydrate sweet tasting protein using an inert peptide linker.
• the fusion protein comprises at least one non-milk bovine protein; at least one low-calorie sweet tasting protein; and at least one inert peptide linker.
• At least one non-milk bovine protein is fused with the low- calorie non-carbohydrate sweetener using an inert peptide linker.
• the fusion protein comprises at least one non-milk non-bovine protein; at least one low-calorie sweet tasting protein; and at least one inert peptide linker.
• At least one non-milk non-bovine protein is fused with the low-calorie non-carbohydrate sweetener using an inert peptide linker.
• the fusion protein comprising milk protein betalactoglobulin is fused with Brazzein using a flexible 12 amino acid chain inert linked peptide.
• the inert peptide linker is selected from flexible linkers or rigid linkers or combination thereof.
• the linkers are a combination of one or more flexible and rigid inert linker peptide chains mentioned above.
• first protein and second protein are joined by the inert linker in the reverse orientation.
• the fusion protein comprising milk protein betalactoglobulin is fused with Brazzein using a rigid 17 amino acid chain inert peptide linker
• a recombinant fusion protein comprising at least one first protein; at least one second protein; and at least one inert peptide linker, wherein the at least one first protein is fused with the second protein using an inert peptide linker.
• nucleic acid molecule encoding the recombinant fusion protein described herein.
• an expression vector comprising the nucleic acid sequence encoding the recombinant fusion protein described herein.
• the milk proteins and sweet tasting proteins are produced in the microbial systems.
• a bovine dairy protein or non-bovine protein are conjugated to a sweet tasting protein via an inert linker.
• composition comprising: a) at least one recombinant fusion protein in the range of 0.1% to 8%; b) at least one non-fusion protein in the range of 1% to 10%; and c) at least one additive in the range of 0.1% to 5%, wherein, at least one recombinant fusion protein comprises a first protein, a second protein, and a linker having an amino acid sequence coded by nucleotide sequence which is 80% similar to sequences selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO : 16, SEQ ID NO :17, SEQ ID NO :18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO :21.
• the at least one non-fusion protein is selected from the group consisting of microbial, animal, plant origin or recombinantly expressed protein.
• the recombinant protein is mixed with at least one additive selected from a group consisting of carbohydrates, lipids, fats, ash, micronutrients, vitamins, and the like; in order to obtain a dairy alternative product with a similar mouth feel.
• the fats are of plant origin, microbially derived, fermentation derived or similar.
• plant origin oils are selected from a group consisting of Sunflower oil, corn oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, rice bran oil, cotton seed oil, olive oil, canola oil, flaxseed oil, coconut oil, algal oil, and microbial produced fats.
• the fats are derived from fermentation of plant origin oil substrates using microbes.
• the recombinant composition comprises a fusion protein in which three proteins are linked with two linkers.
• the recombinant fusion protein or the composition is used in formulating dairy and related products.
• the recombinant fusion protein is mixed with plant-based fats, ash, and water to obtain a dairy alternative beverage with a palatable taste.
• the recombinant fusion protein can be used in other dairy products such as Ice-creams, frozen desserts, cakes, coffee creamer.
• the recombinant fusion protein can be used in but not restricted to vegan protein supplements.
• a method for producing the recombinant fusion protein comprising fermenting a recombinant host cell capable of producing said recombinant fusion protein.
• Figures 1A and IB illustrates a fusion of a representative protein A and protein B using a representative peptide linkage C in accordance with one embodiment of the present invention
• Figure 2 is a vector map of a integrative vector used for production of fusion proteins provided herein, in accordance with representative embodiments of the present invention
• FIGS 3A-3D is an example of various gene cassette representation, in accordance with representative embodiments of the present invention.
• Figure 4 is a gel image for expression of recombinant fusion protein Beta lactoglobulin-Ll-Brazzein, Brazzein-Ll- beta lactoglobulin, Beta lactoglobulin - L2- Brazzein, Brazzein-L2- beta lactoglobulin; and
• Figure 5 is a gel image for expression of recombinant fusion protein Beta lactoglobulin-L3-Brazzein, Beta lactoglobulin - L4- Brazzein.
• the present invention is focused on creation of fused proteins using selected proteins and/or low-calorie sweet tasting proteins by using an inert peptide linker to achieve resultant protein with sweetness, stability and solubility, while ensuring to avoid possible health concerns that currently prevail worldwide.
• Taste receptors on the tongue identify perfectly fitting molecules with a particular structure (common to carbohydrate sugars, artificial low-calorie sweeteners and sweet-tasting proteins) and send signals to the brain indicating it to be sweet in taste.
• the sweet-tasting proteins are usually ⁇ 50 to 200 amino acids in length possessing molecular weight ⁇ 6.5 to 30 Kda (Zhao et al, 2021).
• a recombinant fusion protein comprising at least one first protein; at least one second protein; and at least one inert peptide linker, wherein the at least one first protein is fused with the second protein using an inert peptide linker.
• the disclosure also provides methods for producing the recombinant fusion protein and compositions comprising the same.
• Milk is composed of 2 major classes of proteins - caseins (aSl-casein, aS2-casein, 0- casein and K-casein) and whey proteins (0 -lactoglobulin and a-lactalbumin).
• Caseins are known to interact with each other to give rise to sub-micelles and micelles that play a crucial role in stability and nutritious properties of milk. While the aS 1 -casein, aS2-casein and 0-casein molecules remain embedded inside, K-casein protrudes partially out of the micellar structure. Whey proteins remain suspended and do not participate in formation of micelles.
• milk protein is any protein, or fragment or variant thereof, that is typically found in one or more mammalian milk.
• the milk proteins described herein are casein proteins, such as kappa casein, para - kappa - casein, beta - casein, alpha - SI - casein, and alpha - S2 - casein.
• a protein composition comprising fusion protein for formulating dairy and related product is disclosed. Accordingly, the present invention in one embodiment is focused on creation of fused proteins using selected proteins and/ or a low-calorie sweet tasting protein by using an inert peptide linker to achieve one or more of the goals of resultant protein such as sweetness, stability and solubility.
• fusion genes with components encoding one or more proteins present in milk along with one or more sweet-tasting proteins that have naturally originated from plants would result in fusion protein molecules that would then serve not only as rich source of proteins but also as low-calorie sweet tasting protein thereby overcoming the otherwise associated health concerns posed by alternative sweeteners being used currently.
• composition thereby not only preserves native nutritive properties of targeted milk-based beverages and food items but also imparting desired taste and/or aroma, stability and solubility, while ensuring to avoid possible health concerns that currently prevail worldwide.
• the milk protein part of the resultant fused protein would serve the same nutritive and functional properties as served by the animal-derived milk protein and also act as an apt substitute for all of its applications and products derived thereupon. Furthermore, the sweet-tasting protein part of the resultant fused protein would serve to replace/reduce lactose or other sugars while increasing the overall protein content of such products.
• the present invention discloses a protein composition comprising a fusion protein in which two proteins are linked with one linker.
• the composition comprises a fusion protein in which two proteins are linked with one linker.
• the composition comprises a fusion protein in which three proteins are linked with two linkers.
• the fusion protein comprises any combination of the listed one or more first protein and one or more second protein from any origin using one or more linker.
• Constructing fusion genes with components encoding one or more proteins present in milk along with one or more sweet-tasting proteins that have naturally originated from plants results into the fusion protein molecules. It involves use of an inert peptide linker with desired level of flexibility and stability required during translation and also acts as a bridge connecting the two resulting proteins synthesized as a fusion protein.
• the inert peptide linker comprises at least four amino acids that impart one or more of the following: A) flexibility, or B) Rigidity, or C) Improve solubility, or D) Increase stability of the fused protein.
• the composition thereby not only preserves native nutritive properties of targeted milk-based beverages and food items but also imparting desired taste and/or aroma, stability and solubility, while ensuring to avoid possible health concerns that currently prevail worldwide.
• the milk protein part of the resultant fused protein would serve the same nutritive and functional properties as served by the animal-derived milk protein and also act as an apt substitute for all of its applications and products derived thereupon.
• the sweet-tasting protein part of the resultant fused protein would serve to replace/reduce lactose or other sugars while increasing the overall protein content of such products.
• a composition comprising a fusion protein comprising milk protein Beta-lactoglobulin fused with Brazzein using a flexible 12 amino acid chain inert peptide linker
• the accompanying Figure 1A and Figure IB illustrates fusion of a representative protein A and protein B using a representative inert peptide linkage in accordance with one embodiment of the present invention.
• the protein A can be a milk protein or non-milk bovine protein or non-bovine protein of microbial, animal, plant origin or recombinant protein.
• the protein B is a low-calorie non-carbohydrate sweetener or milk protein or non-milk bovine protein or non-bovine protein of microbial, animal, plant origin or recombinant protein.
• FIG. 2 illustrates the example of vector used for driving the expression.
• the genes were cloned in pPICZA, integrative vector which contain Pichia pastoris alcohol oxidase promoter, alcohol oxidase terminator, zeocin resistance gene.
• Integrative vector containing methanol inducible promoter A0X1, FLD1, M0X1, DAS1 etc
• alpha mating signal factor zeocin resistance cassette
• terminator illustrated in Figure 2
• Further example of vector used herein is integrative vector containing constitutive promoter, alpha mating signal factor, zeocin resistance cassette, terminator. Similar to the above, wherein zeocin is replaced with kanamycin resistance. Similarly, alpha mating factor is replaced by any signal factor driving the expression to extracellular fraction.
• the recombinant molecule in the above embodiments can be expressed in microbial systems used for recombinant molecules such as Bacteria - Escherichia coli, Bacillus subtilis, Cyanobacteria etc. Examples of some of the Fungal expression hosts - Aspergillus niger, Aspergillus nidulans, Trichoderma reesei, Pencillum sps.
• yeasts - Saccharomyces cerevisiae Candida albicans, Kluyveromyces lactis, Kluyveromyces marxianus,Yarrowia sps etc and methylotrophic yeast - Pichia pastoris, Hansenula polymorpha, Candida boidini.
• Transformation protocols like chemical transformation using Lithium acetate and LiCl, transfection, electroporation, PEG mediated, agrobacterium mediated transformation etc can be used. Proteins of interest:
• Milk proteins -Some examples of milk proteins used in the present invention includes Kappa casein, alpha- SI- casein, alpha- S2- casein, Beta- casein, beta-lactoglobulin, alpha lactalbumin, transferrin
• Bovine beta lactoglobulin is a major whey protein of bovine milk which is a part of the few embodiments of the invention.
• the sequence of the gene is as given below.
• Alpha lactalbumin is a whey protein of bovine milk which is a part of the few embodiments of the invention.
• the sequence of the gene is as given below
• Kappa casein is a major casein of the bovine milk protein which is involved in the stabilization of the milk micelles. It is a part of some embodiments of the invention.
• Alpha SI casein is another major casein and is a major component of bovine milk. It is a part of some of the embodiments of the invention.
• Alpha S2 casein is a casein protein and is a component of bovine milk. It is a part of some of the embodiments of the invention.
• Beta casein is a major casein protein and is a component of bovine milk. It is a part of some of the embodiments of the invention.
• Ovalbumin is the main protein found in egg white, making up approximately 55% of the total protein. It is a part of some of the embodiments of the invention.
• Low-calorie non-carbohydrate sweetener examples include Brazzein, Thaumatin, Monellin, Mabinlin, Miraculin, Curculin, Neoculin and Pentadin and like. Brazzein is used herein as low-calorie noncarbohydrate sweetener. Brazzein is a sweet tasting protein from the plant species Pentadiplandra brazzeana and is about 2000 times sweeter than the sucrose on a molar basis. It is found in two forms in nature. The major form starts with a pyroglutamic acid residue and is 54 amino acids long and the minor form starts with the next amino acid Aspartic acid and 53 amino acids long. The minor form was reported to have better sweet tasting capacity therefore, the minor form is selected from the uniprot ID P56552 (sequence is given below), codon optimized for Pichia pastoris and synthesized.
• Brazzein is a sweet tasting protein found in the extracts of the plant Pentadiplandra brazzeana. It tastes 2000 times sweeter than sucrose on a molar basis. It is a part of some of the embodiments of the invention
• Thaumatin is a sweet protein found in the fruits of Thaumatococcus daniellii. It is 100000 times sweeter than sucrose on a molar basis. It is a part of some of the embodiments of the invention
• CTGTTCCTCCACCGTAG [SEQ ID NO: 10]
• Linkers are peptide molecules which are used to link two protein molecules to improve solubility, stability and/or expression. Drugs like Enbrel®, Ontak®, Orencia®, Amevive®, Arcalyst®, and Nplate® contain multiple proteins connected via linkers and have been approved by the FDA. Linkers can be classified as flexible linkers or rigid linkers (Chen et al., 2013; Smith et al. 2003).
• Flexible linkers are predominantly composed of small sized amino acids like Glycine, Alanine and/or Serine which form a disordered loop thus providing relatively higher freedom for folding (Argos, 1990).
• “LI Type Linker” used herein is composed of amino acids (SGG)nSAG [corresponding nucleotide sequence is SEQ ID NO: 14].
• a noted limitation of flexible linkers is the lack of rigidity.
• There are examples in literature where use of flexible linkers yielded relatively lower yield / reduced biological activity (Amet et al., 2009; Maeda et al., 1997). Many alpha helix forming linkers have been used in literature to construct recombinant fusion proteins (Amet et al., 2009; Bai and Shen, 2006).
• “L2 Type Linker” used herein is composed of amino acidsA(EAAAK)nA [corresponding nucleotide sequence is SEQ ID NO: 15].
• L3 Type Linker used herein is composed of amino acids KKKR(SGG) n SAGKREAEA [corresponding nucleotide sequence is SEQ ID NO: 16] and “L4 Type Linker” used herein is composed of amino acids (KKKR)n(EAEA) n [corresponding nucleotide sequence is SEQ ID NO: 17],
• linkers they consist of repetitive sequences of amino acids. This particular repetitive sequence can be repeated “n” number of times to alter the length and property of the linker sequence as per the requirement, wherein “n” could range from 1 to 10. Optimal number of repeats is essential to maintain the functionality and stability of the protein. If the proteins are too close it might affect the 3D structure and hence the functionality. If they are too far, stability of the recombinant molecule is affected.
• Brazzein protein has four intra-disulfide bridges Cys4-Cys52, Cysl6-Cys37, Cys22-Cys47, and Cys26-Cys49. These intra-disulfide bridges make brazzein as the most thermostable sweet protein.
• Vectors The genes were cloned in pPICZA, integrative vectors which contain Pichia pastoris alcohol oxidase promoter, Alcohol oxidase terminator, Zeocin resistance gene.
• Alpha mating signal factor - Yeast alpha mating factor s pro region is a secretory signal factor which has been extensively used in expression of recombinant proteins in extracellular fractions. In few of the embodiments the signal factor which has been optimized for extracellular secretion in Pichia pastoris has been used.
• the sequence is provided below: SEQ ID NO:22
• Human serum albumin signal factor is another commonly used signal peptide for protein expression in Pichia pastoris.
• the signal factor sequence is provided below SEQ ID NO:23
• FIG. 3A-3D illustrates the various gene cassette representations, in accordance with an embodiment of the present invention.
• Figure 3 A illustrates a gene cassette representation in which A0X1 is used as a vector and alpha mating signal factor is a signal peptide used in expression of recombinant proteins.
• the Beta lactoglobulin (Protein A) is linked with brazzein (Protein B) via “LI type linker” and vice versa.
• the “LI type linker” can be replaced by “L2 type linker” in same gene cassette.
• Figure 3 B illustrates a gene cassette representation in which A0X1 is used as a vector and alpha mating signal factor is a signal peptide used in expression of recombinant proteins.
• the Beta casein (Protein A) is linked with brazzein (Protein B) via “LI type linker “and vice versa.
• the “LI type linker” can be replaced by “L2 type linker” in same gene cassette.
• FIG 3 C illustrates a gene cassette representation in which A0X1 is used as a vector and Human Serum Albumin Signal Factor (HAS-SP) is a signal peptide used in expression of recombinant proteins.
• HAS-SP Human Serum Albumin Signal Factor
• the Beta lactoglobulin (Protein A) is linked with brazzein (Protein B) via “L 1 type linker” and vice versa.
• the “L 1 type linker” can be replaced by “L2 type linker” in same gene cassette.
• Figure 3 D illustrates a gene cassette representation in which A0X1 is used as a vector and Human Serum Albumin Signal Factor (HAS-SP) is a signal peptide used in expression of recombinant proteins.
• HAS-SP Human Serum Albumin Signal Factor
• the Beta casein (Protein A) is linked with brazzein (Protein B) via “LI type linker “and vice versa.
• the “LI type linker” can be replaced by “L2 type linker” in the same gene cassette.
• Figure 5 shows the gel image for expression of Beta lactoglobulin-L3 -Brazzein, Beta lactoglobulin - L4- Brazzein, in accordance with an embodiment of the present invention.
• Lanel is Blank
• Lane 2 is Blank
• Lane 3 is -Blank
• Lane 4 is of Milk protein standard
• Lane 5 is of Beta lactoglobulin-L3- Brazzein
• Beta lactoglobulin-L4- Brazzein is of Beta lactoglobulin-L4- Brazzein.
• compositions can be prepared using the recombinant fusion protein molecule.
• the non-limiting examples of the compositions are listed as-
• Milk protein is linked to sweet proteins and combined with fats and ash, wherein the ash comprises essential minerals.
• Non-milk protein is linked to sweet tasting proteins and combined with fats and ash, wherein the ash comprises essential minerals.
• Milk protein and non-milk protein are linked to sweet tasting proteins and combined with fats and ash, wherein the ash comprises essential minerals.
• Milk protein is linked to another milk protein.
• Milk protein is linked to non-milk protein.
• Non-milk protein is linked to non-milk protein.
• proteins are used in the protein composition of the present invention.
• the non-limiting examples of the proteins are listed as:
• the following fats are used in the composition of present invention.
• the non-limiting examples of the fats are listed as:
• Plant based oils such as Sunflower oil, corn oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, rice bran oil, cotton seed oil, olive oil, canola oil, flaxseed oil, coconut oil; algal oil, microbial produced fats and fats derived from microbial fermentation of plant-based oil substrates.
• edible ash is used in the composition of the present invention.
• the non-limiting examples of minerals are Calcium, Phosphorus and magnesium.
• vectors are used in the protein composition of present invention.
• the non-limiting examples of the vectors are listed as:
• the recombinant molecule in the above embodiments can be expressed in microbial systems used for recombinant molecules such as
• Methylotrophic yeast - Pichia pastoris Methylotrophic yeast - Pichia pastoris, Elansenula polymorpha, Candida boidini TRANSFORMATION
• Transformation protocols like chemical transformation using Lithium acetate and LiCl, transfection, electroporation, PEG mediated, agrobacterium mediated transformation and like can be used.
• the gene of interest along with the chosen signal peptide was synthesized.
• the two genes were fused together using linker by overlapping PCR.
• the sequence for the linker was designed within the overlapping PCR primers.
• the fused construct was cloned at the EcoRI and Notl site of the pPICZA and transformed into E. coli NEB5 competent cells through the standard protocols. Transformants were selected on 25pg/ml zeocin LB agar plates. The transformants were confirmed through restriction digestion and sequencing. Host organisms
• Pichia pastoris strain a derivative of BG 16 strain designated and referred to herein as PX-1.
• the plasmids selected for Pichia transformation were isolated in large amounts through midi-prep. 40pg of the plasmid DNA was linearized with Sacl/Pmel enzyme and 5-10pg of linearized vector was used to transform the above PX-1 strain through electroporation.
• the transformation protocol is briefly as follows. 100ml YPD medium was inoculated with 20-50ul inoculum of the PX-1 strain and grown overnight to an O.D of 1-1.3. The cells were harvested and washed twice with cold sterile milliQ water. In the next step they were washed with 2ml of IM sorbitol and finally resuspended in 200pl of 1 M sorbitol.
• the cells were placed on ice and used the same day for electroporation. 80pl of the competent cells were mixed with 5-10pg of linearized DNA and electroporated using preset conditions for Pichia pastoris electroporation in Biorad electroporator. Immediately after electroporation 1ml of IM sorbitol was added and incubated for an hour at 30°C. Following that 1ml YPD was added to the culture and further incubated for 2-3 hours at 30°C while shaking. The colonies were selected on YPD-zeocin plates of varying concentration (100- 2000pg/ml). Integrated positive colonies were confirmed through colony PCR.
• BMGY media 1% yeast extract, 2% peptone, 100 mM potassium phosphate at pH 6.0, 1.34% Yeast Nitrogen Base, 4 x 10-5% biotin 1% glycerol
• BMMY 1% yeast extract, 2% peptone, 100 mM potassium phosphate at pH 6.0, 1.34% YNB, 4 x 10-5% biotin, 0.5% methanol
• Glycerol stock of the strain was inoculated into 50 mL of YPD broth in a 250 mL of baffled flask. This inoculum was then used to inoculate 100 ml of GYP (Yeast extract 1%, Peptone 2% and Glycerol 2%) as adaptation stage. This was then inoculated in 250 ml of BSM (Glycerol 4%, Orthophosphoric acid 2.7%, K2SO41.8%, MgSO4.7H2O 1.4%, KOH 0.413%, CaSO4.2H2O 0.116%). The culture was then used to inoculate a 8L ferementor containing BSM media, maintaining pH at 5 for growth phase and 6 for induction phase. Temperature was maintained at 30 for growth phase and 24 for induction phase.
• the culture supernatant was initially processed through microfiltration to remove cells and other particulate matter.
• the permeate was then concentrated usingultrafiltration/diafiltration followed by freeze drying to obtain powdered protein.
• the gene of interest along with the chosen signal peptide was synthesized.
• the two genes were fused together using linker by overlapping PCR.
• the sequence for the linker was designed within the overlapping PCR primers.
• the fused construct was cloned at the EcoRI and Notl site of the pPICZA and transformed into E. coli NEB5 competent cells through the standard protocols. Transformants were selected on 25pg/ml zeocin LB agar plates. The transformants were confirmed through restriction digestion and sequencing.
• Pichia pastoris strain -A derivative strain of BG16 procured from Atum Biosciences.
• the plasmids selected for Pichia transformation were isolated in large amounts through midi prep protocol. 40pg of the plasmid DNA was linearized with Sacl/Pmel enzyme and 5-1 Opg of linearized vector was used to transform the above PPS-9016 strain through electroporation.
• the transformation protocol is briefly as follows. 100ml YPD medium was inoculated with 20-50ul inoculum of the PX-1 strain and grown overnight to an O.D of 1-1.3. The cells were harvested and washed twice with cold sterile milliQ water. In the next step they were washed with 2ml of IM sorbitol and finally resuspended in 200 pl of 1 M sorbitol.
• the cells were placed on ice and used the same day for electroporation. 80pl of the competent cells were mixed with 5-10pg of linearized DNA and electroporated using present conditions for Pichia pastoris electroporation in Biorad electroporator. Immediately after electroporation 1ml of IM sorbitol was added and incubated for an hour at 30°C. Following that 1ml YPD was added to the culture and further incubated for 2-3 hours at 30°C while shaking. The colonies were selected on YPD-zeocin plates of varying concentration (100-2000pg/ml). Integrated positive colonies were confirmed through colony PCR.
• BMGY 1% yeast extract, 2% peptone, 100 mM potassium phosphate at pH 6.0, 1.34% Yeast Nitrogen Base, 4 x 10-5% biotin 1% glycerol
• BMMY 1% yeast extract, 2% peptone, 100 mM potassium phosphate at pH 6.0, 1.34% YNB, 4 x 10-5% biotin, 0.5% methanol
• O.D O.D of 1-2 for induction.
• Samples were collected at 24, 48 and 72 hrs and were visualized on the SDS page for expression. Positive samples were then serially scaled to 500ml flask, 1ml flask and 8L fermenter.
• Glycerol stock of the strain was inoculated into 50 mL of YPD broth in a 250 mL of baffled flask. This inoculum was then used to inoculate 100 ml of GYP (Yeast extract 1%, Peptone 2% and Glycerol 2%) as adaptation stage. This was then inoculated in 250 ml of BSM (Glycerol 4%, Orthophosphoric acid 2.7%, K2SO41.8%, MgSO4.7H2O 1.4%, KOH 0.413%, CaSO4.2H2O 0.116%). The culture was then used to inoculate an 8L fermenter containing BSM media, maintaining pH at 5 for growth phase and 6 for induction phase. Temperature was maintained at 30 °C for growth phase and 24 °C for induction phase.
• the culture supernatant was initially processed through microfiltration to remove cells and other particulate matter.
• the permeate was then concentrated using ultrafiltration/ diafiltration and freeze dried to obtain protein powder.

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