Purification of a protein

Abstract

The present invention relates to field of proteins, specifically purification of a protein from an aqueous composition by precipitation of the protein.

Description

[0001] Purification of a protein

[0002] Field of the invention

[0003] The present invention relates to field of proteins, specifically purification of a protein from an aqueous composition by precipitation of the protein.

[0004] Background of the invention

[0005] Purification of a protein from an aqueous composition by precipitation of the protein from the aqueous composition has been known in art for a long time. In the art, it is known that several salts can be used to precipitate a protein from an aqueous composition, such as ammonium sulphate, calcium chloride and phosphate salts such as sodium hexametaphosphate (Nae[(PO3)6], E-number E452). It is still a challenge to obtain the protein of interest as pure as possible with a highest yield and / or purity as possible. As an example, proteins produced by precision fermentation need to be separated from the medium components, from the cells that produce the protein, from cell debris and other contaminants. The general term for this process is down-stream processing. Apart from the challenge of obtaining the protein of interest as pure as possible with a highest yield and / or purity as possible, it is desired to limit the amount of down-stream processing steps as much as possible to reduce costs since down-stream processing steps like chromatography are expensive and complex methods. Hoppenreijs et al (Food Research International 176 (2024) 113801) uses sodium hexametaphosphate to purify proteins from yeast precision fermentation. When applied to p-lactoglobulin, a purity of 72% could be achieved. In this purification, the sodium hexametaphosphate is added instantaneously, followed by an incubation for 1 hour.

[0006] Altogether, there remains a need to improve the process of purification of a protein from an aqueous composition.

[0007] Summary of the invention

[0008] The invention relates to a method for the purification of a protein of interest from an aqueous composition comprising a precipitation step using a hexametaphosphate salt, wherein the hexametaphosphate salt is added to the aqueous composition comprising the protein of interest in a time frame of at least 10 minutes.

[0009] The invention further relates to a method for the production of a recombinant protein of interest comprising: production of the protein of interest in a microbial cell, purifying the protein of interest, wherein the purification comprises a method as set forth herein.

[0010] The invention further relates to a protein of interest obtainable by a method as set forth herein.

[0011] The invention further relates to a food product, including a beverage, comprising a protein of interest obtainable by or obtained by a method as set forth herein.

[0012] Description of the invention

[0013] The inventors noticed that when purifying a protein from an aqueous composition by using the state of the art process of precipitation with sodium hexametaphosphate (see e.g. Hidalgo et al, 1973, Hoppenreijs et al, 2004), that the yield and purity were fairly low; only about 70% of the protein originally present in the composition was recovered and a purity of about 70% was achieved. The inventors have solved this problem by improving the overall yield and / or purity of the purified protein. The inventors have established that when purifying a protein of interest from an aqueous composition comprising a precipitation step using a hexametaphosphate salt, the purity of the protein is significantly increased when the hexametaphosphate salt is not added instantaneously but is slowly added to the aqueous composition. The inventors have further established that while the yield increases when slowly adding the hexametaphosphate salt as compared to instantaneously adding the hexametaphosphate salt, an additional incubation after the hexametaphosphate salt has been added, does not result in a higher purity of the protein of interest (data not shown).

[0014] Accordingly, in a first aspect, there is provided for a method for the purification of a protein of interest from an aqueous composition comprising a precipitation step using a hexametaphosphate salt, wherein the hexametaphosphate salt is added to the aqueous composition comprising the protein of interest in a time frame of at least 10 minutes. He person skilled in the art will understand that during addition of the hexametaphosphate salt, the aqueous composition is mixed, e.g. by stirring.

[0015] In the embodiments of the invention, the terms yield and purity are used. Herein, yield means the percentage of protein of interest that is recovered during purification in view of the amount of protein of interest before the process of purification. Herein, purity means the percentage of protein of interest present in the composition after purification in view of the total amount of compounds present in said composition. Other compounds may be, but are not limited to Extracellular Polymeric Substances (EPS), such as polysaccharides, proteins, DNA, and lipids, In examples 1 to 4, the term yield is inadvertently used while the term purity was intended and is appropriate.

[0016] In the embodiments herein, the hexametaphosphate salt may added in any time frame longer than 10 minutes, such as in a time frame of at least 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 1.5 hour, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours or 24 hours. Preferably, the time frame is at most 48 hours, such 36 hours, 30 hours, 24 hours, or 12 hours. Most preferably, the hexametaphosphate salt is added in a time frame of about 4 hours. While it was demonstrated that an additional incubation of the mixture after the hexametaphosphate salt has been added, does not result in a higher purity of the protein of interest, it is preferred in the embodiments herein that after addition of the hexametaphosphate salt, no additional incubation of the mixture is performed,

[0017] In the embodiments herein, the hexametaphosphate salt may be any hexametaphosphate salt known to the person skilled in the art. The person skilled in the art knows that hexametaphosphate salt typically may be a mixture of metaphosphates (empirical formula: NaPCh), of which the hexamer is one, and is usually the compound referred to by this name. Such a mixture may be more correctly termed a polymetaphosphate. A preferred hexametaphosphate salt is sodium hexametaphosphate. In the embodiments herein hexametaphosphate salt may or may not comprise other compounds such as (traces of) other salts.

[0018] In the embodiments herein, the hexametaphosphate salt may be present in a solution which is added to the aqueous composition comprising the protein of interest. In the embodiments herein, the concentration of the hexametaphosphate salt in the solution may be between 5% w / v and 75% w / v, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 20%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% w / v, preferably about 60% w / v, such as 58%, 59%, 60%, 61%, or 62% w / v.

[0019] In the embodiments herein, the pH of the hexametaphosphate salt solution before it is added to the composition comprising the protein of interest may be any pH within a useful range. Preferably, the pH is between pH 3 and pH 5, such as pH 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3,

[0020] 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, more preferably, the pH is 4, even more preferably, the pH is 4.0.

[0021] In the embodiments herein, the method is preferably performed in a batch system wherein the composition comprising the protein of interest is present in a vessel and the hexametaphosphate salt is added to the vessel. The person skilled in the art will comprehend that other embodiments are possible, such as e.g. a fed batch system or a continuous system or a system wherein the hexametaphosphate salt is present in a vessel and the composition comprising the protein of interest is added to said vessel.

[0022] In the embodiments herein, the protein of interest in the aqueous composition may be present in a concentration of between 1 to 100 gram I L composition, preferably between 5 to 75 gram I L composition, more preferably between 10 to 50 gram I L composition, even more preferably between 5 to 25 gram / L composition, such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21 , 22, 23, 24, 25 gram I L composition, most preferably gram I L composition.

[0023] In the embodiments herein, the volume of the aqueous composition comprising the protein of interest may have any volume, such as, but not limited to 1 litre (L), 2 litre, 3litre, 4 litre, 5 litre, 10 litre, 20 litre, 30 litre, 40 litre, 50 litre, 100 litre, 250 litre, 500 litre, 1000 litre, 5000 litre, 10.000 litre,, 20.000 litre,, 50.000 litre, 100.000 litre, or 200.000 litre. In the embodiments herein, the volume of the aqueous composition comprising the protein of interest may be at least 10 litre. In the embodiments herein, the volume of the aqueous composition comprising the protein of interest may be between 1000 and 100,000 litre, such as 5000 litre, 10.000 litre, 25.000 litre, or 50.000 litre.

[0024] In the embodiments herein, preferably, the pH of the aqueous composition comprising the protein of interest is or is brought at between pH 3 and pH 5, such as pH 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, preferably pH 4, before the hexametaphosphate salt is added. A more preferred pH is pH 4.0.

[0025] In the embodiments herein, the pH in the vessel wherein hexametaphosphate salt is added to the aqueous composition is preferably kept at between pH 3 and pH 5, such as pH 3.0, 3.1 , 3.2, 3.3, 3.4,

[0026] 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, preferably at pH 4. A more preferred pH is pH 4.0. In the embodiments herein, the molar ratio between the hexametaphosphate salt and the protein of interest is the final molar ratio, i.e. the molar ratio that is reached when all hexametaphosphate salt has been added. Preferably, the molar ratio between the hexametaphosphate salt and the protein of interest is between 2 : 1 and 10: 1 , such as 2 : 1 , 3 : 1 , 4 : 1 , 5 : 1 , 6 : 1 , 7 : 1 , 8 : 1 , 9 : 1 , or 10 : 1 , more preferably, the molar ratio between the hexametaphosphate salt and the protein of interest is 5 : 1 .

[0027] In the embodiments herein, preferably at least 90%, such as 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most preferably at least 99% of the precipitated particles has a diameter that is at least 3 pm. In the embodiments herein, preferably at least 80% of the precipitated particles has a diameter that is ranged between 3 and 20 pm and / or at least 60% has a diameter that is ranged between 5 and 10 pm. In the embodiments herein, in the method for the purification of the protein of interest preferably at least 80%, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, preferably at least 95% of the protein of interest is precipitated in the precipitation step using the hexametaphosphate salt.

[0028] In the embodiments herein, the purity of the protein of interest after the method has been performed is preferably at least 80%, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, more preferably at least 95%. In the embodiments herein, the method may further comprise a step selected from the group consisting of: solid / liquid separation; filtration, microfiltration, ultrafiltration, diafiltration and / or sterile filtration; centrifugation; chromatography; and, drying.

[0029] In the embodiments herein, the method preferably does not comprise a chromatography step before the step wherein the protein of interest is precipitated by the hexametaphosphate salt.

[0030] In the embodiments herein, the method preferably does not comprise any chromatography step.

[0031] In the embodiments herein, the hexametaphosphate salt may or may not be removed by precipitation using an ionic metal salt, such as calcium chloride.

[0032] In the embodiments herein, the protein of interest may be any protein of interest, such as an enzyme and / or a dairy protein, such as p-lactoglobulin, lactoferrin, a-lactalbumin, or a mixture of two or more proteins, preferably comprising at least p-lactoglobulin, lactoferrin and / or a-lactalbumin.

[0033] A p-lactoglobulin protein is a major whey protein and can be a p-lactoglobulin protein from any mammalian species, e.g., an ape, baboons, bear, buffalo, camel, cat, chimpanzee, cow, dog, donkey, echidna, elephant, fox, gibbons, goat, gorilla, guinea pig, horse, human, lemur, lion, macaque, mandrill, monkey, mountain goat, mouse, opossum, orangutan, panda, pig, rabbit, rat, sheep, squirrel, tiger, wallaby, whale, wolf, or woolly mammoth p-lactoglobulin protein. A p-lactoglobulin protein can also be a protein that is at least 70% identical to a wildtype p-lactoglobulin protein. A nucleic acid encoding a p-lactoglobulin protein can encode a protein that is at least 70% identical to a wildtype p-lactoglobulin protein. p-Lactoglobulin is commonly used in food products for its nutritional and functional properties. A lactoferrin protein is an iron-binding glycoprotein present in various secretory fluids including milk, with antimicrobial, antiviral, and immune-modulating properties and can be a lactoferrin protein from any mammalian species, e.g., an ape, baboons, bear, buffalo, camel, cat, chimpanzee, cow, dog, donkey, echidna, elephant, fox, gibbons, goat, gorilla, guinea pig, horse, human, lemur, lion, macaque, mandrill, monkey, mountain goat, mouse, opossum, orangutan, panda, pig, rabbit, rat, sheep, squirrel, tiger, wallaby, whale, wolf, or woolly mammoth lactoferrin protein. A lactoferrin protein can also be a protein that is at least 70% identical to a wildtype lactoferrin protein. A nucleic acid encoding a lactoferrin protein can encode a protein that is at least 70% identical to a wildtype lactoferrin protein, p-lactoglobulin is the major whey protein in the milk of many mammals. In bovine milk it accounts for approximately 10 - 15% of total milk proteins and about 50 - 54% of whey protein. Bovine p-lactoglobulin is expressed as a precursor protein comprising a 16 amino acid N-terminal signal peptide (referred to herein and elsewhere as the "full- length" p-lactoglobulin protein), which is cleaved to form a mature 162 amino acid protein. There are two primary variants of bovine p-lactoglobulin - variants A and B and a less common variant - variant C. Sequences for both the mature and full-length forms of bovine p- lactoglobulin variants A, B and C, and wild type full length and mature forms of p-lactoglobulin from other species are presented in Table 1 .

[0034] In the embodiments herein, the protein of interest may be recombinant protein, such as a recombinant p-lactoglobulin, lactoferrin and / or a-lactalbumin.

[0035] In the embodiments herein, the term "recombinant p-lactoglobulin" refers to a recombinantly produced polypeptide that comprises a sequence of at least 75 or at least 100 or at least 125 amino acids that is at least 50% or at least 75% or at least 90% identical to a sequence of amino acids in a native p-lactoglobulin, such as a native Bos taurus p-lactoglobulin [e.g., amino acids 17 to 178 of UniProt sequence P02754], a native Ovis aries p-lactoglobulin [e.g., amino acids 19 to 180 of UniProt sequence P67976], a native Capra hircus p-lactoglobulin [e.g., amino acids 19 to 180 of UniProt sequence P02756], a native Equus caballus p-lactoglobulin [e.g. amino acids 19-180 of UniProt sequence P02758], a native Equus asinus p-lactoglobulin [UniProt sequence P13613], a native Equus caballus p-lactoglobulin [amino acids 19 to 181 of UniProt sequence P07380], a native Equus asinus p-lactoglobulin [UniProt sequence PI 9647]).

[0036] Preferred p-lactoglobulins include proteins comprising an amino acid sequence having at least about 70% sequence identity to the sequence of a wild type (native) p-lactoglobulin (either full length or mature p-lactoglobulin lacking a signal sequence, but preferably the mature sequence), but particularly any wild type bovine, ovine, caprine, buffalo, equine, donkey or reindeer p-lactoglobulin sequence, including any sequence of SEQ ID NO: in Table 1 . In some embodiments the amino acid sequence of such variants may comprise a truncation or an elongation at the N-terminus and / or the C-terminus relative to the wildtype sequence, for example, truncations or elongations of from about 1 to about 20 amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, variants or modified p-lactoglobulin proteins may contain from 1 to 20 amino acid insertions, deletions, and / or substitutions (collectively) with respect to the wild-type sequence. Such proteins may be referred to herein as "elongated p-lactoglobulin proteins". In some embodiments the variants or modified p- lactoglobulin proteins may comprise one or more post-translational modifications that differ to a wild type p-lactoglobulin protein, including glycosylation and or phosphorylation at one or more residues. An N-terminal elongation may have a sequence comprising or consisting of amino acid A. An N-terminal elongation may have a sequence comprising or consisting of EA, or two or more repeats of EA, for example three or more repeats of EA, four or more repeats of EA, or five or more repeats of EA. For example, the N-terminal elongation may have a sequence comprising or consisting of A, EA, EAEA (SEQ ID NO: 22), EAEAEA (SEQ ID NO: 23), EAEAEAEA (SEQ ID NO: 24), EAEAEAEAEA (SEQ ID NO: 25, REAEAM (SEQ ID NO: 26), REAEAEAM (SEQ ID NO: 27), REAEAEAEAM (SEQ ID NO: 28), KREAEAM (SEQ ID NO: 29), KREAEAEAM (SEQ ID NO: 30), or KREAEAEAEAM (SEQ ID NO: 31).

[0037] In the embodiments herein, the p-lactoglobulin may be a mixture of heterogenous p-lactoglobulin proteins. In the embodiments herein, the p-lactoglobulin may be a plurality of recombinant p- lactoglobulin proteins heterogeneous in amino acid sequence, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18 or 20 recombinant p -lactoglobulin proteins of differing amino acid sequence and / or of differing elongation. In a non-limiting embodiment, the p-lactoglobulin may comprise a native p- lactoglobulin and a p-lactoglobulin having an N-terminal elongation of the amino acid A. In a non-limiting embodiment, the p-lactoglobulin may comprise a native p-lactoglobulin and a p-lactoglobulin having an N-terminal elongation of the amino acids EAE. In a non-limiting embodiment, the p-lactoglobulin may comprise a p-lactoglobulin having an N-terminal elongation of the amino acid A and a p-lactoglobulin having an N-terminal elongation of the amino acids EAE.

[0038] The elongated p-lactoglobulins and mixes thereof as set forth in WO2022 / 269549 are preferred p- lactoglobulins and are herein incorporated by reference. Accordingly, the recombinant p-lactoglobulins comprising or consisting of an amino acid sequence having at least about 70% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 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, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 61 , 71 , 72, 73, and 74 as set forth in WO2022 / 269549 are preferred p- lactoglobulins herein. In the embodiments herein, the p-lactoglobulin may comprise non-native post- translational modification modulating e.g. the glycosylation and / or phosphorylation of the p- lactoglobulin, as set forth in WQ2020219596A1 and US2022211061 , which are herein incorporated by reference.

[0039] Accordingly, the p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, S21 , S27, S30, S36, T49, T76, T97, S110, S116, T125, S150, N152, and T154 of Bos taurus p-lactoglobulin, and having non-native glycosylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises amino acid residue N 152 of Bos taurus p-lactoglobulin, and having non-native N-glycosylation on such amino acid residue. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, S21 , S27, S30, S36, T49, T97, SI 10, SI 16, T125, S150, and T154 of Bos taurus p-lactoglobulin, and having non-native O- glycosylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from T4, T6, T18, Y20, S21 , S27, S30, S36, Y42, T49, T76, T97, Y99, Y102, SI 10, SI 16, T125, S150, and T154 of Bos taurus p-lactoglobulin, and having non-native phosphorylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from K8, K14, R40, K47, K60, K69, K70, K75, K77, K83, K91 , K100, K101 , R124, K135, K138, K141 , and R148 of Bos taurus p-lactoglobulin, and having non-native methylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C66, C106, C119, C121 , and C160 of Bos taurus p-lactoglobulin, and having non-native palmitoylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from K8, K14, K47, K60, K69, K70, K75, K77, K83, K91 , K100, K101 , K135, K138, and K141 of Bos taurus p-lactoglobulin, and having nonnative sumoylation on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C66, C106, C119, C121 , and C160 of Bos taurus p-lactoglobulin, and having non-native nitrosylation on one or more of such amino acid residues. The p-lactoglobulin n may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from Y20, Y42, Y99, and Y102 of Bos taurus p-lactoglobulin, and having non-native tyrosine nitration on one or more of such amino acid residues. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from F151 of Bos taurus p-lactoglobulin, and having non-native glypiation on such amino acid residue. The p-lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C160 of Bos taurus p-lactoglobulin, and having non-native farnesylation on such amino acid residue. The p- lactoglobulin may be a recombinant p-lactoglobulin comprising an amino acid sequence that comprises one or more of amino acid residues selected from C160 of Bos taurus p-lactoglobulin, and having non- native geranylgeranylation on such amino acid residue.

[0040] In the embodiments herein, the p-lactoglobulin protein may have an attenuated or essentially eliminated allergenicity, such e.g. the p-lactoglobulin as set forth in WO2021168343 and US2023106635, which are herein incorporated by reference. Accordingly, the recombinant p-lactoglobulins comprising or consisting of an amino acid sequence having at least about 70% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10 of WO2021168343 and US2023106635 are preferred p-lactoglobulins herein.

[0041] In the embodiments herein, a more preferred p-lactoglobulin is a p-lactoglobulin that is not of an animal source, a p-lactoglobulin not isolated and / or purified from milk, whey or the like.

[0042] In the embodiments herein, the precipitation step may be performed at useful temperature; the person skilled in the art knows such temperature. A preferred temperature is room temperature, which is usually about 20°C.

[0043] In the embodiments herein, preferably, the protein of interest is p-lactoglobulin, the hexametaphosphate salt is sodium hexametaphosphate, the hexametaphosphate salt is added in a time frame of about 4 hours, no additional incubation is performed after the hexametaphosphate salt is added, the purity of the purified p-lactoglobulin is at least 85%, and at least 90% of the precipitated particles has a diameter that is at least 3 pm.

[0044] In a second aspect, there is provided for a method for the production of a recombinant protein of interest comprising: production of the protein of interest in a microbial cell, purifying the protein of interest, wherein the purification comprises a method according to the first aspect herein.

[0045] The features of this second aspect are preferably the features of the first aspect herein. In a third aspect, there is provided for a protein of interest obtainable by a method according to the first aspect herein.

[0046] In this aspect, there is further provided for a food product, including a beverage comprising a protein according to the second aspect herein or a protein obtained by a method according to the first aspect herein. The features of this third aspect are preferably the features of the first and second aspects herein.

[0047] Table 1 : Overview of sequences Figure legends

[0048] Figure 1 depicts the purification of p-lactoglobulin A by precipitation with sodium hexametaphosphate. The concentration of the p-lactoglobulin A in the aqueous composition was brought to 10 gram I L and the pH was brought to pH 3.0 or 4.0. The abbreviation “LGA” means p-lactoglobulin A.

[0049] Subsequently sodium hexametaphosphate was added in a time frame of 2 hours to the aqueous composition comprising the p-lactoglobulin A, while stirring at 20°C, to a molar ratio sodium hexametaphosphate : p-lactoglobulin A of: 0.5 : 1 , 2 : 1 , 5 : 1 , and 10 : 1.

[0050] Figure 2 depicts the purification of p-lactoglobulin A by precipitation with sodium hexametaphosphate. The concentration of the p-lactoglobulin A in the aqueous composition was brought to 10 gram I L and the pH was brought to pH 4.0.

[0051] Subsequently sodium hexametaphosphate was added in a time frame of 2 hours to the aqueous composition comprising the p-lactoglobulin A, while stirring at 20°C, to a molar ratio sodium hexametaphosphate : p-lactoglobulin A of: 0.5 : 1 , 2 : 1 , 5 : 1 , and 10 : 1. The abbreviation “LGA” means p-lactoglobulin A.

[0052] Definitions

[0053] "Sequence identity" is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" 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. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0054] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., NucleicAcids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity. Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

[0055] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

[0056] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

[0057] A “nucleic acid molecule” or “polynucleotide” (the terms are used interchangeably herein) is represented by a nucleotide sequence. A “polypeptide” is represented by an amino acid sequence. A “nucleic acid construct” is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of the peptide or polypeptide in a cell or in a subject.

[0058] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production / expression of the peptide or polypeptide of the invention in a cell and / or in a subject. “Operably linked” may also be used for defining a configuration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and / or in a subject.

[0059] “Expression” is construed as to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification and secretion.

[0060] A “control sequence” is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

[0061] The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). When the cell is a bacterial cell, as is intended in the present invention, the term usually refers to an extrachromosomal, self-replicating vector which harbors a selectable antibiotic resistance.

[0062] An “expression vector” may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a cell and / or in a subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene. It is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

[0063] A “polypeptide” or “protein" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0064] The term “recombinant polypeptide” or “recombinant protein” as used herein refers to a polypeptide that is produced in a cell of a different species or type as compared to the species or type of cell that produces the polypeptide in nature, or that is produced in a cell at a level at which it is not produced in nature.

[0065] The term "heterogeneous" as used herein with reference to a plurality of recombinant proteins means that the plurality of recombinant proteins comprises at least two or two or more, three or more, four or more, five or more, six or more, or seven or more proteins of differing amino acid sequence.

[0066] The term "mature" as used herein with reference to a protein refers to the protein, or amino acid sequence of the protein, after cleavage of the signal sequence. The term "full length" as used herein with reference to a protein refers to the protein, or amino acid sequence of the protein, comprising the signal sequence. Examples of mature and full-length proteins are provided in Table 1 herein. The term "wild-type" as used herein with reference to proteins or polynucleotides refers to a protein or polynucleotide having an amino acid or nucleotide sequences that is the same as that expressed naturally. This term includes all naturally occurring variants of a particular protein, for example, all naturally occurring variants of p-lactoglobulin. Furthermore, this term includes both full length proteins and mature proteins and polynucleotides that encode wild-type full length and mature protein. The term is generally synonymous with the term "native".

[0067] Sequence identity herein of a polynucleotide, polynucleotide construct or of a polypeptide is preferably at least 70%. Preferably at least 70% is defined as preferably at least 70%, more preferably at least 71 %, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81 %, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, more preferably at least 98%, more preferably at least 99%, or most preferably 100% sequence identity. In case of 100% sequence identity, the polynucleotide or polypeptide has exactly the sequence of the depicted SEQ ID NO:. Sequence identity is preferably determined over the entire length of the subject sequence.

[0068] The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

[0069] In this document and in its claims, the verbs "to comprise", “to contain”, and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist of’ may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; the additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

[0070] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0071] Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

[0072] Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art. Further embodiments of the invention

[0073] 1 . A method for the purification of a protein of interest from an aqueous composition comprising a precipitation step using a hexametaphosphate salt, wherein the hexametaphosphate salt is added to the aqueous composition comprising the protein of interest in a time frame of at least 10 minutes, preferably while mixing during the addition of the hexametaphosphate salt to the aqueous solution.

[0074] 2. A method for the purification of a protein of interest according embodiment 1 , wherein the hexametaphosphate salt is added to the aqueous composition in a time frame of at least 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours or 24 hours.

[0075] 3. A method for the purification of a protein of interest according to embodiment 1 or embodiment 2, wherein the hexametaphosphate salt is sodium hexametaphosphate.

[0076] 4. A method for the purification of a protein of interest according to any one of embodiments 1 to 3, wherein the hexametaphosphate salt is in a solution which is added to the aqueous composition comprising the protein of interest.

[0077] 5. A method for the purification of a protein of interest according to embodiment 4, wherein the concentration of the hexametaphosphate salt in the solution is between 5% w / v and 60% w / v, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 20%, 35%, 40%, 45%, 50%, 55%, or 60% w / v, preferably 10% w / v.

[0078] 6. A method for the purification of a protein of interest according to embodiment 4 or 5, wherein the pH of the hexametaphosphate salt solution is between pH 3 and pH 5, preferably pH 4.

[0079] 7. A method for the purification of a protein of interest according to any one of embodiments 1 to 6, wherein the protein of interest in the aqueous composition is present in a concentration of between 1 to 100 gram I L composition, preferably between 5 to 75 gram I L composition, more preferably between 10 to 50 gram I L composition, even more preferably at 10 gram I L composition .

[0080] 8. A method for the purification of a protein of interest according to any one of embodiments 1 to 7, wherein the volume of the aqueous composition comprising the protein of interest is at least 10 litre (L).

[0081] 9. A method for the purification of a protein of interest according to any one of embodiments 1 to 8, wherein the pH of the aqueous composition comprising the protein of interest is or is brought at between pH 3 and pH 5, preferably pH 4, before the hexametaphosphate salt is added. 10. A method for the purification of a protein of interest according to any one of embodiments 1 to 9, wherein the pH in the vessel wherein hexametaphosphate salt is added to the aqueous composition is kept at between pH 3 and pH 5, preferably at pH 4.

[0082] 11 . A method for the purification of a protein of interest according to any one of embodiments 1 to

[0083] 10, wherein the molar ratio between the hexametaphosphate salt and the protein of interest is between 2 : 1 and 10: 1 , such as 2 : 1 , 3 : 1 , 4 : 1 , 5 : 1 , 6 : 1 , 7 : 1 , 8 : 1 , 9 : 1 , or 10 : 1 , preferably 5: 1.

[0084] 12. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0085] 11 , wherein at least 80%, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, preferably at least 95% of the protein of interest is precipitated in the precipitation step using the hexametaphosphate salt.

[0086] 13. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0087] 12, wherein the purity of the protein of interest after the method is at least 80%, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, preferably at least 95%.

[0088] 14. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0089] 12, wherein at least 90% of the precipitated particles has a diameter that is at least 3 pm and / or wherein at least 80% of the precipitated particles has a diameter that is ranged between 3 and 20 pm and / or wherein at least 60% has a diameter that is ranged between 5 and 10 pm.

[0090] 15. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0091] 13, wherein the method further comprises a step selected from the group consisting of: solid / liquid separation; filtration, microfiltration, ultrafiltration, diafiltration and / or sterile filtration; centrifugation; chromatography; and, drying.

[0092] 16. A method for the purification of a protein of interest according to any one of embodiments 1 to 15, wherein the method does not comprise a chromatography step before the step wherein the protein of interest is precipitated by the hexametaphosphate salt.

[0093] 17. A method for the purification of a protein of interest according to embodiment 16, wherein the method does not comprise any chromatography step.

[0094] 18. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0095] 17, wherein the hexametaphosphate salt is removed by precipitation using an ionic metal salt, such as calcium chloride, or wherein the hexametaphosphate salt is not removed by precipitation using an ionic metal salt, such as calcium chloride.

[0096] 19. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0097] 18, wherein the protein of interest is a dairy protein, such as p-lactoglobulin, lactoferrin, a-lactalbumin, or a mixture of two or more proteins, preferably comprising at least p-lactoglobulin, lactoferrin and / or a-lactalbumin.

[0098] 20. A method for the purification of a protein of interest according to any one of embodiments 1 to

[0099] 19, wherein the protein of interest is a recombinant protein.

[0100] 21 . A method for the purification of a protein of interest according to any one of embodiments 1 to

[0101] 20, wherein the precipitation step is performed at room temperature..

[0102] 22. A method for the production of a recombinant protein of interest comprising: production of the protein of interest in a microbial cell, purifying the protein of interest, wherein the purification comprises a method according to any one of embodiments 1 to 21 .

[0103] 23. A protein of interest obtainable by a method according to any one of embodiments 1 to 22.

[0104] 24. A food product comprising a protein according to embodiment 23 or a protein obtained by a method according to any one of embodiments 1 to 22.

[0105] Examples

[0106] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention. In examples 1 to 4, the term yield is inadvertently used, while the term purity was intended and is appropriate.

[0107] The inventors have established that when purifying a protein of interest from an aqueous composition comprising a precipitation step using a hexametaphosphate salt, the purity of the protein is significantly increased when the hexametaphosphate salt is not added instantaneously but is slowly added to the aqueous composition. The inventors have further established that while the yield increases when slowly adding the hexametaphosphate salt as compared to instantaneously adding the hexametaphosphate salt, an additional incubation after the hexametaphosphate salt has been added, does not result in a higher purity of the protein of interest (data not shown).

[0108] Materials and methods p-lactoglobulin A was produced using fermentation as disclosed in WO 2022 / 269549, Example 1. WO 2022 / 269549 is herein incorporated by reference.

[0109] A 12.0 kg fermentation broth was obtained after fermentation during 7 days at a temperature of 30°C. After separation of the solid and liquid phases, an aqueous composition comprising between 10 to 50 gram I L p-lactoglobulin A was obtained.

[0110] Quantification of compounds was performed using methods known to the person skilled in the art. Example 1. Comparative purification of p-lactoglobulin A using precipitation with sodium hexametaphosphate.

[0111] The concentration of the p-lactoglobulin A in the aqueous composition was brought to 10 gram I L and the pH was brought to pH 4.0.

[0112] Subsequently, sodium hexametaphosphate was instantaneously added to the aqueous composition comprising the p-lactoglobulin A, while stirring at 20°C, to a molar ratio sodium hexametaphosphate : p-lactoglobulin A of 5 : 1 .

[0113] The yield of precipitated p-lactoglobulin A was 72%.

[0114] Example 2. Purification of p-lactoglobulin A using precipitation with sodium hexametaphosphate (1),

[0115] The concentration of the p-lactoglobulin A in the aqueous composition was brought to 10 gram I L and the pH was brought to pH 3.0 or 4.0.

[0116] Subsequently, sodium hexametaphosphate was added in a time frame of 2 hours to the aqueous composition comprising the p-lactoglobulin A, while stirring at 20°C, to a molar ratio sodium hexametaphosphate : p-lactoglobulin A of: 0.5 : 1 , 2 : 1 , 5 : 1 , and 10 : 1.

[0117] The yield of precipitated p-lactoglobulin A was > 90% for molar ratio’s sodium hexametaphosphate : p- lactoglobulin A of 5 : 1 ; and 10 : 1 at pH 3.0 and 4.0 and at molar ratio 5 : 1 and pH 4.0, the yield was even > 95% (see Figure 1).

[0118] Example 3. Purification of p-lactoglobulin A using precipitation with sodium hexametaphosphate (2).

[0119] The concentration of the p-lactoglobulin A in the aqueous composition was brought to 10 gram I L and the pH was brought to pH 4.0.

[0120] Subsequently, sodium hexametaphosphate was added in a time frame of 2 hours to the aqueous composition comprising the p-lactoglobulin A, while stirring at 20°C, to molar ratio’s sodium hexametaphosphate : p-lactoglobulin A of: 0.5 : 1 , 2 : 1 , 5 : 1 , and 10 : 1.

[0121] The yield of precipitated p-lactoglobulin A was > 90% for molar ratio’s sodium hexametaphosphate : p- lactoglobulin A of 5 : 1 ; and 10 : 1 (see Figure 2).

[0122] Example 4. Purification of p-lactoglobulin A using precipitation with sodium hexametaphosphate (3).

[0123] The concentration of the p-lactoglobulin A in the aqueous composition was brought to 10 gram I L and the pH was brought to pH 4.0.

[0124] Subsequently, sodium hexametaphosphate was added in a time frame of 4 hours to the aqueous composition comprising the p-lactoglobulin A, while stirring at 20°C, to molar ratio’s sodium hexametaphosphate : p-lactoglobulin A of 5 : 1 .

[0125] The yield of precipitated p-lactoglobulin A was > 95% and 87% of the carbohydrates were removed during the process. References:

[0126] Hidalgo et al, 173: Recovery of Whey Proteins with Sodium Hexametaphosphate; Journal of Dairy

[0127] Science, Vol 56, No.8, pages 988 - 993.

[0128] Hoppenreijs et al Food Research International 176 (2024) 113801.

Claims

CLAIMS1 . A method for the purification of a protein of interest from an aqueous composition comprising a precipitation step using a hexametaphosphate salt, wherein the hexametaphosphate salt is added to the aqueous composition comprising the protein of interest in a time frame of at least 10 minutes, preferably while mixing during the addition of the hexametaphosphate salt to the aqueous solution.

2. A method for the purification of a protein of interest according claim 1 , wherein the hexametaphosphate salt is added to the aqueous composition in a time frame of at least 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours or 24 hours.

3. A method for the purification of a protein of interest according to claim 1 or claim 2, wherein the hexametaphosphate salt is sodium hexametaphosphate.

4. A method for the purification of a protein of interest according to any one of claims 1 to 3, wherein the protein of interest in the aqueous composition is present in a concentration of between 1 to 100 gram I L composition, preferably between 5 to 75 gram I L composition, more preferably between 10 to 50 gram I L composition, even more preferably at 10 gram I L composition .

5. A method for the purification of a protein of interest according to any one of claims 1 to 4, wherein the pH of the aqueous composition comprising the protein of interest is or is brought at between pH 3 and pH 5, preferably pH 4, before the hexametaphosphate salt is added.

6. A method for the purification of a protein of interest according to any one of claims 1 to 5, wherein the pH in the vessel wherein hexametaphosphate salt is added to the aqueous composition is kept at between pH 3 and pH 5, preferably at pH 4.

7. A method for the purification of a protein of interest according to any one of claims 1 to 6, wherein the molar ratio between the hexametaphosphate salt and the protein of interest is between 2 : 1 and 10: 1 , such as 2 : 1 , 3 : 1 , 4 : 1 , 5 : 1 , 6 : 1 , 7 : 1 , 8 : 1 , 9 : 1 , or 10 : 1 , preferably 5 : 1.

8. A method for the purification of a protein of interest according to any one of claims 1 to 7, wherein at least 80%, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, preferably at least 95% of the protein of interest is precipitated in the precipitation step using the hexametaphosphate salt.

9. A method for the purification of a protein of interest according to any one of claims 1 to 8, wherein the purity of the protein of interest after the method is at least 80%, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, preferably at least 95%10. A method for the purification of a protein of interest according to any one of claims 1 to 9, wherein the method does not comprise a chromatography step before the step wherein the protein of interest is precipitated by the hexametaphosphate salt.

11. A method for the purification of a protein of interest according to any one of claims 1 to 10, wherein the protein of interest is a dairy protein, such as p-lactoglobulin, lactoferrin, a-lactalbumin, or a mixture of two or more proteins, preferably comprising at least p-lactoglobulin, lactoferrin and / or a-lactalbumin.

12. A method for the purification of a protein of interest according to any one of claims 1 to 11 , wherein the protein of interest is a recombinant protein.

13. A method for the purification of a protein of interest according to any one of claims 1 to 12, wherein the precipitation step is performed at room temperature..

14. A method for the production of a recombinant protein of interest comprising: production of the protein of interest in a microbial cell, purifying the protein of interest, wherein the purification comprises a method according to any one of claims 1 to 13.

15. A food product comprising a protein obtainable by or obtained by a method according to any one of claims 1 to 14.

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