Field of the invention

The present invention relates to a recombinant fusion protein, and methods for preparing proteins which utilise a recombinant fusion protein. More particularly, the invention relates to a recombinant fusion protein comprising a casein, and the use of the recombinant fusion protein in preparing caseins.

Background

Milk (e.g. cows’ milk) is an oil-in-water emulsion which comprises dissolved carbohydrates, minerals, lipids and proteins. The proteins present in milk include caseins (a-S1 , a-S2, p and K-caseins) and whey proteins (including p-lactoglobulin, a-lactalbumin, serum albumin and immunoglobulins).

The production of milk proteins, particularly caseins, through the expression of recombinant DNA in a heterologous host system is desirable for multiple ecological, health and animal welfare reasons. For it to be economically viable, the process must occur with high titre (g protein I L culture) and/or high productivity (g protein I number of viable cells in culture).

Well established methods to achieve high level titre and productivity of recombinant proteins in heterologous expression systems, such as expression under a strong promoter or insertion of multiple copies of the recombinant DNA, are not equally effective for all proteins. Specifically, K-casein responds well to these strategies while a-S1 casein, a-S2 casein and p-casein do not.

Accordingly, there is a need to provide milk proteins such as caseins in a more sustainable and/or economically viable manner.

Summary of the invention

According to a first aspect of the present invention there is provided a recombinant fusion protein comprising :

(a) a casein selected from a-S1 casein, a-S2 casein and p-casein; and

(b) a peptide comprising a caseinomacropeptide (CMP), or a fragment thereof. The peptide may increase the hydrophilicity of the recombinant fusion protein relative to the hydrophilicity of the casein alone. In other words, the peptide may decrease the hydrophobicity of the recombinant fusion protein relative to the hydrophobicity of the casein alone.

According to a second aspect of the invention there is provided a nucleic acid molecule comprising a sequence encoding the recombinant fusion protein according to the first aspect of the invention.

According to a third aspect of the invention, there is provided a vector comprising the nucleic acid molecule of the second aspect of the invention.

According to a fourth aspect of the invention, there is provided a host cell comprising the nucleic acid molecule of the second aspect of the invention, or the vector of the third aspect of the invention.

In a fifth aspect, the invention provides a method of producing a recombinant protein, the method comprising culturing the host cell of the fourth aspect of the invention under conditions suitable for expression of the recombinant fusion protein of the first aspect.

In a sixth aspect, the invention provides a method of making a food composition, the method comprising:

(i) providing a recombinant fusion protein according to the first aspect of the invention;

(ii) separating the casein from the peptide; and

(iii) using the separated casein to make a food composition.

In a further aspect, the invention provides a recombinant casein obtained by the method of the fifth aspect.

In yet a further aspect, the invention provides a food composition obtained by the method of the sixth aspect.

In another aspect, the invention provides the use of the recombinant fusion protein of the first aspect in the production of a recombinant casein, and/or in the production of a food composition.

Embodiments of the invention will now be described with reference to the accompanying figures in which: Figure 1 shows the amino acid sequence of the CMP of bovine K-casein (uniprot accession number P02668) variant B (referred to herein as SEQ ID NO. 12), excluding the chymosin recognition sequence, with the potential glycosylation sites indicated by arrows;

Figure 2a is a schematic drawing of a recombinant fusion protein in accordance with embodiments of the invention;

Figure 2b is a schematic drawing of a recombinant casein obtained from the recombinant fusion protein of Figure 2a;

Figure 3 is a graph showing the quantity of recombinant a-S1 casein, a-S2 casein, p-casein and K-casein expressed under a strong promoter in Pichia pastoris, and

Figure 4 is a graph showing the quantity of casein (CSN) - caseinomacropeptide (CMP) recombinant fusion proteins expressed using the same promoter, host cell and conditions as in the example shown in Figure 3;

Figure 5a is an image of a Western blot showing the generation of recombinant a-S1 casein (referred to as “CSN aS1”) by chymosin cleavage of a recombinant fusion protein comprising a-S1 casein fused to CMP (“CSN aS1-CMP”);

Figure 5b is a graph showing the generation of recombinant CSN aS1 by chymosin cleavage of CSN aSI-CMP;

Figure 6 is a graph showing the titre for expression in P. pastoris using two culture conditions (A and B) for CSN aS1 and CSN aS1-CMP relative to CSN aS1-CMP (condition A);

Figure 7 is a graph showing the titre of CSN aS1 , CSN aS1-CMP, CSN aS1-CtruncCMP, and CSN aS1-NtruncCMP relative to CSN aS1-CMP, expressed in P. pastoris,

Figure 8 is a graph showing the titre of CSN aS1 , CMP-CSN aS1 (N-terminal CMP), and CSN aS1-CMP (C-terminal CMP) relative to CSN aS1-CMP, expressed in P. pastoris,

Figure 9 is a graph showing the titre for expression in Saccharomyces cerevisiae (Sc) for CSN aS1 relative to expression of CSN aS1-CMP in the same host; and

Figure 10 is a graph showing the titre of CSN aS1 , CSN aS1-CSN K, and CSN aS1-CMP relative to CSN aS1-CMP, expressed in P. pastoris.

In this specification, the amino acids are designated by the one-letter code, for example, G stands for glycine, P stands for proline, R stands for arginine, etc.

The present invention provides a recombinant fusion protein comprising :

(a) a casein selected from a-S1 casein, a-S2 casein and p-casein; and

(b) a peptide comprising a caseinomacropeptide (CMP), or a fragment thereof.

In some embodiments, the casein is a-S1 casein. In some embodiments, the casein is a-S2 casein.

In some embodiments, the casein is p-casein.

The a-S1 casein, the a-S2 casein and/or the p-casein may comprise or be constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of the equivalent wild-type casein protein.

The casein may be derived from any suitable species. For example, the casein (e.g. a-S1 casein, the a-S2 casein and/or the p-casein) may be that of cow, goat, sheep, camel, donkey, horse, human, rat, mouse, guinea pig, pig, water buffalo, rabbit, whale (e.g. fin whale, beluga whale), giraffe or elephant (e.g. African elephant). The accession numbers of casein proteins of representative species are shown in Table 1 .

In some embodiments, the a-S1 casein comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of bovine (Bos taurus) a-S1 casein (Uniprot accession no. P02662). The bovine a-S1 casein sequence may be variant B.

A representative sequence for bovine a-S1 casein is shown below and is referred to herein as SEQ ID NO. 1. Thus, in some embodiments, the a-S1 casein comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO 1.

RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDI KQMEA ESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHS MKE GIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNP I GSENSEKTTMPLW (SEQ ID NO:1)

In some embodiments, the a-S2 casein comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of bovine (Bos taurus) a-S2 casein (Uniprot accession no. P02663). The bovine a-S2 casein sequence may be the reference sequence.

A representative sequence for bovine a-S2 casein is shown below and is referred to herein as SEQ ID NO. 2. Thus, in some embodiments, the a-S2 casein comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO 2.

KNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEVVRNANEEEYSIGSS SEESA EVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIVLNPWDQVKRNAVPITPT LN REQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLKKISQRYQKFALPQYLKTVY QH QKAMKPWIQPKTKVIPYVRYL (SEQ ID NO:2).

In some embodiments, the p-casein comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of bovine (Bos taurus) casein (Uniprot accession no. P02666). In some embodiments, the bovine p casein sequence is variant A2 (the reference). In some embodiments, the bovine p casein sequence is variant A2 (the reference) with the substitution S137R .

A representative sequence for bovine p-casein is shown below and is referred to herein as SEQ ID NO. 3. Thus, in some embodiments, the p casein comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO 3.

RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQS LVYPFP GPIPNSLPQNIPPLTQTPWVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTERQS LTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRD MPIQAFLLYQEPVLGPVRGPFPIIV (SEQ ID NO:3).

The term “identity”, with reference to amino acid and nucleic acid sequences, is used herein to describe the level of similarity between two sequences. Sequence similarity or identity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection. Another suitable algorithm is the BLAST algorithm, described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993).

The recombinant fusion protein of the invention comprises a casein which is fused, directly or indirectly, to a peptide which comprises, or is constituted by, a caseinomacropeptide (CMP), or a fragment thereof.

As used herein, the term “caseinomacropeptide” or “CMP” refers to the C-terminal portion of K-casein (i.e. the portion which is released upon cleavage of K-casein by chymosin, which may also be referred to as the “CMP domain”) but excludes the chymosin recognition sequence. As is known in the art, bovine chymosin cuts K-casein between an F residue and an M residue (residues 105 and 106 in bovine K-casein) to release an N-terminal portion (para-kappa casein) and a C-terminal portion (comprising the caseinomacropeptide). The FM motif is contained within a recognition sequence, which in bovine K-casein is HPHPHLSFMAIPPK (SEQ ID NO:4). Although, post-cleavage, a part of this recognition sequence resides in the C-terminal portion, this part of the recognition sequence is not considered to form a part of the CMP as defined in the context of this specification. However, in some embodiments the recombinant fusion protein comprises a linker which may include some or all of the recognition sequence.

In some embodiments, the peptide consists of a caseinomacropeptide (CMP), or a fragment thereof.

Thus, in some embodiments, the recombinant fusion protein comprises: (a) a casein selected from a-S1 casein, a-S2 casein and p-casein; and (b) a caseinomacropeptide (CMP), or a fragment thereof.

In some embodiments, the recombinant fusion protein, or the peptide comprising the CMP, comprises a sequence of no more than 150, no more than 120, no more than 100, no more than 90, no more than 80, no more than 75, no more than 72, no more than 70, no more than 65, no more than 64, no more than 60, or no more than 58 consecutive amino acids derived from the C-terminus of kappa casein. In other words, the recombinant fusion protein, or the peptide comprising the CMP, may not comprise the full length kappa casein protein. In some embodiments, the recombinant fusion protein, or the peptide comprising the CMP, comprises a sequence of 72 consecutive amino acids derived from the C-terminus of kappa casein.

In some embodiments, the recombinant fusion protein, or the peptide comprising the CMP, comprises a sequence of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 consecutive amino acids derived from the C-terminus of kappa casein.

In some embodiments, the recombinant fusion protein, or the peptide comprising the CMP, comprises a sequence of from 20 to 120, from 25 to 110, from 30 to 100, from 35 to 90, from 40 to 85, from 50 to 80, from 55 to 75, from 60 to 70 consecutive amino acids derived from the C-terminus of kappa casein.

The terms “cleavage site” and “recognition sequence” may be used interchangeably herein to refer to a sequence or chemical motif which is recognised by an enzyme ora chemical capable of cleaving the recombinant fusion protein so as to separate the casein from the CMP.

Based only on their amino acid composition, all caseins are relatively hydrophobic. In addition, their lack of tertiary structure results in exposure of the hydrophobic residues and, therefore, a highly hydrophobic surface. This leads to relatively low solubility in aqueous environments and tendency to form aggregates through hydrophobic interactions. The presence of post- translational modifications (e.g. phosphorylations and glycosylations) introduces additional parameters and changes the behaviour of the proteins. Caseins a-S1 , a-S2 and p have multiple phosphorylations which introduce a negative charge and increases their solubility. However, within certain pH ranges, the charge is neutralized eliminating the solubilizing behaviour. Furthermore, in the presence of calcium, calcium phosphate nanoclusters are formed, resulting in an even higher tendency to form aggregates.

With reference to Figure 1 , the CMP of bovine K-casein has multiple potential glycosylation sites (indicated by arrows) making it highly hydrophilic. The carbohydrates attached to the glycosylation sites decrease the protein’s hydrophobicity, and therefore increase its solubility, in a manner that is independent of calcium and tolerant to a wider pH range. Without being bound by theory, it is believed that by fusing the CMP of K-casein, or a fragment thereof, to other casein proteins (a-S1 , a-S2 or p-casein), the resulting fusion protein has improved solubility relative to that of the native caseins, thereby improving the expression of recombinant caseins in a heterologous system. The glycosylated form of CMP may also be referred to in the art as “GMP” or “gCMP” (glyco- caseino-macropeptide), while the non-glycosylated form is sometimes referred to as “aCMP” (aglyco-caseino-mactopeptide). It is also known in the art to use the term “CMP” for a heterogeneous mixture of non-glycosylated peptides and peptides with different degrees of glycosylation. In the context of the present specification, the term “CMP” is used to refer to both the non-glycosylated peptide and to glycosylated forms, unless otherwise stated.

In some embodiments, the peptide increases the hydrophilicity of the recombinant fusion protein relative to the hydrophilicity of the casein alone. The CMP is a highly hydrophilic sequence, particularly in its glycosylated states. This is evident from the CMP sequence itself. Thus, in some embodiments the peptide is capable of conferring on the recombinant fusion protein an increased hydrophilicity, relative to the hydrophilicity of the (equivalent) casein alone. In other words, the hydrophilicity of the recombinant fusion protein may be higher than the hydrophilicity of the (equivalent) casein alone. For example, a recombinant fusion protein comprising a-S1 casein fused to the peptide may have a higher hydrophilicity than the a-S1 casein alone. By fusing the casein to a CMP or fragment thereof, a more hydrophilic protein is created. This may interfere with aggregation based on hydrophobic interactions and decrease the dependence of the protein’s solubility on pH and on the concentration of other ions. This can result in more efficient biosynthesis, less intracellular degradation, better hostcell fitness and/or more efficient secretion.

It will be understood that increasing the hydrophilicity of the recombinant fusion protein is equivalent to decreasing its hydrophobicity. In some embodiments the peptide decreases the hydrophobicity of the recombinant fusion protein relative to the hydrophobicity of the casein alone. Thus, the peptide may be capable of conferring on the recombinant fusion protein a reduced hydrophobicity, relative to the hydrophobicity of the (equivalent) casein alone. In other words, the hydrophobicity of the recombinant fusion protein may be lower than the hydrophobicity of the (equivalent) casein alone. For example, a recombinant fusion protein comprising a-S1 casein fused to the peptide comprising the CMP or fragment thereof may have a lower hydrophobicity than the a-S1 casein alone.

The hydrophobicity of the recombinant fusion protein and/or the casein may be determined by Hydrophobic Partition, as described in Nakai S. 2003 Measurement of Protein Hydrophobicity, Current Protocols in Food Analytical Chemistry 9(1), Basic Protocol 5.

In some embodiments, the peptide is capable of increasing the solubility of the recombinant fusion protein in a medium, relative to the solubility of the (equivalent) casein alone in the same medium. In other words, the solubility of the recombinant fusion protein in a medium may be greater than the solubility of the (equivalent) casein alone in the medium. For example, a recombinant fusion protein comprising a-S1 casein may have a greater solubility in a medium than the a-S1 casein alone.

The effect of fusion to a peptide comprising the CMP or fragment thereof on the solubility of a casein protein may be determined according to the following protocol:

1 . Prepare multiple separate aliquots of recombinant fusion protein and casein ³ alone at the same concentration (e.g. 2.7% w/w) in demineralised water adjusted to high pH with NaOH (e.g. pH 11);

2. Adjust the pH of each aliquot to a different (lower) value (e.g. decrease in 1 pH unit increments down to pH 1) with HCI, and incubate (e.g. 30 min with stirring) for equilibration;

3. Determine the resulting concentration in each condition by centrifugation of the equilibrated sample (e.g. 4000 g, 15 min) and of the original solution (pH 11), then remove the supernatant and add 1 volume of 2x SDS PAGE buffer to the supernatant. Run the samples on an SDS-PAGE gel and divide the intensity of the band obtained for each condition by the intensity of the band obtained at pH 11.

³ Since it is difficult to express casein alone, the casein may be expressed in the recombinant fusion protein which can then be cleaved to release the casein. Alternatively, the casein may be isolated from animal milk.

In some embodiments, the peptide is capable of increasing the yield of the recombinant fusion protein relative to the yield of the (equivalent) casein alone when expressed under identical conditions (e.g. from the same expression cassette, in the same host, under the same environmental conditions). In other words, the yield of the recombinant fusion protein may be greater than the yield of the (equivalent) casein alone when expressed under identical conditions.

In some embodiments, the CMP or fragment thereof comprises a sequence of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 consecutive amino acids.

In some embodiments, the CMP or fragment thereof comprises at least 3, at least 4, at least 5 glycosylation sites. In some embodiments, the CMP or fragment thereof comprises 6 glycosylation sites. In some embodiments, the CMP comprises an N-terminal or a C-terminal truncation, relative to the full length CMP (as defined herein). In some embodiments, the CMP comprises both an N-terminal and a C-terminal truncation, relative to the full length CMP (as defined herein).

In some embodiments the CMP or fragment thereof comprises:

(i) the amino acid sequence:

X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2 X ₃ (SEQ ID NO:5) wherein each Xi is independently selected from S and T, each X2 is independently selected from D and E, and each X3 is independently selected from A, I, L and V; or

(ii) an amino acid sequence which is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 5.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 5, with the proviso that at least 3, at least 4 or at least 5 of the Xi residues are retained. In some embodiments, all of the Xi residues are retained.

In some embodiments the CMP or fragment thereof comprises:

(i) an amino acid sequence selected from:

X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2 X3X3X2SPPX2X3NT X3QX3X1STX3X3 (SEQ ID NO:6); and

KNQX2KTX2X3PX1 X3NTX3X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2 X ₃ (SEQ

ID NO:7), or

(ii) an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 6 or SEQ ID NO:7.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 6, with the proviso that at least 3, at least 4, at least 5 or at least 6 of the Xi residues are retained. In some embodiments, all of the Xi residues are retained.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 7, with the proviso that at least 3, at least 4, at least 5 or at least 6 of the Xi residues are retained. In some embodiments, all of the Xi residues are retained. In some embodiments the CMP or fragment thereof comprises:

(i) the amino acid sequence

KNQX2KTX2X3PX1 X3NTX3X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2

X3X3X2SPPX2X3NT X3QX3X1STX3X3 (SEQ ID NO:8); or

(ii) an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 8.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 8, with the proviso that at least 3, at least 4, at least 5, at least 6 or at least 7 of the Xi residues are retained. In some embodiments, all of the Xi residues are retained.

In some embodiments the CMP or fragment thereof comprises:

(i) the amino acid sequence ASGEPT STPTI EAVES TVATLEASPE V (SEQ ID NO:9); or

(ii) an amino acid sequence which is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 9.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 9 with the proviso that at least 3, at least 4 or at least 5 glycosylation sites are retained (i.e. present). In some embodiments, 6 glycosylation sites are retained.

In some embodiments the CMP or fragment thereof comprises:

(i) an amino acid sequence selected from:

ASGEPT STPTI EAVES TVATLEASPE VIESPPEINT VQVTSTAV (SEQ ID NO: 10); and KNQDKTEIPT INTIASGEPT STPTIEAVES TVATLEASPE V (SEQ ID NO:11), or

(ii) an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 10 or SEQ ID NO:11.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ I D NO: 10, with the proviso that at least 3, at least 4, at least 5 or at least 6 glycosylation sites are retained (i.e. present). In some embodiments, 7 glycosylation sites are retained.

In some embodiments, the CMP or fragment thereof comprises an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ I D NO: 11 , with the proviso that at least 3, at least 4, at least 5 or at least 6 glycosylation sites are retained (i.e. present). In some embodiments, 7 glycosylation sites are retained.

In some embodiments the CMP or fragment thereof comprises or consists of:

(i) the amino acid sequence

KNQDKTEIPT INTIASGEPT STPTIEAVES TVATLEASPE VIESPPEINT VQVTSTAV (SEQ ID NO:12); or

(ii) an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96% or at least 98% identical to SEQ ID NO: 12.

In some embodiments the CMP or fragment thereof comprises (an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96% or at least 98% identical to SEQ ID NO: 12, with the proviso that at least 3, at least 4, at least 5, at least 6 or at least 7 glycosylation sites are retained (i.e. present). In some embodiments, 8 glycosylation sites are retained.

In some embodiments, the CMP comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the CMP of cow (Bos ta urns') K- casein (Uniprot accession no. P02668). The cow K-casein may be variant B, or it may be the reference variant A or any one of the other variants (B2, E, G, or F).

The amino acid sequence of the CMP from cow K-casein protein (variant B) is designated herein as SEQ ID NO:12.

The CMP, or fragment thereof, may be derived from any suitable mammal. In some embodiments, the CMP comprises or is constituted by an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the wild-type CMP of K-casein from goat, sheep, camel, donkey, horse, human, rat, mouse, guinea pig, pig, water buffalo, rabbit, whale (e.g. fin whale, beluga whale), giraffe or elephant. The accession numbers of K-casein for exemplary species are shown in Table 1. Table 1

As used herein, the term “fusion” will be understood to mean that the casein and the peptide comprising the caseinomacropeptide (CMP) orfragmentthereof are joined directly or indirectly via peptide bonds such that they form two portions of the same recombinant protein.

In some embodiments, the recombinant fusion protein comprises a linker between the casein and the peptide. The linker may comprise an amino acid sequence of from 3 to 50, from 5 to 40, from 8 to 30, from 10 to 20 or from 12 to 15 residues in length.

In some embodiments, the linker comprises a cleavage site. Advantageously, the cleavage site can be used to cleave the recombinant fusion protein so as to separate the casein from the peptide. The cleavage site may be a chemical cleavage site or an enzyme cleavage site.

In some embodiments, the cleavage site is an enzyme cleavage site. The enzyme cleavage site may be a hydrolase cleavage site (such as a protease cleavage site) or a peptide lyase cleavage site (such as an amidine lyase cleavage site).

In some embodiments the cleavage site is a protease cleavage site. The cleavage site may be a chymosin cleavage site, or an enteropeptidase cleavage site.

A suitable enteropeptidase cleavage site may be that of bovine enteropeptidase (Uniprot accession number P98072).

In some embodiments, the cleavage site is a chymosin cleavage site. The chymosin cleavage site may be that of any mammal. For example, the chymosin cleavage site may be bovine, goat, sheep, or camel. In some embodiments, the cleavage site is derived from the same species as the CMP.

In some embodiments the chymosin cleavage site comprises the amino acid sequence HLSFXAIP (SEQ ID NO.13), wherein X is L, M or F. Cleavage occurs between the F and X residues.

Some additional residues are believed to make the interaction with the enzyme more stable. Therefore, in some embodiments the chymosin cleavage site comprises the sequence HPHPHLSFMAIPPK (SEQ ID NO.4).

In some embodiments, the chymosin cleavage site comprises the sequence FLQKQQYGISSKFX wherein X is selected from: W, Y, F, C, R, I, M, H, L, V, K, N, T or A (SEQ ID NO.14). Preferably, X is W, Y or F.

In some embodiments, the chymosin cleavage site comprises the sequence RPRPRPSFIAIPPK (SEQ ID NO.15). Other suitable cleavage sites which may be incorporated into the recombinant fusion protein will be known to those skilled in the art, such as those described in WO2022/072718.

The casein may be located at or towards the N-terminal end of the recombinant fusion protein, and the peptide may be located at or towards the C-terminal end of the recombinant fusion protein. Alternatively, the casein may be located at or towards the C-terminal end of the recombinant fusion protein, and the peptide may be located at or towards the N-terminal end of the recombinant fusion protein.

In some embodiments, the casein is located at or towards the N-terminal end, and the peptide at or towards the C-terminal end of the recombinant fusion protein.

The recombinant fusion protein may further comprise additional features, such as one or more tags (e.g. a HIS tag or a FLAG tag), and/or a signal sequence.

It will be appreciated that the components of the recombinant fusion protein (i.e. the casein, the peptide, and any tag(s) and/or signal sequences, if present) may be directly joined to each other via peptide bonds, or they may be joined via one or more linkers.

In some embodiments, the recombinant fusion protein comprises a signal sequence or a fragment thereof. As is known in the art, a signal sequence (also referred to as a “signal peptide”, “leader sequence”, “signal leader”, “targeting signal”, “localization signal” or “localization sequence”) is a short amino acid sequence that is normally present at the N- terminus of a protein, and which prompts the producing cell to translocate the protein through a membrane. A secretory signal sequence prompts translocation through the membrane of the endoplasmic reticulum (in eukaryotic organisms) and therefore into the secretory pathway. Signal sequences may be cleaved during or after translocation of the protein through the target membrane to generate a free signal peptide and a mature protein. Complex leader sequences can comprise multiple processing (i.e. cleavage) positions. A first (N-terminal) portion, which strictly speaking is the localization sequence, is referred to as pre-sequence (or “pre-signal”, “pre-region”, “pre-peptide”) and is cleaved during translocation or immediately after translocation. A further portion, referred to as pro-sequence (or “pro-signal”, “pro-peptide”, “pro-region”), is believed to modulate progression through the pathway and is cleaved at later stages of transit through the secretory pathway. Incomplete processing of a complex leader sequence may result in the secreted protein retaining some amino acids from the prosequence at its N terminus. Thus, in some embodiments the recombinant fusion protein comprises at its N terminus at least some amino acids from the pro-region of the signal sequence.

The signal sequence may be any suitable signal that is known in the art. The skilled person is capable of selecting a signal sequence which is appropriate for the host cell in which the recombinant fusion protein is intended to be expressed.

In some embodiments, the signal sequence is selected from: S. cerevisiae a-mating factor (MF-a) signal sequence or a chimera, fragment or variant (e.g. comprising point mutations) thereof.

In some embodiments, the signal sequence is the S. cerevisiae a-mating factor (MF-a) signal sequence.

In some embodiments, the signal sequence is a chimeric signal sequence comprising the preregion of the S. cerevisiae Ost1 signal sequence and the pro-region of the S. cerevisiae MF- a signal sequence.

In some embodiments, the signal sequence comprises a sequence which is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO. 16.

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSN STNNGL LFINTTIASIAAKEEGVSLEKREAEA (SEQ ID NO: 16).

In some embodiments, the recombinant fusion protein comprises or has the structure, from N- terminus to C-terminus:

Signal sequence (or fragment thereof) - tag - casein - linker - CMP (or fragment thereof) - tag, wherein the linker comprises a cleavage site (e.g. a chymosin cleavage site).

In some embodiments, the recombinant fusion protein comprises or has the structure, from N- terminus to C-terminus:

Signal sequence (or fragment thereof) - casein - linker - CMP (or fragment thereof), wherein the linker comprises a cleavage site (e.g. a chymosin cleavage site).

In some embodiments, the recombinant fusion protein comprises or has the structure, from N- terminus to C-terminus: Casein - linker - CMP (or fragment thereof), wherein the linker comprises a cleavage site (e.g. a chymosin cleavage site).

In an embodiment, the recombinant fusion protein comprises or consists of an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17 or SEQ ID NO: 18. In an embodiment, the recombinant fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO: 17 or SEQ ID NO: 18. The recombinant fusion protein comprises bovine a-S1 casein fused via a linker to the CMP peptide of bovine k-casein, wherein the linker comprises the bovine chymosin cleavage site. The sequence of SEQ ID NO: 17 comprises the S. cerevisiae a-mating factor (MF-a) signal sequence at its N- terminus. During secretion of the recombinant fusion protein by the host cell, this signal sequence is cleaved, leaving behind a small fragment thereof on the secreted protein. The secreted protein is represented by SEQ ID NO:18.

Expressed fusion protein comprising a-S1 casein-linker-CMP:

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSN STNNGL LFINTTIASIAAKEEGVSLEKREAEAHHHHHHRPKHPIKHQGLPQEVLNENLLRFFVAPF PEV FGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERY LG YLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQF YQ LDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLWHPHPHLSFMAIPPKKN Q DKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAVGSGSH HHHH H (SEQ ID NO:17)

Secreted fusion protein comprising a-S1 casein-linker-CMP:

EAEAHHHHHHRPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSE STED QAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEI VPN SAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYT DAPSFSDIPNPIGSENSEKTTMPLWHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTS TPTI EAVESTVATLEASPEVI ESPPEI NTVQVTSTAVGSGSH H H H H H (SEQ ID NO:18).

In an embodiment, the recombinant fusion protein comprises or consists of an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO. 19 or SEQ ID NO. 20. In an embodiment, the recombinant fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO. 19 or SEQ ID NQ:20. The recombinant fusion protein comprises bovine a-S2 casein fused via a linker to the CMP peptide of bovine k-casein, wherein the linker comprises the bovine chymosin cleavage site. The sequence of SEQ ID NO: 19 comprises the S. cerevisiae a-mating factor (MF-a) signal sequence at its N- terminus. During secretion of the recombinant fusion protein by the host cell, this signal sequence is cleaved, leaving behind a small fragment thereof on the secreted protein. The secreted protein is represented by SEQ ID NO:20.

Expressed fusion protein comprising a-S2 casein-linker-CMP:

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSN STNNGL LFINTTIASIAAKEEGVSLEKREAEAHHHHHHKNTMEHVSSSEESIISQETYKQEKNMAI NPS KENLCSTFCKEVVRNANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQK FP QYLQYLYQGPIVLNPWDQVKRNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKL TE EEKNRLNFLKKISQRYQKFALPQYLKTVYQHQKAMKPWIQPKTKVIPYVRYLHPHPHLSF MA I PPKKNQDKTEI PTI NTI ASGEPTSTPTI EAVESTVATLEASPEVI ESPPEI NTVQVTSTAVGSG SHHHHHH (SEQ ID NO: 19).

Secreted fusion protein comprising a-S2 casein-linker-CMP:

EAEAHHHHHHKNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEVVRNA NEEE YSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIVLNPWDQ VK RNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLKKISQRYQK FAL PQYLKTVYQHQKAMKPWIQPKTKVIPYVRYLHPHPHLSFMAIPPKKNQDKTEIPTINTIA SGE PTSTPTI EAVESTVATLEASPEVI ESPPEI NTVQVTSTAVGSGSH H H H H H (SEQ ID NQ:20).

In an embodiment, the recombinant fusion protein comprises or consists of an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO. 21 or SEQ ID NO:22. In an embodiment, the recombinant fusion protein comprises or consists of an amino acid sequence according to SEQ ID NO. 21 or SEQ ID NO:22. The recombinant fusion protein comprises bovine p-casein fused via a linker to the CMP peptide of bovine k- casein, wherein the linker comprises the bovine chymosin cleavage site. The sequence of SEQ ID NO:21 comprises the S. cerevisiae a-mating factor (MF-a) signal sequence at its N- terminus. During secretion of the recombinant fusion protein by the host cell, this signal sequence is cleaved, leaving behind a small fragment thereof on the secreted protein. The secreted protein is represented by SEQ ID NO:22.

Expressed fusion protein comprising p-casein-linker-CMP:

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSN STNNGL LFI NTTI ASI AAKEEGVSLEKREAEAHHHHHH RELEELN VPGEI VESLSSSEESITRI N KKI EKF QSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVM GV SKVKEAMAPKHKEMPFPKYPVEPFTERQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTV MFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIVHPHPHL SF MAI PPKKNQDKTEI PTI NTI ASGEPTSTPTI EAVESTVATLEASPEVI ESPPEI NTVQVTSTAVG SGSHHHHHH (SEQ ID N0:21).

Secreted fusion protein comprising p-casein-linker-CMP:

EAEAHHHHHHRELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQD KIHPFA QTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPK Y PVEPFTERQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVP Q KAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIVHPHPHLSFMAIPPKKNQDKTEIPTIN TIAS G EPTSTPTI EAVESTVATLEASPEVI ESPPEI NTVQVTSTAVGSGSH H H H H H (SEQ ID NO:22).

The invention further provides a nucleic acid molecule comprising a sequence encoding a recombinant fusion protein as described herein.

The nucleic acid molecule may be DNA or RNA. In some embodiments the nucleic acid molecule is DNA.

In some embodiments the nucleic acid molecule comprises a sequence selected from SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25:

DNA sequence encoding fusion protein comprising a-S1 casein-linker-CMP: ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCTGCT C CAGTTAACACTACTACTGAAGATGAGACTGCTCAAATTCCTGCTGAAGCTGTTATTGGTT ACTCTGATTTGGAGGGAGATTTTGATGTTGCTGTTTTGCCTTTCTCTAACTCTACTAACA ACGGTTTGTTGTTCATTAACACTACTATCGCTTCTATCGCTGCTAAGGAAGAGGGTGTTT CTTTGGAAAAGAGAGAAGCTGAGGCTCACCATCACCACCATCACAGACCAAAGCATCC TATTAAACACCAAGGTTTGCCACAAGAAGTTTTGAACGAGAATTTGTTGAGATTTTTCGT TGCTCCATTTCCTGAAGTTTTCGGTAAAGAAAAAGTTAACGAGTTGTCTAAGGATATTGG TTCTGAATCTACTGAGGATCAAGCTATGGAAGATATCAAGCAAATGGAAGCTGAGTCTA TCTCTTCTTCTGAAGAGATTGTTCCAAACTCTGTTGAACAAAAGCATATCCAAAAGGAAG ATGTTCCTTCTGAGAGATACTTGGGTTATTTGGAACAATTGTTGAGATTGAAGAAATACA AGGTTCCACAATTGGAGATTGTTCCTAACTCTGCTGAAGAGAGATTGCATTCTATGAAG GAAGGTATTCACGCTCAACAAAAAGAGCCAATGATTGGTGTTAATCAAGAATTGGCTTA CTTCTACCCTGAGTTGTTTAGACAATTCTACCAATTGGATGCTTATCCATCTGGTGCTTG GTACTATGTTCCTTTGGGTACTCAATACACTGATGCTCCATCTTTCTCTGATATTCCAAA CCCTATTGGTTCTGAAAATTCTGAGAAAACTACTATGCCTTTGTGGCATCCACACCCTCA TTTGTCTTTCATGGCTATTCCACCTAAGAAAAACCAAGATAAGACTGAAATCCCAACTAT TAATACTATTGCTTCTGGAGAGCCAACTTCTACTCCTACTATTGAAGCTGTTGAGTCTAC TGTTGCTACTTTGGAAGCTTCTCCTGAAGTTATTGAGTCTCCACCTGAGATTAATACTGT TCAAGTTACTTCTACTGCTGTTGGTTCTGGATCCCATCACCATCACCATCACTAA (SEQ ID NO:23).

DNA sequence encoding fusion protein comprising a-S2 casein-linker-CMP:

ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCT GCTC CAGTTAACACTACTACTGAAGATGAGACTGCTCAAATTCCTGCTGAAGCTGTTATTGGTT ACTCTGATTTGGAGGGAGATTTTGATGTTGCTGTTTTGCCTTTCTCTAACTCTACTAACA

ACGGTTTGTTGTTCATTAACACTACTATCGCTTCTATCGCTGCTAAGGAAGAGGGTG TTT CTTTGGAAAAGAGAGAAGCTGAGGCTCACCATCACCACCATCACAAGAACACTATGGAA CATGTTTCTTCTTCTGAAGAGTCTATCATCTCTCAAGAAACTTACAAGCAAGAGAAAAAC

ATGGCTATTAATCCATCTAAGGAAAATTTGTGTTCTACTTTCTGTAAGGAGGTTGTT AGA AACGCTAATGAAGAGGAATACTCTATTGGTTCTTCTTCTGAGGAATCTGCTGAAGTTGCT ACTGAGGAAGTTAAGATCACTGTTGATGATAAGCACTACCAAAAGGCTTTGAACGAGAT TAATCAATTCTACCAAAAGTTCCCTCAATATTTGCAATACTTGTATCAAGGTCCAATTGT T TTGAACCCTTGGGATCAAGTTAAGAGAAACGCTGTTCCAATCACTCCTACTTTGAACAG AGAACAATTGTCTACTTCTGAGGAAAACTCTAAGAAAACTGTTGATATGGAATCTACTGA GGTTTTTACTAAGAAAACTAAGTTGACTGAGGAAGAGAAAAACAGATTGAACTTTTTGAA GAAGATTTCTCAAAGATACCAAAAGTTCGCTTTGCCACAATACTTGAAGACTGTTTACCA ACATCAAAAGGCTATGAAACCATGGATTCAACCTAAGACTAAAGTTATTCCTTACGTTAG ATATTTGCATCCACACCCTCATTTGTCTTTCATGGCTATTCCACCTAAGAAAAACCAAGA TAAGACTGAAATCCCAACTATTAATACTATTGCTTCTGGAGAGCCAACTTCTACTCCTAC TATTGAAGCTGTTGAGTCTACTGTTGCTACTTTGGAAGCTTCTCCTGAAGTTATTGAGTC TCCACCTGAGATTAATACTGTTCAAGTTACTTCTACTGCTGTTGGTTCTGGATCCCATCA CCATCACCATCACTAA (SEQ ID NO:24).

DNA sequence encoding fusion protein comprising p-casein-linker-CMP:

ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCT GCTC CAGTTAACACTACTACTGAAGATGAGACTGCTCAAATTCCTGCTGAAGCTGTTATTGGTT ACTCTGATTTGGAGGGAGATTTTGATGTTGCTGTTTTGCCTTTCTCTAACTCTACTAACA

ACGGTTTGTTGTTCATTAACACTACTATCGCTTCTATCGCTGCTAAGGAAGAGGGTG TTT CTTTGGAAAAGAGAGAAGCTGAGGCTCACCATCACCACCATCACAGAGAATTGGAAGA GTTGAACGTTCCAGGTGAAATTGTTGAGTCTTTGTCTTCTTCTGAAGAGTCTATCACTAG AATTAATAAGAAAATTGAGAAGTTTCAATCTGAAGAGCAACAACAAACTGAAGATGAGTT GCAAGATAAGATCCATCCATTCGCTCAAACTCAATCTTTGGTTTACCCATTCCCTGGTCC AATTCCTAACTCTTTGCCTCAAAATATTCCACCTTTGACTCAAACTCCAGTTGTTGTTCC A CCTTTCTTGCAACCTGAAGTTATGGGTGTTTCTAAGGTTAAAGAAGCTATGGCTCCAAA GCATAAAGAGATGCCATTTCCTAAGTATCCAGTTGAACCTTTCACTGAGAGACAATCTTT GACTTTGACTGATGTTGAAAACTTGCACTTGCCATTGCCTTTGTTGCAATCTTGGATGCA TCAACCACACCAACCTTTGCCACCTACTGTTATGTTTCCACCTCAATCTGTTTTGTCTTT GTCTCAATCTAAGGTTTTGCCAGTTCCTCAAAAAGCTGTTCCATACCCTCAAAGAGATAT GCCAATTCAAGCTTTCTTGTTGTATCAAGAACCAGTTTTGGGTCCTGTTAGAGGTCCATT TCCTATTATTGTTCATCCACACCCTCATTTGTCTTTCATGGCTATTCCACCTAAGAAAAA C CAAGATAAGACTGAAATCCCAACTATTAATACTATTGCTTCTGGAGAGCCAACTTCTACT CCTACTATTGAAGCTGTTGAGTCTACTGTTGCTACTTTGGAAGCTTCTCCTGAAGTTATT GAGTCTCCACCTGAGATTAATACTGTTCAAGTTACTTCTACTGCTGTTGGTTCTGGATCC CATCACCATCACCATCACTAA (SEQ ID NO:25).

In some embodiments, the sequence encoding the fusion protein is operably linked to a promoter. The skilled person will be aware of suitable promoters for expression of recombinant proteins in a given host. Preferred promoters may include those which are strong constitutive promoters in the presence of a particular carbon source (e.g. glucose, glycerol) or those which are strong regulated promoters (e.g. induced or derepressed under specific conditions such as presence or absence of a particular compound).

In some embodiments, the promoter is the GAP promoter (GAPp). The GAP promoter is the promoter from the Pichia pastoris TDH3 gene (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH). In some embodiments the promoter is that of the Pichia pastoris GCW14 gene (GCW14p). In some embodiments the promoter is that of the Pichia pastoris A0X1 gene (AOX1 p) or that of the Pichia pastoris HSP12 gene (HSP12p, also known a DHp).

In some embodiments, the sequence further comprises a terminator. Preferred terminators may include those which are associated with high levels of expression. Suitable terminators include those of the A0X1 and TDH3 genes of Pichia pastoris, and the terminators of the PGK1 , CYC1 , RPL3, and BNA4 genes of Saccharomyces cerevisiae. In some embodiments, the terminator is that of the Pichia pastoris A0X1 gene.

The promoter, the sequence which encodes the recombinant fusion protein and the terminator (if present), which are comprised within the nucleic acid molecule, are together referred to as an “expression cassette”. The invention thus also provides an expression cassette. In some embodiments, the nucleic acid molecule further comprises one or more homology regions (e.g. two homology regions). These can be used to direct integration of the sequence encoding the recombinant fusion protein, or the expression cassette, into the genome of a host cell. The skilled person will be capable of designing suitable homology regions using their common general knowledge. For example, the homology regions may be designed based on integration loci that have been successfully used previously, loci that are known to result in higher expression, and/or loci where integration does not disturb endogenous expression.

In some embodiments, the nucleic acid molecule, or the sequence encoding the recombinant fusion protein, is codon-optimised for expression in a desired host cell, such as a bacterial cell or a eukaryotic cell (e.g. a yeast or a plant cell). In some embodiments the nucleic acid molecule, or the sequence encoding the recombinant fusion protein, is codon-optimised for expression in Pichia pastoris.

The invention further provides a vector comprising the nucleic acid molecule described herein.

The vector may be a plasmid, a cosmid, or a viral vector.

In some embodiments, the vector is an expression vector for expression of the recombinant fusion protein in a host cell. The expression vector may be a plasmid or a cosmid. It will be appreciated that an expression vector is a vector (e.g. a DNA vector) which can be introduced into a host cell and which will remain autonomous (i.e. it will replicate within the host cell and remain as an individual entity rather than be integrated in the genome) and from which proteins will be expressed.

In some embodiments, the vector is a cloning vector, such as a plasmid.

Also provided is a host cell comprising a nucleic acid molecule (e.g. an expression cassette) or a vector (e.g. an expression vector or a cloning vector) as described herein.

In some embodiments, the host cell comprises an expression cassette which is integrated into the genome of the host cell, wherein the expression cassette comprises a promoter, a sequence encoding the recombinant nucleic acid molecule operably linked to the promoter and, optionally, a terminator. Integration of the expression cassette into the host cell genome can be carried out using standard techniques known to those skilled in the art, and as described herein. For example, the host cell may be co-transformed with: (i) a donor DNA molecule comprising the expression cassette located between two homology regions; and (ii) a plasmid comprising an RNA-guided endonuclease (CRISPR) system. The plasmid may further comprise an antibiotic resistance gene, enabling the selection of host cells which have been successfully transformed with the plasmid DNA. The proportion of plasmid and donor DNA in the transformation mix may be controlled so as to achieve a high probability that any host cell transformed with the plasmid is also transformed with the donor DNA molecule. The RNA-guided endonuclease (CRISPR) system ensures that the host cell genome has a double-strand cut at a target location, thereby promoting homology-driven recombination of the donor DNA molecule such that the expression cassette is integrated into the host cell genome.

In some embodiments, the host cell comprises multiple expression cassettes which are integrated into the host cell genome. In some embodiments, the host cell genome comprises two, three, four or more expression cassettes. All of the expression cassettes may be the same. For example, each expression cassette may comprise a sequence encoding the same recombinant fusion protein. Insertion of multiple copies of the same gene (i.e. the sequence encoding the recombinant fusion protein) can be useful to increase protein yield. In some embodiments, each expression cassette is different. For example, each expression cassette may comprise a sequence encoding a different recombinant fusion protein.

In embodiments wherein the host cell comprises multiple expression cassettes, each expression cassette may comprise a different promoter (and, optionally a different terminator). This may be advantageous to reduce the risk of unwanted recombinations. The relative abundance of each recombinant protein expressed may also be controlled through the use of different promoters.

Such host cells may be prepared by transforming the host cell with: (i) a first donor DNA molecule comprising a first expression cassette located between a first pair of homology regions; and (ii) a first plasmid. Once integration of the first expression cassette into the host cell genome is complete, the first plasmid can be lost by culturing the host cells without selective pressure (i.e. in the absence of the antibiotic which the first plasmid confers resistance to). The host cell can then be transformed with: (i) a second donor DNA molecule comprising a second expression cassette located between a second pair of homology regions; and (ii) a second plasmid. The first and second expression cassettes may be the same (e.g. for integration of a second copy of DNA encoding the same recombinant fusion protein) or they may be different (e.g. for integration of DNA encoding a different recombinant fusion protein). The first and second pairs of homology regions can be different, so that the first and second expression cassettes are integrated into different regions of the host cell genome. The first and second plasmids can encode different guide RNAs.

In some embodiments, the host cell comprises an expression vector. The expression vector may comprise an expression cassette comprising a promoter, a sequence encoding the recombinant nucleic acid molecule operably linked to the promoter and, optionally, a terminator. The expression vector may be a plasmid. In some embodiments, the expression vector comprises multiple expression cassette, e.g. two, three, four or more expression cassettes. The expression cassettes may all be the same, or they may be different. For example, each expression cassette may comprise a sequence encoding a different recombinant fusion protein.

The host cell may be a bacterial cell or a eukaryotic cell. A eukaryotic host can be a plant cell, an animal cell or a fungal cell. A fungal cell can be a yeast cell.

In some embodiments, the host cell is a eukaryotic cell selected from Saccharomyces spp., Kluyveromyces spp., Pichia spp., Aspergillus spp., Tetrahymena spp., Yarrowla spp., Hansenula spp., Blastobotrys spp., Candida spp., Zygosaccharomyces spp., Debaryomyces spp., Fusarium spp., and Trichoderma spp.

In some embodiments, the host cell is Pichia spp, such as Pichia pastoris.

In some embodiments, the host cell is a plant cell, such as soy.

The invention also provides the use of the recombinant fusion protein described herein in the production of recombinant casein.

A method of producing a recombinant protein comprises culturing a host cell as described herein under conditions suitable for expression of a recombinant fusion protein as described herein.

Suitable conditions for culturing and expressing recombinant proteins in a given host cell will be known to those skilled in the art. For example, the expression of the recombinant fusion protein in a eukaryotic host cell, such as Pichia pastoris, may be carried out under the conditions described herein. In some embodiments, a method of culturing the host cell (e.g. a yeast such as Pichia pastoris) comprises: a) Culturing the host cell at a first temperature; b) Diluting the culture; and c) Culturing the diluted culture at a second temperature.

In some embodiments, the first temperature is 30 °C.

The host cell may be cultured at the first temperature until mid-exponential phase. In some embodiments, the host cell is cultured at the first temperature for 6-12 hours (e.g. about 8 hours)

In some embodiments, the culture is diluted to an ODeoo of from 0.01 to 0.2, or from 0.05 to 0.15 (e.g. 0.1).

In some embodiments, the second temperature is from 24 to 30 °C, or from 25 to 28 °C. In some embodiments the second temperature is 25 °C.

In some embodiments, the diluted culture (or a portion thereof) is cultured for 12-30 hours, or from 16-24 hours. In some embodiments, the diluted culture (or a portion thereof) is cultured for 16 hours.

In some embodiments, the method comprises: a) Culturing the host cell at 30 °C, optionally until the culture reaches mid-exponential phase; b) Diluting the culture to an ODeoo of 0.1 ; and c) Culturing the diluted culture at 25 °C, optionally for 16-24 hours.

In some embodiments, the host cell expresses a recombinant fusion protein which comprises a cleavage site between the casein and the peptide. In such embodiments, the method may further comprise treating the recombinant fusion protein with an enzyme or a chemical which is capable of cleaving the recombinant fusion protein at the cleavage site, so as to separate the casein from the peptide.

In some embodiments, the method further comprises separating the recombinant fusion protein from the host cell culture, prior to treating the recombinant fusion protein with the enzyme or chemical. Alternatively, the method may comprise treating the recombinant fusion protein with the enzyme or chemical in the host cell culture (i.e. by adding the enzyme or chemical to the culture), and then separating the cleaved casein from the culture.

The enzyme may be any suitable enzyme, such as those described herein. In some embodiments, the enzyme is chymosin.

In some embodiments, the method may further comprise inactivating the cleaving enzyme. This may be achieved by thermally treating the enzyme e.g. by heating. For example, the method may comprise heating a suspension comprising the recombinant fusion proteins. The suspension may be the host cell culture, or it may comprise the recombinant fusion proteins after separating the proteins from the cell culture, containing the enzyme. Inactivating the cleaving enzyme may help to prevent or reduce degradation of the separated casein. In some embodiments in which the separated casein is isolated (e.g. from the cell culture) rapidly after cleavage is performed, thermal inactivation of the cleaving enzyme may not be required.

The method may further comprise isolating and/or purifying the separated casein.

Isolating the separated casein from the host cell culture may comprise one or more of the following techniques: centrifugation; precipitation (e.g. by adjusting the pH and/or salt composition of the culture medium); ultrafiltration; size exclusion chromatography; affinity chromatography; ion exchange chromatography; reverse phase chromatography; and immunoprecipitation.

The invention thus provides a recombinant casein obtained by the method described hereinabove. The recombinant casein may be recombinant a-S1 casein, recombinant a-S2 casein or recombinant p-casein.

It will be appreciated that the recombinant casein may comprise, in addition to the amino acid sequence of the casein protein itself, one or more additional features that were present in the recombinant fusion protein, such as one or more tags, a signal sequence fragment, and/or a linker or portion thereof.

In some embodiments, the recombinant casein comprises at least a portion of a linker. The linker, or portion thereof, may be located at the C-terminus or the N-terminus of the recombinant casein. In some embodiments the linker, or portion thereof, is located at the C- terminus of the recombinant casein. In some embodiments, the recombinant casein comprises at least a portion of a cleavage site, such as an enzyme cleavage site (e.g. a chymosin cleavage site).

In some embodiments, the recombinant casein comprises a fragment of a signal sequence. Such a fragment may be generated by cleavage of the signal sequence during or immediately after translocation of the recombinant fusion protein into the host cell’s secretory pathway. The signal sequence fragment may be located at the N-terminus of the recombinant casein.

Thus, in some embodiments the recombinant casein may comprise the structure:

Signal sequence fragment - casein - linker (or portion thereof).

In some embodiments, the recombinant a-S1 casein comprises the sequence of SEQ ID NO: 26:

EAEAHHHHHHRPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSE STED QAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEI VPN SAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYT

DAPSFSDIPNPIGSENSEKTTMPLWHPHPHLSF (SEQ ID NO:26).

In some embodiments, the recombinant a-S2 casein comprises the sequence of SEQ ID NO: 27:

EAEAHHHHHHKNTMEHVSSSEESIISQETYKQEKNMAINPSKENLCSTFCKEWRNAN EEE YSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPIVLNPWDQ VK RNAVPITPTLNREQLSTSEENSKKTVDMESTEVFTKKTKLTEEEKNRLNFLKKISQRYQK FAL PQYLKTVYQHQKAMKPWIQPKTKVIPYVRYLHPHPHLSF (SEQ ID NO:27).

In some embodiments, the recombinant p-casein comprises the sequence of SEQ ID NO: 28: EAEAHHHHHHRELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIH PFA QTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPK Y PVEPFTERQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVP Q KAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIVHPHPHLSF (SEQ ID NO:28).

In the sequences of SEQ ID NOs 17-28 shown above, the underlined portions indicate tags or, in the case of the nucleic acid sequences, a sequence encoding a tag. It will be appreciated that one or both of the tags may be omitted from the fusion protein. Accordingly, the present invention also provides corresponding sequences without one or both of the tags (i.e. the same sequence but lacking one or both of the underlined portions).

The invention further provides the use of the recombinant fusion protein described herein in the production of a food composition.

The invention further provides a food composition comprising a recombinant fusion protein or a recombinant casein as described herein.

A method of making a food composition comprises:

(i) providing a recombinant fusion protein as described herein;

(ii) separating the casein from the peptide; and

(iii) using the separated casein to make a food composition.

In some embodiments, the recombinant fusion protein comprises a cleavage site between the casein and the peptide. In such embodiments, the step of separating the casein from the peptide comprises treating the recombinant fusion protein with an enzyme or a chemical capable of cleaving the recombinant fusion protein so as to separate the casein from the peptide.

In some embodiments, the food composition is an alternative food composition, such as an alternative dairy food composition.

As used herein, the term “alternative” refers to a composition that comprises at least one isolated, recombinant casein protein. The recombinant casein may be present in the composition instead of, or in addition to, its equivalent animal-derived protein. For example, an alternative dairy composition may comprise a recombinant a-S1 casein according to the invention instead of an animal-derived a-S1 casein. The “animal derived” will be understood as referring to a natural casein protein that comes from an animal, e.g. in animal milk.

In some embodiments, the alternative food composition does not comprise any animal-derived caseins.

In some embodiments, the alternative food composition comprises one, two or three recombinant casein proteins in accordance with the invention. For example, the alternative food composition may comprise recombinant a-S1 casein and recombinant a-S2 casein. The alternative composition may comprise recombinant a-S1 casein and recombinant p-casein. The alternative food composition may comprise recombinant a-S2 casein and recombinant - casein. In some embodiments, the alternative food composition comprises recombinant a-S1 casein, recombinant a-S2 casein and recombinant p-casein.

The alternative food composition may further comprise recombinant K-casein.

The alternative food composition may comprise one or more additional ingredients selected from: beta-lactoglobulin, alpha-lactalbumin, serum albumin, lactoferrin, immunoglobulin, vegetal protein, salt (e.g. sodium chloride or potassium chloride), starch, sugar (e.g. lactose, sucrose, glucose, fructose and/or galactose), water, flavouring agent, preservative, emulsifier (e.g. lecithin), fats.

In some embodiments, the alternative dairy composition is a milk composition, a butter composition, a yoghurt composition, a sour cream composition, a creme fraiche composition, a baby formula composition, a cream composition, an ice-cream composition, a custard composition, or a cheese composition.

The invention thus provides a food composition obtained by the method described herein above.

It will be appreciated that any aspect or embodiment described herein may be combined with any other aspect or embodiment as appropriate, unless otherwise stated.

Example 1 : Expression of recombinant fusion protein

Preparation constructs for expression of recombinant fusion proteins

1. Protein and DNA sequences of bovine caseins were obtained from databases. The codon usage of protein coding DNA was optimized for expression in Pichia pastoris.

2. DNA sequences coding for a promoter (GAPp: promoter of the Pichia pastoris TDH3 gene), S. cerevisiae a-mating factor (MF-a) signal sequence (SS), caseins (aS1 ,aS2, p, K), tags and terminator (AOX1t: terminator of the Pichia pastoris A0X1 gene) were produced by in vitro DNA synthesis and onboarded in a cloning vector (a derivative of pUC19 which retains the AmpR-Linker-Ori sequence, and in which the LacZ and multiple cloning site (MCS) region is replaced by a MCS partly derived from pllCrnu (Staal et al. 2019 Biotechniques 66(6); 254- 259) modified to include at its centre Aarl and Bsgl sites repeated and separated by a Sall site.). 3. A ca. 1000 bp region upstream of the genomic loci targeted for insertion was amplified from genomic DNA by PCR using a standard PCR protocol, thereby creating a first homology region (HRL).

4. A ca. 1000 bp region downstream of the genomic loci targeted for insertion was amplified from genomic DNA by PCR using a standard PCR protocol, thereby creating a second homology region (HRR).

The primers used in steps 3 and 4 to create the homology regions were designed with overhangs to allow assembly of the resulting fragments and a linearized cloning vector through a standard Gibson Assembly protocol (i.e. the reverse primer for HRL has an overhang that overlaps the forward primer for HRR, the forward primer for HRL has an overhang that overlaps the reverse primer from step 5 and the reverse primer for HRR has an overhang that overlaps the forward primer from step 5). Furthermore the forward primer for HRL and the reverse primer for HRR were designed to introduce type IIS restriction sites (e.g. Aarl, Bsgl) such that recognition occurs outside the homology region but cleavage occurs within.

5. A cloning vector (pUC19) was linearized by PCR with primers that amplify the entire vector except the MCS.

6. The DNA fragments resulting from steps 3, 4 and 5 where assembled to form a circular vector pUC19-HRL-HRR by a standard Gibson Assembly protocol.

7. A DNA fragment encoding the expression cassette Promoter-SS-Casein-Tag-Terminator was amplified by PCR for each of casein aS1 , aS2, p and K, from the vectors of step 2, wherein “SS” refers to a signal sequence.

8. The vector of step 6 was linearized by PCR with primers containing overhangs designed to overlap the first bases of the promoter and last bases of the terminator from step 7 (i.e. a reverse primer binding the 3’ end of HRL with an overhang complementary to the 5’ end of the promoter and a forward primer binding the 5’ end of HRR with an overhang complementary to the 3’ end of the terminator).

9. The linearized vector from step 8 was assembled into a circular vector with each of the DNA fragments from step 7 by a standard Gibson Assembly protocol, resulting in the circular vectors: a) pUC19-H RL-Promoter-SS-CSN 1 S1 -T ag-T erminator-H RR b) pUC19-H RL-Promoter-SS-CSN 1 S2-T ag-T erminator-H RR c) pUC19-HRL-Promoter-SS-CSN2-Tag-Terminator-HRR d) pUC19-HRL-Promoter-SS-CSN3-Tag-Terminator-HRR where CSN1S1 is the gene encoding aS1 casein, CSN1S2 is the gene encoding aS2 casein, CSN2 is the gene encoding casein, CSN3 is the gene encoding K casein and where K casein comprises the regions: para-casein K - chymosin recognition sequence (CRS) - caseinomacropeptide (CMP). 10. Vector (d) from step 9 was linearized by PCR with a reverse primer binding on the 3’ end of the SS and containing an overhang coding for a tag and a forward primer binding on the 5’ end of the CRS resulting in a linear DNA molecule that excludes the para Kappa Casein fragment: CRS-CMP-Tag-Terminator-HRR-pUC19-HRL-Promoter-SS-Tag

11 . DNA encoding caseins aS1 , aS2, and p (without SS or Tag) was amplified by PCR from the plasmids of step 9. The primers used were designed to introduce an N-terminal tag and contain overhangs to allow Gibson Assembly with the linear DNA molecule of step 10 (i.e. a forward primer binding on the 5’ end of each casein with an overhang complementary to the tag introduced by the reverse primer of step 10, and a reverse primer binding the 3’ end of each casein with an overhang complementary to the 5’ end of the CRS).

12. The linearized vector from step 10 was assembled into a circular vector with each of the DNA molecules from step 11 by a standard Gibson Assembly protocol resulting in the circular vectors: e) pUC19-H RL-Promoter-SS-T ag-CSN 1 S1 -linker-CM P-T ag-T erminator-H RR f) pUC19-H RL-Promoter-SS-T ag-CSN 1 S2-linker-CM P-T ag-T erminator-H RR g) pUC19-HRL-Promoter-SS-Tag-CSN2-linker-CMP-Tag-Terminator-HRR where the linker comprises the chymosin recognition sequence (CRS).

13. Donor DNA was separated from backbone DNA (pUC19) by restriction of the vectors of steps 9 and 12 on the Aarl or Bsgl restriction sites introduced in steps 3 and 4 (on the 5’ end of HRL and the 3’ end of HRR) followed by agarose gel electrophoresis and extraction of the pertinent DNA band from the gel.

At all pertinent steps, correct amplification and assembly of DNA was verified by restriction analysis and/or sequencing.

Note that Gibson Assembly of only two fragments (backbone plus a single insert) can be inefficient compared to assembly of three or more fragments. To counter this, in steps 8 and 10 the backbone can be linearized into two partially overlapping fragments rather than a single full-length fragment.

Transformation of host cells

14. Electro-competent Pichia pastoris cells (strain NRRL Y-11430 also known as CBS7435 or ATCC 76273) were prepared from exponentially growing cells by treatment with lithium acetate and sorbitol as described by Wu and Letchworth 2004 BioTechniques 36:152.

15. The electro-competent cells of step 14 were mixed with the donor DNA molecules of step 13 (individually) and with an episomal vector expressing an RNA guided endonuclease, guide RNA specific for the genomic loci targeted for insertion and an antibiotic resistance gene.

16. The mix of step 15 was transferred to an electroporation cuvette, subjected to a pulse (2 kV, 25 pF, 200 ohm), diluted with YPD growth media (Yeast extract 1 %, Peptone 2%, Dextrose 2%), transferred to a fresh vial, allowed to recover for 2 hours at 30°C and spread on a YPD- Agar (2%) plate with an antibiotic for selection according to the resistance provided by the vector of step 15.

17. After 2-3 days of growth at 30°C single transformed colonies were picked from the selective YPD-Agar plate of step 16 and analysed by a standard colony PCR protocol with primers specific for the targeted genomic locus and the donor DNA.

18. Clones identified as positive (i.e. with a correctly integrated expression cassette) in step 17 were spread on non-selective YPD-Agar plates.

19. After 2 days at 30°C single colonies were picked from the plates of step 18 and spread on selective and non-selective plates. This step was repeated, passing cells from the non- selective plates onto selective and non-selective plates, until there was no growth on selective plates (indicating loss of the episomal vector).

Host cell culture

20. Cells from step 19 were inoculated in 3 ml YPD and grown at 30 °C for ca. 8 h until midexponential phase.

21. The pre-cultures of step 20 were diluted to OD600 0.014 in 30 ml of BMDY media (2% peptone, 1 % yeast extract, 100 mM potassium phosphate pH 6, 13.4 g/L YNB with ammonium sulphate, 2 % glucose 0.4 mg/L biotin) and grown for 25 h at 30 °C.

22. Samples from the cultures of step 21 where collected at various time points. Results are reported for samples at 23 hours of growth. For sampling, 4 ml was removed from the culture.

Protein recovery

23. Secreted proteins in the samples of step 22 were separated from the cells by centrifugation, 5 min 1500 ref.

Determination of expression levels.

24. Recombinant casein or casein - CMP hybrids in a fraction of the supernatants from step 23 (e.g. 200 pl) were diluted as required (e.g. 500x) in PBS and quantified by a standard ELISA protocol using an HRP conjugated antibody specific against the tag (HIS), an HRP substrate (ABTS) and a calibration curve with a tagged protein of known concentration (HIS tagged FKBP12). To calculate protein concentration from detected absorbance, the differences in molecular weight of the standard and the various recombinant proteins as well as the number of tags on a single recombinant protein were taken into account.

Results Caseins (CSN) alpha S1 , alpha S2, beta and kappa were expressed, under the same conditions, from a strong promoter. As shown in Figure 3, the titre of casein kappa after 23 hours of culture was found to be four times that of casein alpha S1. The titres of caseins alpha S2 and beta were so low that they were almost at the detection limit (21 and 37 times less than that of casein kappa, respectively).

Constructs were prepared for expression of recombinant fusion proteins. With reference to Figure 2a, constructs were designed to include a DNA sequence encoding a recombinant fusion protein 10 comprising, from the C-terminus to the N-terminus: a MF-a signal sequence from S. cerevisiae 12; a first HIS tag 14; a casein protein 16; a linker 18 comprising a cleavage site; a caseinomacropeptide 20; and a second HIS tag 22. The DNA sequence was operably linked to promoter and terminator sequences. Homology regions to direct chromosomal integration are also fused to these DNA molecules. Separate constructs were prepared for the expression of each of a-S1 casein, a-S2 casein and p-casein as fusion proteins.

The a-S1 casein - CMP fusion protein expressed in Example 1 is designated herein as SEQ ID NO: 17. The corresponding DNA sequence is designated as SEQ ID NO: 23.

The a-S2 casein - CMP fusion protein expressed in Example 1 is designated herein as SEQ ID NO: 19. The corresponding DNA sequence is designated as SEQ ID NO: 24.

P-casein - CMP fusion protein expressed in Example 1 is designated herein as SEQ ID NO: 21. The corresponding DNA sequence is designated as SEQ ID NO: 25.

It will be appreciated that upon secretion of the fusion proteins from the host cell the signal sequence will be cleaved (indicated in Figure 2 by the dotted line), leaving behind a short fragment of the signal sequence 12’ in the expressed protein. While the fusion proteins shown in the embodiment of Figure 2 comprised HIS tags, it will be appreciated that in other embodiments the recombinant fusion protein may contain different tags or no tags.

The resulting DNA constructs were separately transformed into Pichia pastoris host cells, which were cultured. This resulted in the host cells expressing the recombinant fusion proteins a-S1 casein-CMP, a-S2 casein-CMP or p-casein-CMP under the same conditions and from the same promoter as the (non-fusion) recombinant caseins from Figure 3. After 23 hours the titre of casein kappa was only 1.1 times that of a-S1 casein-CMP or a-S2 casein-CMP, and 1 .4 times that of p-casein-CMP (Figure 4). Example 2: cleavage of recombinant fusion protein

With reference to Figure 2, the recombinant fusion proteins may be cleaved at a cleavage site (CS) (e.g. using chymosin) so as to separate the caseinomacropeptide from the casein protein. In the embodiment shown in Figure 2b, the resulting cleaved casein protein 24 retains at least a portion of the linker 18’. Since cleavage of the CMP is carried out after secretion, the cleaved casein protein 24 comprises only a fragment 12’ of the signal sequence 12.

Cleavage may be carried out according to the following protocol:

1. To supernatants containing a recombinant casein - CMP fusion, prepared as described in Example 1 , a solution of cleavage enzyme (e.g. chymosin) in water is added to a final concentration of ca. 1 CU (coagulating unit) per gram of recombinant protein (as an example a standard commercial chymosin is provided as a solid with 1700 Cll/g).

2. The mixture of step 1 is incubated for 20 min at 40 °C

3. For evaluation of cleavage 1 ml of the reaction from step 2 is stopped by addition of 250 pl trichloroacetic acid (TCA) 100%.

4. For recovery of recombinant protein the remainder of the reaction from step 2 is stopped by thermal treatment (2 min 60 °C).

5. Proteins and peptides are precipitated from the mix of step 3 by centrifugation at 4 °C, 10000 ref for 15 minutes.

6. The supernatant from step 5 is completely removed.

7. The pellet from step 6 is washed once with ice cold acetone. Acetone is completely removed after centrifugation as in steps 5 and 6 and the resulting pellet is dried.

8. The dry pellet from step 7 is resuspended in Laemmli sample buffer with 0.1M dithiothreitol (DTT).

9. The mix of hybrids I proteins I peptides in the suspension from step 8 are separated through a standard SDS-PAGE electrophoresis protocol.

10. The separated hybrids I proteins I peptides from step 9 are analysed through a standard western blot protocol with an antibody specific for the tags (HIS) to evaluate the ratios of uncleaved I cleaved I degraded proteins.

11 . Proteins in the mix from step 4 are recovered in purified form from the growth/reaction media through: salt and pH-induced precipitation and/or ultrafiltration and/or a combination of chromatographic methods. Example 3: FLAG-tagged constructs

A recombinant fusion protein comprising a-S1 casein similar to that described in Example 1 was prepared, except that instead of two HIS tags the protein had an N-terminal FLAG tag and a C-terminal HIS tag.

The a-S1 casein - CMP fusion protein expressed in this example is designated herein as SEQ ID NO: 29. The corresponding secreted protein is designated herein as SEQ ID NO: 30. The corresponding DNA sequence is designated as SEQ ID NO: 31 :

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSN STNNGL

LFINTTIASIAAKEEGVSLEKREAEADYKDDDDKGSGSRPKHPIKHQGLPQEVLNEN LLRFFV

APFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQ KEDVP

SERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAY FYPEL

FRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLWHPHPHLS FMAI

PPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVT STAVGSG

SHHHHHH (SEQ ID NO: 29).

EAEADYKDDDDKGSGSRPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELS KDIG

SESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLK KYKVP

QLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAW YYVP

LGTQYTDAPSFSDIPNPIGSENSEKTTMPLWHPHPHLSFMAIPPKKNQDKTEIPTIN TIASGE

PTSTPTI EAVESTVATLEASPEVI ESPPEI NTVQVTSTAVGSGSH H H H H H (SEQ ID NO: 30).

ATGAGATTTCCATCTATTTTCACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCT GCTC

CAGTTAACACTACTACTGAAGATGAGACTGCTCAAATTCCTGCTGAAGCTGTTATTG GTT

ACTCTGATTTGGAGGGAGATTTTGATGTTGCTGTTTTGCCTTTCTCTAACTCTACTA ACA

ACGGTTTGTTGTTCATTAACACTACTATCGCTTCTATCGCTGCTAAGGAAGAGGGTG TTT

CTTTGGAAAAGAGAGAAGCTGAGGCTGATTATAAGGACGATGACGATAAAGGTTCTG G

ATCCAGACCAAAGCATCCTATTAAACACCAAGGTTTGCCACAAGAAGTTTTGAACGA GA

ATTTGTTGAGATTTTTCGTTGCTCCATTTCCTGAAGTTTTCGGTAAAGAAAAAGTTA ACG

AGTTGTCTAAGGATATTGGTTCTGAATCTACTGAGGATCAAGCTATGGAAGATATCA AG

CAAATGGAAGCTGAGTCTATCTCTTCTTCTGAAGAGATTGTTCCAAACTCTGTTGAA CAA

AAGCATATCCAAAAGGAAGATGTTCCTTCTGAGAGATACTTGGGTTATTTGGAACAA TTG

TTGAGATTGAAGAAATACAAGGTTCCACAATTGGAGATTGTTCCTAACTCTGCTGAA GA

GAGATTGCATTCTATGAAGGAAGGTATTCACGCTCAACAAAAAGAGCCAATGATTGG TG

TTAATCAAGAATTGGCTTACTTCTACCCTGAGTTGTTTAGACAATTCTACCAATTGG ATG

CTTATCCATCTGGTGCTTGGTACTATGTTCCTTTGGGTACTCAATACACTGATGCTC CAT

CTTTCTCTGATATTCCAAACCCTATTGGTTCTGAAAATTCTGAGAAAACTACTATGC CTTT

GTGGCATCCACACCCTCATTTGTCTTTCATGGCTATTCCACCTAAGAAAAACCAAGA TAA

GACTGAAATCCCAACTATTAATACTATTGCTTCTGGAGAGCCAACTTCTACTCCTAC TAT

TGAAGCTGTTGAGTCTACTGTTGCTACTTTGGAAGCTTCTCCTGAAGTTATTGAGTC TCC

ACCTGAGATTAATACTGTTCAAGTTACTTCTACTGCTGTTGGTTCTGGATCCCATCA CCA

TCACCATCACTAA (SEQ ID NO: 31). Methods

1 . Vector (a) from step 9 in example 1 was linearized by PCR with a forward primer annealing on the 5’ end of CSN1S1 and a reverse primer annealing on the 3’ end of SS. Both primers were designed with overhangs, complementary to each other, coding for a FLAG tag.

2. The linear DNA molecule of step 1 was circularized by a standard Gibson Assembly protocol resulting in the circular vector: h) pUC19-HRL-Promoter-SS-Tag-CSN1S1-Tag-Terminator-HRR

3. Vector (d) from step 9 in example 1 was linearized by PCR with a reverse primer binding on the 3’ end of the SS and containing an overhang coding for a FLAG tag and a forward primer binding on the 5’ end of the CRS resulting in a linear DNA molecule that excludes the para Kappa Casein fragment: CRS-CMP-Tag-Terminator-HRR-pUC19-HRL-Promoter-SS- Tag

4. DNA encoding casein a-S1 (without SS or Tag) was amplified by PCR from vector (a) of step 9 in example 1. The primers used were designed to introduce an N-terminal FLAG tag and contain overhangs to allow Gibson Assembly with the linear DNA molecule of step 3 (i.e. a forward primer binding on the 5’ end of the casein with an overhang complementary to the tag introduced by the reverse primer of step 3, and a reverse primer binding the 3’ end of casein aS1 with an overhang complementary to the 5’ end of the CRS).

5. The linearized vector from step 3 was assembled into a circular vector with the DNA molecule from step 4 by a standard Gibson Assembly protocol resulting in the circular vector: i) pUC19-HRL-Promoter-SS-Tag-CSN1S1-linker-CMP-Tag-Terminator-H RR

6. Donor DNA was prepared from the vectors (h) and (i) of steps 2 and 5 and transformed into competent Pichia pastoris cells as described in example 1.

Results

Figure 5a shows the generation of recombinant a-S1 casein by chymosin cleavage of the recombinant fusion protein a-S1 casein-CMP. Figure 5b shows that almost 80% of the fusion protein was effectively cleaved by chymosin.

Example 4: Variation of culture conditions

1. Host cells (P. pastoris) expressing a-S1 casein or a-S1 casein-CMP were inoculated in 3 ml Yeast Extract-Peptone-Dextrose (YPD) and grown at 30 °C for ca. 8 h until midexponential phase.

2. The pre-cultures of step 1 were diluted to various GD600 values (e.g. 0.014, 0.05, 0.1) in 30 ml of BMDY media (2% peptone, 1 % yeast extract, 100 mM potassium phosphate, 13.4 g/L YNB with ammonium sulphate, 2 % glucose 0.4 mg/L biotin) buffered at various pH values (e.g. pH5, pH6, pH7) and grown at various temperatures (e.g. 25 °C, 28 °C, 30 °C).

3. Samples were collected at different intervals (e.g. 16 h, 20 h, 23 h). For sampling, 4 ml was removed from the culture.

4. Secreted proteins in the samples of step 3 were separated from the cells by centrifugation and quantified by a standard ELISA protocol as described in example 1.

Results

Figure 6 compares the results from example 1 (host cells grown at 30 °C for 23 h after dilution to OD600 0.014 — condition A) to the result from host cells grown at 25 °C for 16 h after dilution to OD600 0.1 — condition B. In both conditions media was buffered at pH 6.

The titre of recombinant a-S1 casein was marginally improved by variation of culture conditions (10%). In contrast, the titre of the recombinant fusion protein a-S1 casein - CMP increased over 6-fold, resulting in a-20 fold higher titre than that of the non-fusion protein.

Example 5: CMP truncations

Preparation of constructs for expression of recombinant fusion proteins

C-terminal truncation

1. A Linear DNA molecule was produced by PCR amplification of the vector (i) from step 5 in Example 3 with a reverse primer annealing on the CMP immediately upstream of the region to be eliminated and a forward primer annealing immediately downstream of the CMP and containing a 5’ overhang homologous to the reverse primer.

2. The DNA molecule of step 1 was circularized by a standard Gibson Assembly protocol, resulting in the circular vector: j) pUC19-H RL-Promoter-SS-T ag-CSN 1 S1 -linker-CtruncCM P-T ag-T erminator-H RR where CtruncCMP indicates a CMP with a C-terminal truncation, in which the last 17 amino acids were removed. CtruncCMP has a sequence as shown in SEQ ID NO: 11.

N-terminal truncation

3. A linear DNA molecule was produced by PCR amplification of the vector (i) from step 5 of Example 3 with a reverse primer annealing on the linker immediately upstream of the CMP and a forward primer annealing on the CMP immediately downstream of the region to be eliminated and containing a 5’ overhang homologous to the reverse primer. 4. The DNA molecule of step 3 was circularized by a standard Gibson Assembly protocol, resulting in the circular vector: k) pUC19-HRL-Promoter-SS-Tag-CSN1S1-linker-NtruncCMP-Tag-Termin ator-HRR where NtruncCMP indicates a CMP with an N-terminal truncation, in which the first 14 amino acids of the N-terminus were removed. NtruncCMP has a sequence as shown in SEQ ID NO: 10.

5. Donor DNA was separated from backbone DNA (pUC19) by restriction of the vectors of steps 2 and 4 on the Aarl or Bsgl restriction sites on the 5’ end of HRL and the 3’ end of HRR followed by agarose gel electrophoresis and extraction of the pertinent DNA band from the gel.

At all pertinent steps, correct amplification and assembly of DNA was verified by restriction analysis and/or sequencing.

Transformation of host cells

6. Pichia pastoris cells were transformed and selected as described in example 1.

Host cell culture

7. Cells from step 6 of this example and cells from step 6 of Example 3 (expressing a-S1 casein and a-S1 casein - CMP) were inoculated in 3 ml YPD and grown at 30 °C for ca. 8 h until mid-exponential phase.

8. The pre-cultures of step 7 were diluted to OD600 0.1 in 30 ml of BMDY media (2% peptone, 1 % yeast extract, 100 mM potassium phosphate pH 6, 13.4 g/L YNB with ammonium sulphate, 2 % glucose 0.4 mg/L biotin) and grown at 25 °C.

9. Samples from the cultures of step 8 where collected after 16 hours.

Protein recovery and determination of expression levels.

10. Secreted proteins in the samples of step 9 were separated from the cells by centrifugation and quantified by a standard ELISA protocol as described in example 1.

Results

Proteins a-S1 casein, a-S1 casein-CMP, a-S1 casein-CtruncCMP, a-S1 casein-NtruncCMP were expressed, under the same conditions, from a strong promoter. As shown in Figure 7, fusion to either a C-terminal truncated or an N-terminal truncated CMP improved the expression of alpha S1 casein. Example 6: N-terminal CMP

Preparation of constructs for expression of recombinant fusion proteins

1 . Using the vector (i) from step 5 of Example 3 as a template pUC19-HRL-Promoter-SS-Tag-CSN1S1-linker-CMP-Tag-Terminator-H RR, a DNA fragment comprising

Terminator-HRR-pUC19-HRL-Promoter-SS-Tag was generated by a standard PCR protocol with a forward primer annealing on the terminator and a reverse primer annealing on the N-terminal TAG. The reverse primer was designed with an overhang complimentary to the 5’ portion of the linker.

2. Using the same vector as template a DNA fragment comprising

Iinker-CMP-Iinker2 was generated by a standard PCR protocol with a forward primer annealing on the 5’ end of the linker and a reverse primer annealing on the 3’ end of the CMP. The reverse primer was designed with an overhang to introduce a flexible linker (Iinker2).

3. Using vector (a) from step 9 in example 1 as a template pUC19-H RL-Promoter-SS-CSN 1 S1 -Tag-Terminator-H RR a DNA fragment comprising

CSN1S1 -Tag-Terminator was generated by a standard PCR protocol with a forward primer annealing on the 5’ end of CSN1S1 and a reverse primer annealing on the terminator downstream from the forward primer of step 1. The forward primer was designed with an overhang complimentary to the overhang of the reverse primer from step 2.

4. The DNA fragments from steps 1-3 were assembled, by a standard Gibson Assembly protocol, into a circular vector:

I) pUC19-HRL-Promoter-SS-Tag-linker-CMP-linker2-CSN1S1-Tag-Term inator-HRR

Transformation of host cells

5. Donor DNA was prepared from vector (I) of step 4, host cells (P. pastoris) were transformed and selected as described in Example 1 .

Host cell culture, protein recovery and determination of expression levels.

6. Transformed cells were cultured, recombinant proteins were recovered from cultures and quantified as described in Example 5. Results

Protein a-S1 casein, and the fusion proteins a-S1 casein with an N terminal CMP (CMP - CSN a-S1) and alpha S1 with a C terminal CMP (CSN a-S1 - CMP) were expressed, under the same conditions, from a strong promoter. As shown in Figure 8, fusion of the CMP on the N termini of the protein increased its expression by more than 2 fold (2.4). Under the same conditions, fusion of the CMP on the C termini of the protein was almost 10 times more effective.

Example 7: Expression in different species

Preparation of constructs for expression of recombinant fusion proteins in Saccharomyces cerevisiae

1 . A ca. 600 bp region upstream of the genomic loci targeted for insertion was amplified from S. cerevisiae genomic DNA by PCR using a standard PCR protocol, thereby creating a first homology region (HRL).

2. A ca. 700 bp region downstream of the genomic loci targeted for insertion was amplified from S. cerevisiae genomic DNA by PCR using a standard PCR protocol, thereby creating a second homology region (HRR).

The primers used in steps 1 and 2 to create the homology regions were designed with overhangs to allow assembly of the resulting fragments and a linearized cloning vector through a standard Gibson Assembly protocol

3. A cloning vector (pUC19) was linearized by PCR with primers that amplify the entire vector except the MCS.

4. The DNA fragments resulting from steps 1-3 where assembled to form a circular vector pUC19-HRL-HRR by a standard Gibson Assembly protocol.

5. A DNA sequence coding for MFaSS-TAG-CSN aS1-TAG was generated by a standard PCR protocol from the vector (h) of step 2 in Example 3.

6. A DNA sequence coding for MFaSS-TAG-CSN aS1-linker-CMP-TAG was generated by a standard PCR protocol using the vector (i) from step 5 in Example 3 as a template.

7. A DNA sequence coding for a promoter (ScGAPp: promoter of the Saccharomyces cerevisiae TDH3 gene) was amplified from S. cerevisiae genomic DNA by a standard PCR protocol.

8. A DNA sequence coding for a terminator (ScCYCIt: terminator of the Saccharomyces cerevisiae CYC1 gene) was amplified from S. cerevisiae genomic DNA by a standard PCR protocol. 9. A DNA sequence coding for a Kanamycin/G418 resistance cassette was amplified by PGR from a common yeast expression plasmid.

10. The vector of step 4 was linearized using a standard PGR protocol.

The primers used in steps 5-10 were designed with overhangs to create overlaps between the resulting DNA fragments.

11. The linearized vector from step 10 was assembled, by a standard Gibson Assembly protocol, into a circular vector with the DNA fragments from steps 7-9 and either the fragment from step 5 or the fragment from step 6, resulting in the circular vectors: m) pUC19-HRL-Promoter-SS-Tag-CSN1S1-Tag-Terminator-KanMX-HRR n) pUC19-HRL-Promoter-SS-Tag-CSN1S1-linker-CMP-Tag-Terminator-K anMX-HRR where CSN1S1 is the gene encoding aS1 casein, the linker is the chymosin recognition sequence and KanMX is a cassette conferring resistance to the antibiotic G418.

12. Donor DNA was generated from the vectors of step 11 by a standard PGR protocol using a forward primer at the 5’ end of the upstream homology region (HRL) and a reverse primer at the 3’ end of the downstream homology region (HRR).

At all pertinent steps, correct amplification and assembly of DNA was verified by restriction analysis and/or sequencing.

Transformation of host cells

13. Electro-competent Saccharomyces cerevisiae cells (isogenic to strain S288C) were prepared from exponentially growing cells by treatment with lithium acetate and sorbitol as described by Wu and Letchworth 2004 BioTechniques 36:152.

14. The electro-competent cells of step 13 were mixed with the donor DNA molecules of step 12 (individually) and transferred to an electroporation cuvette, subjected to a pulse (2 kV, 25 pF, 200 ohm), diluted with YPD growth media (Yeast extract 1%, Peptone 2%, Dextrose 2%), transferred to a fresh vial, allowed to recover for 2 hours at 30°C and spread on a YPD-Agar (2%) plate with the antibiotic G418 for selection.

15. After 2-3 days of growth at 30°C single transformed colonies were picked from the selective YPD-Agar plate of step 14 and analysed by a standard colony PGR protocol with primers specific for the targeted genomic locus and the donor DNA.

Host cell culture

16. Cells from step 15 were inoculated in 3 ml YPD and grown at 30 °C for ca. 8 h until midexponential phase.

17. The pre-cultures of step 16 were diluted to GD600 0.1 in 30 ml of YPD media and grown at 25 °C. 18. Samples from the cultures of step 17 where collected after 16 hours of growth. For sampling, 4 ml was removed from the culture.

Protein recovery and determination of expression levels.

19. Recombinant proteins were recovered from cultures and quantified as described in example 1 .

Results a-S1 casein and recombinant fusion protein a-S1 casein-CMP (CSN alpha S1-CMP) were expressed in S. cerevisiae, under the same conditions, from a strong promoter. As shown in Figure 9, equivalent to the results obtained in P. pastoris, the titre of the fusion protein CSN alpha S1-CMP was over 7 fold that of (non-fusion) CSN alpha S1.

Comparative Example 8: Use of whole kappa casein instead of the CMP

Preparation of constructs for expression

1 . Using as a template the vector (i) from step 5 in Example 3, pUC19-HRL-Promoter-SS-Tag-CSN1S1-linker-CMP-Tag-Terminator-H RR, a DNA fragment comprising

Promoter-SS-Tag-CSN 1 S1 was generated by a standard PCR protocol with a forward primer annealing on the promoter and a reverse primer annealing on the 3’ end of CSN1S1. The reverse primer was designed with an overhang complementary to the 5’ end of CSN3.

2. Using the same vector as template a DNA fragment pUC19-HRL-Promoter was generated by a standard PCR protocol with a forward primer annealing on the vector’s backbone and a reverse primer annealing on the Promoter downstream from the forward primer of step 1

3. Using vector (d) from step 9 in example 1 pUC19-HRL-Promoter-SS-CSN3-Tag-Terminator-HRR a DNA fragment

CSN3-Tag-Terminator-HRR-pUC19 was generated by a standard PCR protocol with a forward primer annealing on the 5’ end of CSN3 and a reverse primer annealing on the vector’s backbone downstream from the forward primer of step 2.

4. The DNA fragments from steps 1-3 were assembled, by a standard Gibson Assembly protocol, into a circular vector: (o) pUC19-HRL-Promoter-SS-Tag-CSN1S1-CSN3-Tag-Terminator-HRR

Transformation of host cells

5. Donor DNA was prepared from vector (o) of step 4, host cells (P pastoris) were transformed and selected as described in example 1.

Host cell culture, protein recovery and determination of expression levels.

6. Transformed cells were cultured, recombinant proteins were recovered from cultures and quantified as described in Example 5.

Results

The recombinant protein a-S1 casein (CSN a1) and the recombinant fusion proteins comprising a-S1 casein fused to the CMP (CSN cd-CMP) or fused to the complete casein kappa protein (CSN c -CSN K) were expressed, under the same conditions, from a strong promoter. As shown in Figure 10, while fusion to the CMP resulted in a 20-fold increase in the titre of the recombinant protein, fusion to the complete kappa casein resulted in only a 1 .2 fold increase.

The invention may be described by any of the following clauses:

1. A recombinant fusion protein comprising:

(a) a casein selected from a-S1 casein, a-S2 casein and p-casein; and

(b) a peptide comprising a caseinomacropeptide (CMP), or a fragment thereof.

2. The recombinant fusion protein of clause 1 , wherein the recombinant fusion protein does not comprise a full-length kappa casein protein.

3. The recombinant fusion protein of clause 1 or clause 2, wherein the recombinant fusion protein comprises a sequence of no more than 150, no more than 120, no more than 100, no more than 90, no more than 80, no more than 75, no more than 70, no more than 65 or no more than 60 consecutive amino acids derived from the C-terminus of kappa casein.

4. The recombinant fusion protein of any preceding clause , wherein the peptide is capable of decreasing the hydrophobicity of the recombinant fusion protein relative to the hydrophobicity of the casein alone. 5. The recombinant fusion protein of any preceding clause, wherein the solubility of the recombinant fusion protein in a medium is greater than the solubility of the casein in the medium.

6. The recombinant fusion protein of any preceding clause, wherein the CMP or fragment thereof comprises an amino acid sequence of at least 20 residues.

7. The recombinant fusion protein of any preceding clause, wherein the CMP comprises an N-terminal and/or a C-terminal truncation.

8. The recombinant fusion protein of any preceding clause, wherein the CMP or fragment thereof comprises:

(i) the amino acid sequence:

X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2 X ₃ (SEQ ID NO:5) wherein each Xi is independently selected from S and T, each X2 is independently selected from D and E, and each X3 is independently selected from A, I, L and V, or

(ii) an amino acid sequence which is at least 80% identical to SEQ ID NO: 5, optionally wherein at least 3 of the Xi residues are retained.

9. The recombinant fusion protein of any preceding clause, wherein the CMP or fragment thereof comprises

(i) an amino acid sequence selected from:

X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2 X3X3X2SPPX2X3NT X3QX3X1STX3X3

(SEQ ID NO:6); and

KNQX2KTX2X3PX1 X3NTX3X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2 X ₃ (SEQ ID NO:7), or

(ii) an amino acid sequence which is at least 80% identical to SEQ ID NO:6, optionally wherein at least 3 of the Xi residues are retained, or

(iii) an amino acid sequence which is at least 80% identical to SEQ ID NO:7, optionally wherein at least 3 of the Xi residues are retained.

10. The recombinant fusion protein of any preceding clause, wherein the CMP or fragment thereof comprises

(i) the amino acid sequence KNQX2KTX2X3PX1 X3NTX3X3SGX2PX1 X1X1PTX3X2X3X3X2S X1X3X3X1X3X2X3X1PX2

X3X3X2SPPX2X3NT X3QX3X1STX3X3 (SEQ ID N0:8); or

(ii) an amino acid sequence which is at least 80% identical to SEQ ID NO:8, optionally wherein at least 3 of the Xi residues are retained.

11. The recombinant fusion protein of part (ii) or (iii) of any one of clauses 5 to 8, wherein all of the Xi residues are retained.

12. The recombinant fusion protein of any preceding clause, wherein the CMP or fragment thereof comprises

(i) the amino acid sequence

ASGEPT STPTI EAVES TVATLEASPE V (SEQ ID NO:9), or

(ii) an amino acid sequence which is at least 80% identical to SEQ ID NO: 9..

13. The recombinant fusion protein of any preceding clause, wherein the CMP or fragment thereof comprises:

(i) an amino acid sequence selected from:

ASGEPT STPTI EAVES TVATLEASPE VIESPPEINT VQVTSTAV (SEQ ID NO: 10);

KNQDKTEIPT INTIASGEPT STPTIEAVES TVATLEASPE V (SEQ ID NO:11); and KNQDKTEIPT INTIASGEPT STPTIEAVES TVATLEASPE VIESPPEINT VQVTSTAV (SEQ ID NO:12), or

(ii) an amino acid sequence which is at least 80% identical to SEQ ID NO: 10, SEQ ID NO:11 or SEQ ID NO:12.

14. The recombinant fusion protein of any preceding clause wherein the CMP or fragment thereof comprises at least 3 glycosylation sites.

15. The recombinant fusion protein of any preceding clause wherein the peptide is capable of increasing the yield of the recombinant fusion protein relative to the yield of the equivalent casein alone when expressed under identical conditions.

16. The recombinant fusion protein of any preceding clause wherein the casein is a-S1 casein, optionally wherein the a-S1 casein comprises an amino acid sequence which is at least 70% identical to SEQ ID NO. 1. 17. The recombinant fusion protein of any one of clauses 1 to 15, wherein the casein is a- S2 casein, optionally wherein the a-S2 casein comprises an amino acid sequence which is at least 70% identical to SEQ ID NO. 2.

18. The recombinant fusion protein of any one of clauses 1 to 15, wherein the casein is p- casein, optionally wherein the p-casein comprises an amino acid sequence which is at least 70% identical to SEQ ID NO 3.

19. The recombinant fusion protein of any preceding clause, which further comprises a linker between the casein and the peptide.

20. The recombinant fusion protein of clause 19, wherein the linker comprises a cleavage site, optionally an enzyme cleavage site.

21. The recombinant fusion protein of clause 20, wherein the enzyme cleavage site is a protease cleavage site, optionally a chymosin cleavage site.

22. A nucleic acid molecule comprising a sequence encoding the recombinant fusion protein of any one of claims 1 to 21.

23. A vector comprising the nucleic acid molecule of clause 22.

24. A host cell comprising the nucleic acid of clause 22 or the vector of clause 23.

25. The host cell of clause 24, wherein the host cell is a plant cell, a bacterial cell, a fungal cell, a yeast cell or a mammalian cell, optionally wherein the yeast cell is Pichia pastoris.

26. The use of a recombinant fusion protein according to any one of clauses 1 to 21 in the production of a recombinant casein.

27. The use of a recombinant fusion protein according to any one of clauses 1 to 21 in the production of a food composition.

28. A method of producing a recombinant casein, the method comprising culturing the host cell of claim 24 under conditions suitable for expression of the recombinant fusion protein of any one of claims 1 to 21. 29. The method of clause 28, wherein the recombinant fusion protein comprises a linker comprising a cleavage site, and wherein the method further comprises treating the recombinant fusion protein with an enzyme or a chemical which is capable of cleaving the recombinant fusion protein at the cleavage site, so as to separate the casein from the peptide.

30. A recombinant casein obtained by the method of clause 29, optionally wherein the recombinant casein comprises at least a portion of a cleavage site.

31. A method of making a food composition, the method comprising:

(i) providing a recombinant fusion protein according to any one of clauses 1 to 21 ;

(ii) separating the casein from the peptide; and

(iii) using the separated casein to make a food composition.

32. A food composition obtained by the method of any one of clauses 28 to 31.

33. A food composition comprising a recombinant fusion protein according to any one of clauses 1 to 21 , or a recombinant casein according to clause 30.