Field of the Invention

The present invention relates to a polynucleotide encoding an ice binding protein having increased pepsin digestibility, to a modified ice binding protein encoded by the polynucleotide, and to a method of making and using the modified ice binding protein.

Background of the Invention

Ice Structuring Proteins (ISPs), also sometimes referred to as “Antifreeze Proteins” (AFPs) or Ice Binding Proteins (IBPs) are a class of polypeptides which are found to occur naturally in many different organisms (e.g. fish, insects, plants, bacteria) which have evolved to survive in very cold (sub-zero °C) environments. For present purposes, the terms “ISP” and “IBP” are considered to be substantially synonymous.

ISPs are found to have two properties which are relevant to their role in nature: a) thermal hysteresis; and b) ice recrystallization inhibition (“IRI”). Thermal hysteresis refers to the ability of the ISPs to create a difference, or temperature gap, between the melting point and the freezing point of a liquid. IRI refers to the ability of ISPs to inhibit the growth of existing ice crystals in a liquid (which growth would otherwise tend to occur as being energetically favoured - a phenomenon known as “Ostwald ripening”). The thermal hysteresis and IRI properties of ISPs are not necessarily linked in any obvious way - for example, an ISP may have little thermal hysteresis effect but possess considerable IRI activity, or vice versa.

Many ISPs are known. Some of the best characterised are those from fish, such as Winter flounder, Northern cod, Atlantic herring and Antarctic eelpout. At least four types of fish ISPs have been classified, types I-IV AFP. In addition, as noted above, ISPs have been found in insects (e.g. the beetles Tenebrio sp. and Dendroides sp.), plants, bacteria (e.g. Pseudomonas sp. AFP), and other organisms.

The properties of ISPs make them of potential interest and use in a number of different applications (e.g. producing transgenic crops expressing ISPs to improve their freezetolerance and resistance to frost damage). In particular, it is known to use ISPs as a food ingredient. Unilever has used a type III AFP from ocean pout, known as HPLC12, in the manufacture of certain frozen food products.

WO 2015/082488 (DSM IP Assets B.V.) discloses a nucleic acid sequence encoding an ISP (AFP 19 of Leucosporidium, which protein is also known as LelBP) which is expressible at high levels in Aspergillus niger. The document also discloses the use of the ISP in the manufacture of a frozen food to inhibit hardening of the product and to reduce the deterioration of the product as a result of thermal abuse (e.g. in supply chains where temperature control is sub-optimal). The high level of expression of the ISP described in WO 2015/082488 means that it is comparatively cheap to produce the protein in large amounts, which was a problem with the manufacture of HPLC12.

Protease resistance (and pepsin resistance in particular) is thought to be associated with allergenicity, so protease-resistant polypeptides are unsuited for incorporation into food products for human consumption or for use in the production of foods for human consumption. US 2002/0098277 aims to render proteins less allergenic and discloses a method in which a protein containing disulphide bridges is treated with a reducing agent, such as dithiothreitol, in order to reduce the disulfide bonds. The reduced protein is then contacted with a “physiological disulfide” such as cystamine, to prevent the disulfide bridges being reformed. Proteins treated in this way were shown to have increased pepsin digestibility and reduced allergenicity. However, the breaking of disulphide bridges in a protein is likely to have substantial effects on the conformation and three-dimensional structure of the protein, such that the biological properties of the protein might be significantly altered in a manner which cannot necessarily be predicted.

WO 2021/168343 proposes modification of a milk protein, which modification renders the milk protein less allergenic by, for example, the introduction of an exposed protease site, such as a pepsin cleavage site. A very large number of amino acid substitutions are proposed, but there is nothing in the way of actual worked examples to demonstrate the usefulness of the approach, and little if any discussion of what other (possibly undesirable) effects on the protein such modification may induce. Summary of the Invention

In a first aspect, the invention provides a recombinant polynucleotide encoding a modified ice binding protein having at least 80% amino acid sequence identity with wild type LelBP having SEQ ID NO 1, the modified ice binding protein comprising an altered amino acid at position 75 relative to LelBP which confers increased pepsin digestibility compared to wild type LelPB, and wherein the modified ice binding protein substantially retains the ice structuring functionality of the wild type protein.

Typically the ice binding protein (IBP) retains at least 50% of the ice structuring functionality of the wild type protein, preferably at least 60%, more preferably at least 65%, and most preferably at least 70% of the ice structuring activity of the wild type IBP. The ice structuring function can be measured using assays of thermal hysteresis and/or ice recrystallization inhibition known to those skilled in the art. For example, an assay for measuring ice recrystallization inhibition is described by Pudney et al., (Arch. Biochem. Biophys. 2003 Feb.15; 410(2), p238-245). An assay method for measuring thermal hysteresis properties of IBPs is described by Gaede-Koehler et al., (Anal Chem. 2012 Dec 4;84(23): 10229-35).

Pepsin digestibility can be measured using the assay method described by Minekus et al., (Food Func. 2014, 5: 1114-1124). Typically the modified IBP will be at least 50% digestible by pepsin (that is, at least 50% of the protein will be digested by pepsin, as judged by SDS PAGE analysis, after a 60 minute incubation with pepsin using the method described by Minekus et al.,), whilst the wildtype IBP will generally be less than 10% digested, more usually less than 5% digested, under the same conditions. Where the modified IBP contains a single mutation relative to the wild type IBP, the single mutation results in increased pepsin digestibility. Where the modified IBP contains two or more mutations relative to the wild type IBP, the net effect of those mutations is to increase pepsin digestibility, whilst substantially retaining the ice structuring functionality of the wild type IBP. Thus, for example, it is possible that 2 or more mutations may co-operate to increase pepsin digestibility relative to the wild type. Alternatively, some mutations in the modified IBP may increase pepsin digestibility, whilst one or more other mutations may be important in substantially retaining the ice structuring functionality (e.g. may offset detrimental effects in that regard arising from the pepsin digestibility-inducing mutation/s).

The modified IBP is a modified LeIBP.

Accordingly, in a preferred embodiment, the invention provides a polynucleotide encoding an ice binding protein having at least 80% amino acid sequence identity with wild type LeIBP, the modified ice binding protein comprising an altered amino acid at position 75 relative to the amino acid sequence of wild type LeIBP, and wherein said modified ice binding protein has increased pepsin digestibility compared to wild type LeIBP.

LeIBP is the ice binding protein made by Leucosporidium (Park et al., 2012 Cryobiology 64; 286-296). The amino acid sequence of wild type LeIBP (SEQ ID NO 1) is shown in Figure 1. In wild type LeIBP, the amino acid residue at position 75 is a proline. The polynucleotide of the first aspect of the invention encodes an ice binding protein in which the proline is preferably altered to, or substituted by, an amino acid residue which confers pepsin digestibility, typically selected from the group consisting of aspartic acid (D), leucine (L), valine (V) and tyrosine (Y). The applicant has found that a protein comprising any one of these substitutions at position 75 has increased pepsin digestibility compared to wild type LeIBP. Advantageously, the substitution at position 75 creates a pepsin cleavage site in the modified ice binding protein.

Pepsin is a well-characterised enzyme. Pepsin is most active in acidic environments between 37°C and 42°C. Accordingly, its primary site of synthesis and activity is in the stomach (pH 1.5 to 2). Pepsin cleaves amide bonds preferentially at the C-terminal side of aromatic amino acids such as phenylalanine, tryptophan, and tyrosine. Pepsin exhibits preferential cleavage for hydrophobic, preferably aromatic, residues in Pl and PL positions. Increased susceptibility to hydrolysis occurs if there is a sulfur- containing amino acid close to the peptide bond, which has an aromatic amino acid.

The pepsin cleavage site could be created in the modified ice binding protein by altering the nucleic acid coding sequence to cause the deletion or the insertion of one or more selected amino acid residues relative to the amino acid sequence of the unmodified polypeptide. For the purposes of the present specification, “one or more selected amino acid residues” means any number from 1 to 10 amino acid residues, preferably 5 or fewer amino acid residues, more preferably 3 or fewer amino acid residues and in some embodiments, a single amino acid residue.

More preferably, the pepsin cleavage site is created in the modified ice binding protein by altering the nucleic acid coding sequence to cause substitution of one or more selected amino acid residues (this latter term having the meaning defined above), including or consisting of the residue at position 75.

Advantageously, the ice binding protein encoded by the polynucleotide of the invention may be used as an ingredient in a food product for human consumption, or is to be used in the manufacture of such a food product, such that traces of the polypeptide might remain in the food product after manufacture.

The term "polynucleotide" as used herein refers to a polymeric form of at least 500 nucleotides, more typically at least 600, and preferably at least 700 nucleotides. The term includes both nucleic acid sequences composed entirely of naturally occurring nucleotides and also those polynucleotide sequences which may comprise one or more non-natural nucleotide analogues, and/or chemical modifications. A polynucleotide may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels; methylation; and substitution of one or more of the naturally occurring nucleotides with an analogue. Examples of modified nucleotides are well known to those skilled in the art and are described in the literature (e.g. Li et al. 2014. J. Am. Chem. Soc. 136:826).

Methods of altering the nucleic acid sequence encoding a polypeptide of interest are well known to those skilled in the art and include, for example, site-directed mutagenesis (see for example, Hutchison et al., 1978 J. Biol. Chem, 253 (18) 6551- 6560) and CRISPR/Cas9 (see e.g. Doudna & Mali 2016 CRISPR-Cas: A Laboratory Manual, Cold Spring Harbor, New York.), although alternative nucleases (e.g. Cpfl) may also be utilised in the CRISPR technique in place of Cas9. The polynucleotide of the invention will typically be a "recombinant polynucleotide" i.e. one which is removed from its naturally occurring environment, or a polynucleotide that is not associated with all or a portion of a polynucleotide abutting or proximal to the polynucleotide when it is found in nature, or a polynucleotide that is operatively linked to a polynucleotide that it is not linked to in nature, or a polynucleotide that does not occur in nature, or a polynucleotide that contains a modification that is not found in that polynucleotide in nature (e.g., insertion, deletion, or point mutation introduced as a result of human intervention), or a polynucleotide that is integrated into a chromosome at a heterologous site.

The term can be used, e.g., to describe cloned DNA isolates, or a polynucleotide comprising a chemically synthesized nucleotide analogue. A polynucleotide is also considered "recombinant" if it contains a genetic modification that does not naturally occur. For instance, an endogenous polynucleotide is considered a "recombinant polynucleotide" if it contains an insertion, deletion, or substitution of one or more nucleotides that is introduced artificially (e.g., by human intervention). Such modification can introduce into the polynucleotide especially a substitution mutation, but other changes such as a point mutation, deletion mutation, insertion mutation, or duplication mutation may be present. A recombinant polynucleotide in a host cell or organism may replicate using the in vivo cellular machinery of the host cell; however, such recombinant polynucleotide, although subsequently replicated intracellularly, is still considered recombinant for purposes of this invention.

In particular, the polynucleotide of the invention will preferably be operably associated with a promoter, so as to permit the expression of the ice binding protein coding sequence. The promoter will preferably be a heterologous promoter to allow higher levels of expression than would be obtained with the natural wild type LelBP promoter.

The polynucleotide sequence will typically possess a high level of identity with the wild type LelBP coding sequence, i.e. at least 80% nucleotide sequence identity, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity, as determined by standard computer algorithms known to those skilled in the art. However, because of the redundancy of the genetic code, it will be appreciated that the polynucleotide sequence could vary from that of the wildtype LelBP sequence quite considerably, and yet still encode a polypeptide which has 80% or more amino acid sequence identity with the wild type LelBP amino acid sequence. For example, the polynucleotide sequence of the first aspect of the invention might be “codon optimised” for better expression in a particular host cell.

Typically the polynucleotide sequence encoding the modified ice binding protein will be inserted or cloned in a vector. The term "vector" as used herein refers to a nuclei acid that can carry a polynucleotide sequence to be introduced into a host cell. Nonlimiting examples of vectors include plasmids, phage particles, viral vectors, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).

Many suitable vectors are known to those skilled in the art. Specialised expression vectors for causing expression of the polynucleotide sequence are also well known and widely available to those skilled in the art. The expression vector, comprising the polynucleotide sequence, may be introduced into a suitable host cell to cause the host cell to express the modified ice binding protein. Both prokaryotic (bacterial) and eukaryotic (yeast, filamentous fungi, mammalian cell culture) host expression systems are known, and simple trial and error experiments can be performed to find a suitable expression vector and host cell to express the modified ice binding protein.

Equally, once the ice binding protein has been expressed by the chosen expression system, it is generally straightforward to purify, if desired, the modified polypeptide. Numerous purification techniques are known and common general knowledge for the person skilled in the art and include, for example, affinity chromatography, ion exchange chromatography, and the like. "Purifying", for present purposes, means the process of substantially separating the polypeptide from chemicals and cellular components (e.g., cell debris, membrane lipids, nucleic acids, and other proteins). The term does not necessarily require (but allows) that the protein be entirely separated from all other chemicals and cellular components, although it will be generally be preferred to achieve as high a degree of purity of the polypeptide as may be feasibly attained at reasonable cost.

The pepsin digestibility of the modified ice binding protein can be easily compared to that of the wild type LelBP protein. For example, samples of both the modified and wild type polypeptides can be contacted with equal amounts of pepsin under identical suitable conditions (e.g. pH 2.0-3.0; temperature 37°C) and for an equal amount of time. The samples can then be analysed and the amount of digestion of the respective polypeptides can be compared by a suitable analytical technique, such as polyacrylamide gel electrophoresis (and, optionally, Western blotting). The respective tracks of the samples on a polyacrylamide gel can be analysed using a densitometer to provide a numerical value or comparison of the amount of digestion of the respective polypeptides by the pepsin. Detailed guidance for an in vitro method of determining pepsin digestibility is given by Minekus et al., (Food Funct. 2014, 5, 1113-1124) and in Example 4 below, either of which may be used to measure the pepsin digestibility of an ice binding protein in accordance with the invention. Typically, an ice binding protein in accordance with the invention, when treated in the manner described in Example 4 below, will be completely digested by pepsin (i.e. no full length ice binding protein will be detectable upon SDS PAGE analysis) after a 60 minute incubation with pepsin, more preferably after a 45 minute incubation, and more preferably after a 30 minute incubation.

LelBP has properties which render it useful for incorporation in, or production of, foodstuffs for human consumption, especially where the foodstuff is stored at a temperature below 0°C, and/or is subjected to manufacture or processing at a temperature below 0°C. In particular LelBP has the ability to inhibit the formation of ice crystals (“ice recrystallization inhibition” activity). However, wild type LelBP is largely resistant to digestion by pepsin and therefore is considered potentially allergenic. The modified ice binding protein encoded by the polynucleotide of the invention has been shown, in preferred embodiments, substantially to retain the desirable ice recrystallization inhibition activity whilst losing the undesirable resistance to pepsin digestion. “Substantially retaining ice recrystallization inhibition activity” for present purposes, means that the modified IBP has at least 50% of the IRI activity of the wildtype ice binding protein, as measured by an appropriate assay. Preferably the modified IBP will have at least 60%, more preferably at least 65%, and most preferably at least 70% of the IRI activity of the wildtype IBP.

The alteration of just one or more selected amino acid residues in the polypeptide is important in minimising changes to, or loss of, the desirable ice recrystallization inhibition activity of the polypeptide. By avoiding major alterations to the amino acid sequence of the polypeptide, the chances of substantially retaining the desired property or functionality are increased.

Selection of appropriate sites in the polypeptide for alteration of the amino acid sequence other than, or in addition to, amino acid residue 75, can be assisted by the use of computer modelling software, in combination with the teachings and guidance provided by the present specification.

In order to reduce the risk of unwanted changes in the structure of the ice binding protein, and hence reduce the risk of inadvertently introducing unwanted changes in the properties of the protein, it will be generally be preferred that the amino acid sequence alterations of the ice binding protein, relative to the wild type LelBP sequence, will be as few in number, and/or as conservative in structure, as possible whilst still conferring the necessary pepsin sensitivity. Desirably therefore any alterations will be selected point mutations, and conveniently the number of such selected point mutations will be less than 5 residues, preferably less than 4 residues, more preferably less than 3 residues, and most preferably less than 2 residues, although the person skilled in the art will appreciate that conservative amino acid substitutions are less likely to result in a major alteration of the structure of the protein, such that up to 5 amino acid residue substitutions may be permitted. In some embodiments, the ice binding protein of the invention may differ from the wild type sequence of LelBP only at position 75.

Desirably, the polynucleotide encodes an ice binding protein which has at least 91% sequence identity with wild type LelBP, preferably at least 92%, more preferably at least 93%, 94% or 95%; and most preferably at least 96, 97 or 98% sequence identity with wild type LeIBP.

The term "sequence identity" in the present context refers to amino acid residue sequences that are the same when the two (or more) polypeptide sequences are aligned for maximum correspondence. For present purposes, the comparison is made over essentially the full length of the wild type LeIBP amino acid sequence which acts as a reference sequence to which one or more test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, and a sequence algorithm program used to calculate the percent sequence identity for the test sequence(s) relative to the reference sequence. Optimal alignment of sequences for comparison can be conducted using any one of algorithms known to those skilled in the art, such as that described by Needleman & Wunsch, (1970 J. Mol. Biol. 48:443), Smith & Waterman (1981 Adv. Appl. Math. 2:482), or Pearson & Lipman, (1988 Proc. Natl. Acad. Sci. USA 85:2444), by computerized implementations of these algorithms (GAP, BESTFIT, FAST A, and FASTA in the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, which can be used with default parameters), or by simple visual inspection. One algorithm that is widely used for determining percent sequence identity is the BLAST algorithm (described by Altschul et al. 1990 J. Mol. Biol. 215:403-410; Gish & States. 1993 Nature Genet. 3:266-272; and Madden et al. 1996 Meth. Enzymol. 266: 131-141; Altschul et al. 1997 Nucleic Acids Res. 25:3389-3402; Zhang 7 Madden. 1997 Genome Res. 7:649-656). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In cases where two or more polypeptide sequences differ from each other by conservative substitutions (i.e., substitutions of amino acids with chemically similar amino acids; conservative substitution tables providing functionally similar amino acids are well known in the art), the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. (e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89). As mentioned above, the modified ice binding protein encoded by the polynucleotide of the first aspect of the invention preferably retains substantial ice recrystallization inhibition (IRI) activity. This activity can be measured or determined by, for example, performing the ice recrystallization inhibition assay essentially as described by Pudney et al., (Arch. Biochem. Biophys. 2003 Feb.15; 410(2), p238-245).

In a second aspect, the invention provides a modified ice binding protein having at least 80% amino acid sequence identity with wild type LelBP, wherein the modified ice binding protein has a substitution at position 75 relative to wild type LelBP, and wherein the modified ice binding protein has increased pepsin digestibility compared to wild type LelBP. Preferably the modified ice binding protein has at least 85% amino acid sequence identity with wildtype LelBP, more preferably at least 90%, and most preferably at least 95% sequence identity. Clearly the modified ice binding protein must have less than 100% amino acid sequence identity with the wildtype LelBP. A single amino acid residue difference, in a polypeptide of 241 residues, would equate to about 99.6% amino acid sequence identity, so this would be about the maximum % sequence identity between the modified ice binding protein and the wildtype LelBP which could be expected.

The modified polypeptide is conveniently encoded by, and expressed from, a polynucleotide in accordance with the first aspect of the invention, and the description of preferred features and embodiments of the first aspect apply, mutatis mulandem. to the modified ice binding protein of the second aspect.

In a third aspect, the invention provides a method of producing an ice binding protein in accordance with the second aspect of the invention defined above, the method comprising the steps of (a) using a polynucleotide sequence in accordance with the first aspect of the invention to express, in a suitable host cell, the ice binding protein; and (b) at least partially purifying the expressed ice binding protein.

In a fourth aspect, the invention provides a method of making a food product for human consumption, the method comprising the step of using a modified ice binding protein in accordance with the second aspect of the invention, preferably produced by a method in accordance with the third aspect of the invention, as an ingredient in the manufacture of the food product.

The food product will typically be one which is subjected to processing (during manufacture), and/or storage, at a temperature below 0°C. Examples of such food products include ice cream, sorbets, frozen yoghurt, and other frozen desserts and frozen processed foods generally.

The features of the invention will now be further described by way of illustrative embodiment and with reference to the accompanying drawing figures, in which:

Figure 1 shows the amino acid sequence of wild type LelBP;

Figure 2 is an alignment of the amino acid sequence of wild type LelBP with the sequence of four different variants of the protein made by the applicant;

Figure 3 is a diagram showing the results of electrophoretic analysis of culture supernatants obtained from P. pastoris strains expressing wild type LelBP or a modified form of the protein;

Figures 4-6 are pictures of SDS-PAGE analysis of wild type or modified LelBP, to assess digestibility with pepsin;

Figures 7-8 are pictures showing results of analysis of wild type LelBP and modified LelBP proteins to inhibit ice recrystallization; and

Figure 9 is a map of the expression vector pPZalpha with inserted LelBP coding sequence. Examples

Example 1 : Modification of LelBP coding sequence

DNA sequences corresponding to wild type LelBP and four variants, along with linker sequences that introduced cloning restriction sites, were chemically synthesised. The amino acid sequence of wildtype LelBP is shown in Figure 1.

Figure 2 is an amino acid sequence alignment of the wild type sequence with each of the four variant LelBP sequences. It will be apparent that the variants were identical to the wild type sequence, other than for a substitution at amino acid residue 75. This is a proline in the wild type sequence, but is either D (aspartic acid), L (leucine), V (valine) or Y (tyrosine) in the variants.

The five sequences were digested with Xhol/Notl restriction enzymes and ligated into the expression plasmid pPZ-alpha, digested with the same enzymes. A map of pPZalpha (with inserted wildtype or modified LelBP coding sequence) is shown in Figure 9. pPZ-alpha carries the S. cerevisiae alpha-mating factor pre-protein secretion leader sequence (without the EAEA amino acid sequence) along with the wildtype- A0X1 promoter for methanol-induced expression (giving 5 plasmids in total). After transformation into E. coli TOPI OF' cells, re-cultivation of transformants and plasmid preparations, correct insertion of the target genes was checked by restriction pattern analysis, and authenticity of the gene sequences was confirmed by DNA sequencing.

Plasmids carrying the target DNA sequences target were linearized via the restriction enzyme SacI and were desalted for transformation into P. pastoris using standard methodologies easily repeatable by those skilled in the art. Transformation was via electroporation of the plasmids using a modified standard procedure and standard equipment for electroporation. Competent cells comprised the basic production strain CBS7435 muts (genotype A aoxl, phenotype muts).

Single colonies (30 in total per target protein) were picked from transformation plates into single wells of a 96- deep well plate filled with optimized cultivation media. After an initial growth phase to generate biomass, expression from the wildtype- AOXl- promoter was induced by addition of an optimized liquid mixture containing methanol. At defined points of time, further induction with methanol was performed. After a total of 72 hours from the initial methanol induction, the deep well plate was centrifuged, and supernatants of all wells were harvested into stock microtiter plates for subsequent analysis. De-glycosylation was performed for 1 hour on expression samples using EndoHf (NEB, USA) under denaturing and heat-treated conditions (10 minutes at 70°C) prior to addition of 0.5pL EndoHf.

Example 2 : Analysis of expression of LelBP and its P75 variants

A high throughput screening method involving microfluidic capillary electrophoretic (mCE) separation (GXII, CaliperLS, now Perkin Elmer) and subsequent identification of the target protein based on its size was established. Briefly, several pL of all culture supernatants were fluorescently labelled and analysed according to protein size, using the electrophoretic microfluidics system. Internal standards enabled approximate allocations to size in kDa and approximate concentrations of detected signals. As calibrator for molecular weight and concentration, Cytochrome c (nominal molecular weight of 12 kDa) was applied, diluted to 100 mg/L in mock strain matrix. Supernatants were analysed by mCE after 72 hours of methanol induction under reducing conditions against Cytochrome c as calibrator.

Figure 3 shows an electropherogram overlay of selected EndoHf-treated screening supernatants of mutant strains producing: wild type LelBP (dotted line ...), LelBP- P75D (solid line), LeIBP-P75L (small dashed line — ), LeIBP-P75V (dashed/dotted line -•-•) and LeIBP-P75Y (bold dashed line - ).

Table 1 below shows the LelBP production levels of selected clones after induced expression in P. pastoris. Concentrations are based on peak area of mCE analysis, using Cytochrome c as a calibration control. It can be seen that the levels of expression of the variant LelBP proteins are lower than that for the wild type, but are still at useful levels.

Table 1

Example 3 : Theoretical digestion of LelBP and variants by pepsin

The Freeware Expasy PeptideCutter tool was used to predict the sites of LelBP and LelBP variants susceptible to pepsin degradation (https://web.expasy.org/cgi- bin/peptide_cutter/peptidecutter.pl). The tool indicated a total of 73 cleavage sites for pepsin at a pH above 2.0 (at amino acid positions 3 7 8 13 14 16 17 30 31 42 43 46 47 48 49 56 57 65 66 67 68 71 72 79 80 90 91 96 97 102 103 104 105 109 110 114 119 120 121 122 131 132 133 134 141 142 143 144 149 150 151 152 157 158 159 160 170 171 184 185 188 189 190 196 197202 203 207 214 215 223 239 and 241). The proline at position 75 is non-digestible by pepsin using this analysis tool.

Investigating the digestion of LeIBP-P75D and LeIBP-P75V using this tool did not alter the predicted cleavage pattern (relative to that of the wild type) by pepsin. The predicted digestion of LeIBP-P75L and LeIBP-P75Y by pepsin added a single cleavage site at position 74-75 (amino acids T and L/Y). As such it is clearly demonstrated that tools designed to determine the digestibility of a protein in silico are not sufficient to predict digestibility of proteins with an innate resistance to in vitro digestion.

Example 4: In vitro digestibility of LelBP and P75 variants by pepsin

The digestibility of LelBP and the P75 variants was determined by in vitro digestion using standard methodology, as described by Minekus et al 2014 (Food Funct., 2014,5, 1113-1124), with minor modifications. Essentially, a 2x concentrated simulated gastric fluid (SGF) buffer was prepared and adjusted to pH 3.0 using HC1, and containing 10,000U/ml of porcine pepsin. LelBP and variants were desalted into distilled water and adjusted to Img/ml of protein. Protein content of the desalted protein samples was determined by BCA assay using bovine serum albumin as calibrant, according to the manufacturer’s instructions for microplate assays (ThermoPierce). lOOpl of LelBP protein was incubated for 1 hour in SGF buffer plus pepsin and with SGF buffer alone. Incubation was at 37°C and with vigorous mixing using an Eppendorf thermomixer. 1 Opl samples were removed at time zero, 15 and 60 minutes and placed in Eppendorf tubes containing 3.5 pl of stop solution (200mM NaHCO3 pHl l). 4x LDS buffer plus reducing agent (ThermoPierce) was added to each aliquot which were subsequently heated to 70°C for 10 minutes. The digestibility of LelBP and LelBP variants was assessed by SDS-PAGE, loading 7.5pl sample per lane, using NuPAGE bis-tris mini gels and lx MES or lx MOPS buffer (ThermoFisher). Gels were stained using Oriole fluorescent protein stain (Bio-Rad) and molecular weight of protein bands was estimated using PrecisionPlus unstained protein standard (Bio-Rad).

Figure 4 shows the results of SDS-PAGE (MES buffer) illustrating the effect of pepsin on LeIBP-P75D (lanes 2 to 5) and LeIBP-P75L (lanes 6 to 9). The profile of pepsin alone is shown in lane 10. TO, T15 and T60 denotes the incubation time of the pepsin with target protein, in minutes. C denotes control lanes in which pepsin is absent (after 60 minutes incubation). Arrows denote the location of the non-digested LelBP variants in control lanes 4 and 7. Boxed areas in T15 and T60 samples (lanes 3, 4, 7 and 8) highlight digestion products of a high m.w. protein present in the expression supernatant, that does not correspond to the LelBP variants. As can be clearly seen, the band corresponding to LelBP variants are present in control lanes and in TO lanes but are almost completely digested by T15 and are absent in T60 lanes.

Figure 5 shows the results of SDS-PAGE illustrating the effect of pepsin on LelBP- P75V (lanes 2 to 5) and LeIBP-P75Y (lanes 6 to 9). The profile of pepsin alone is shown in lane 10. TO, T15 and T60 denotes the incubation time of the pepsin with target protein, in minutes. C denotes control lanes in which pepsin is absent (after 60 minutes incubation). Arrows denote the location of the non-digested LelBP variants in control lanes 4 and 7. As can be clearly seen, the band corresponding to LelBP variants are present in control lanes and are almost completely digested at TO. By T15 the digestions are complete.

Figure 6 shows the results of SDS-PAGE illustrating the effect of pepsin on wild type LelBP (lanes 2 to 4). TO and T60 denotes the incubation time of the pepsin with target protein, in minutes. C denotes the control lane, in which pepsin is absent (after 60 minutes incubation). Arrow denotes the location of the non-digested LelBP in the control lane. As can be clearly seen, the band corresponding to LelBP is present in the control lane and in the TO and T60 lanes, with no indication of digestion of LelBP by pepsin.

Example 5 : Assessment of ice recrystallization inhibition activity

The ice recrystallization inhibition (IRI) assay has been developed to measure the ice structuring activity of ice binding proteins such as LelBP and results are characterised by reduced size and/or shape of ice crystals compared to those obtained for a 30% sucrose solution.

LelBP and LeIBP-P75 variant test samples (as shown in figure 4) were prepared in a 30% sucrose solution by 1 in 4 dilution with 40% sucrose prepared in distilled water. A 5 pl drop of solution to be studied was placed on a 22 mm diameter slide inside a 20pm thick spacer ring. A 14 mm diameter coverslip was then placed on top of the spacer ring, to produce a uniform thin film of sample. The slide was placed on a Linkam LTS120 temperature-controlled microscope stage, which was subsequently cooled to approximately -30°C at a rate of 30°C/min. This cooling produced a large population of small ice crystals. The stage temperature was then raised rapidly (30°C/min) to - 12°C and then raised more slowly (5°C/min) to -8°C and more slowly still (l°C/min) to -6°C. The stage was held at this temperature for 30 minutes. The ice phase was observed using a Zeiss Axiostop2 plus microscope. The state of the ice phase was recorded by capturing images with a Zeiss axiocam digital camera once the target temperature of -6°C was reached and after 30 minutes of holding at that temperature. The results are shown in Figures 7 & 8. Figures 7A and 7B are images of the ice crystal morphology of a 30% sucrose solution at time 0 (Figure 7A) and after 30 minutes (Figure 7B) at -6°C. The images clearly show that, in the absence of ice structuring protein, the ice phase alters from one of many small crystals at the start of incubation, to fewer, much larger ice crystals at the end.

Figures 8A-8E are images showing the ice crystal morphology of a 30% sucrose solution containing, respectively, wild type LelBP (Figure 8A), LeIBP-P75D (Figure 8B), LeIBP-P75L (Figure 8C), LeIBP-P75V (Figure 8D) or LeIBP-P75Y (Figure 8E) after 30 minutes at to -6°C. The images clearly show that in the presence of the wild type LelBP protein, or of the LelBP variants, the ice crystals remain small after 30 minutes incubation at -6°C when compared to a solution of 30% sucrose alone (Figure 7B). The LelBP variants thus retain the desirable ice recrystallization inhibition (IRI) property of the wild type LelBP.