WO2008040059A1

WO2008040059A1 - Anti-bacterially active agents and related methods

Info

Publication number: WO2008040059A1
Application number: PCT/AU2007/001477
Authority: WO
Inventors: Ross Lindsay Tellam; Roger Daniel Pearson; Kritaya Kongsuwan; Lillian Sando; Phillip David Parker; Stuart Craig Smith; Christian Paul Gray
Original assignee: Innovative Dairy Products Pty Ltd As Trustee For The Participants Of The Cooperative Research Centre For Innovative Dairy Products
Priority date: 2006-10-04
Filing date: 2007-10-04
Publication date: 2008-04-10

Abstract

The invention relates to isolated purified recombinantly produced anti-bacterially active bovine MUC1 glycoprotein or active fragments or derivatives thereof as well as to products comprising them, methods and uses involving them and to processes for their purification. Methods of screening mammals to determine the VNTR number of MUC1 glycoprotein produced by the animal, particularly within the animal's milk are also provided along with methods of increasing the VNTR number of MUC1 glycoprotein within milk from a herd of cows and methods of up-regulating MUC1 production in the milk of individual animals.

Description

ANTI-BACTERIALLY ACTIVE AGENTS AND RELATED METHODS

FIELD OF THE INVENTION

The present invention relates to an isolated, purified or recombinantly produced anti- bactierially active bovine MUCl glycoprotein or active fragment or derivative thereof, to products comprising them, methods and uses involving them and to processes for their purification. The invention also relates to methods of screening mammals to determine the VNTR number of MUCl glycoprotein produced by the animal, and particularly within the animal's milk, to methods of increasing the VNTR number of MUCl glycoprotein within milk from a herd of cows and to methods of up-regulating MUCl production in the milk of individual mammals. The invention further relates to methods of measuring MUCl production levels in the milk of mammals and to methods of determining low and high MUCl producing animals.

BACKGROUND OF THE INVENTION

Milk contains a variety of substances that inhibit the infection of pathogens (1). This is of benefit to the mother, safeguarding the integrity of the lactating mammary gland, but also of huge importance for protection of the suckling offspring. The anti-bacterial substances in milk can be classified into two categories. First, substances like antibodies that actively provide protection against pathogen infections or modulate immune responses and have developed during the mother's lifetime. The second category of substances in milk that possess anti-microbial activity is the non-specific, broad-spectrum defence molecules such as lactoferrin and a range of other proteins and glycolipids (2). The present inventors have considered whether it may be possible to alter some of these molecules or their amounts in milk (or milk related products) to result in improved anti-bacterial properties. These alterations of milk composition, especially in relation to anti-bacterial activities, have applications in human and animal health.

The present inventors have shown that enhancement of the anti-bacterial properties of milk can lead to a "designer milk" product that is able to prevent, and to treat, microbial diseases. Such new milk products will be important for treatment of recalcitrant microbial diseases that cannot be cured or which are difficult to treat with current therapies, especially antibiotics. This will help decrease the therapeutic use of antibiotics and lessen the emergence of microbial resistance among human and veterinary bacterial pathogens. Antibiotic resistance is a major public health threat. Approaches to preventing diseases that utilise the broad-range properties of milk substances, especially anti-bacterial properties, will be extremely beneficial as functional food components. The potential of milk bioactives to enhance the maturation of the gut epithelium will also be useful in the context of human and animal applications that promote the repair, maintenance and improved function of the gut.

Natural fat globules in milk are coated with a protective layer generally known as the milk fat globule membrane (MFGM) (3). The MFGM is intimately involved in natural processes in milk (e.g. creaming and agglutination), and the composition and structure of the MFGM and its associated proteins are important parameters affecting processing and physical properties, such as mouth-feel and flavour of milk. Among the distinctive components of MFGM is the glycoprotein mucin, MUCl. MUCl is a very large and highly glycosylated membrane bound protein with an extended extracellular domain. It is synthesised by mammary epithelial cells and shed into milk as a component of the MFGM (4). It is found in high concentrations in MFGM and in freshly drawn milk is associated with the cream fraction (estimated concentration of 40 mg/1) (5). However after storage, agitation or temperature fluctuations it may be found in the whey component of milk. Multiple copies of the extracellular domain of MUC 1 produce numerous, mostly radially oriented filaments (~ 0.2- 0.5 μm) that protrude from the cell surface and are visible by electron microscopy. The extensive oligosaccharides on the protein, which are the predominant architectural feature, are highly negatively charged due to the presence of sialic acid. However, the MUCl glycoprotein itself, and particularly the associated oligosaccharides, vary substantially in structural detail between mammalian species.

It has been speculated that human MUCl may function to protect exposed epithelial cell surfaces from physical damage and from invasive pathogenic bacteria (6). For example, the mucin fraction from the human milk fat globule membrane has been shown to inhibit the adhesion of S-fimbriated Escherichia coli to buccal epithelial cells (7). In addition, human MUCl may have a role in protecting the suckling neonate by binding to and sequestering pathogenic bacteria (8).

Previous studies have shown that gastric and airway mucins (MUC2, MUC3, MUC5AC, MUC6 and MUC7), as well as MUCl isolated from human milk, can bind several bacterial species. Moreover, this binding can facilitate inhibition of the binding of bacteria to cells and prevent infection. Although this information pertains to MUCl orthologs in general it cannot be inferred that it is relevant to bovine MUCl, because of its unusually low sequence identity with MUCl from other mammalian species. Further, many of these studies are restricted to non-milk MUCl activities, such as in non-mammary and non- gastrointestinal tract environments. Although it has been speculated upon, the inventors are not aware of any earlier studies demonstrating anti-bacterial activities of bovine MUCl derived from mammary and gastrointestinal tract tissues.

The present inventors have determined that bovine MUCl is characterised by a variable number of tandem repeats (VNTR) region within its extracellular domain, which is variable across sub-species, breeds and individuals and have shown that a higher VNTR repeat number (an increased level of repetition of the tandem repeat sequence) is associated with increasing anti-bacterial activity of the bovine MUCl glycoprotein. These and other related determinations have important implications for human and animal health, as will be further described below.

SUMMARY OF THE INVENTION According to a preferred embodiment of the present invention there is provided an isolated, purified or recombinantly produced anti-bacterially active bovine MUCl glycoprotein or active fragment or derivative thereof.

Preferably the glycoprotein or active fragment or derivative thereof comprises a variable number of tandem repeats (VNTR) region with a repeat number of between about 7 and about 23. Preferably the repeat number is at least 10, preferably at least 14, more preferably at least 16 and most preferably at least 20. In a preferred embodiment of the invention the glycoprotein or active fragment or derivative thereof comprises an amino acid sequence selected from any one of SEQ ID NOS. 1 to 14.

Preferably the glycoprotein or active fragment or derivative thereof is O-link glycosylated at between about 51 to about 161 sites, preferably at at least about 70 sites, more preferably at at least about 90 sites, more preferably at at least about 110 sites, more preferably still at at least about 130 sites and most preferably at at least about 150 sites.

In other embodiments of the invention there are provided:

(a) an anti-bacterially active food or feed comprising the glycoprotein or active fragment or derivative thereof according to the invention and one or more food or feed compatible components;

(b) an anti-bacterially active pharmaceutical composition comprising the glycoprotein or active fragment or derivative thereof according to the invention and one or more pharmaceutically acceptable additives;

(c) an anti-bacterially active veterinary composition comprising the glycoprotein or active fragment or derivative thereof according to the invention and one or more veterinary acceptable additives;

In another embodiment of the invention there is provided a bovine milk or milk derived product enriched with the glycoprotein or active fragment or derivative thereof according to the invention. For example the enriched milk derived product can be a modified milk, a flavoured milk, a milk based drink, an infant formula, a geriatric food/formula, a yogurt, a cheese, a cream, a butter, a junket, a custard, a baked good, a dessert, an ice-confection or a milk powder.

In a further embodiment of the invention there is provided a non-food product comprising the glycoprotein or active fragment or derivative thereof according to the invention and other conventional ingredients. Examples of such products include chewing gum, oral moisturiser, mouth spray, lozenges, medical plastics and contact lenses. According to another embodiment of the invention there is provided a process for purification of MUCl glycoprotein (extracellular domain) from milk fat globule membranes (MFGM), comprising the steps of:

(a) exposing an aqueous suspension of MFGM to digestion with protease at an appropriate temperature for a suitable period;

(b) inactivating said protease and recovering a soluble fraction obtained from said digestion:

(c) exposing said soluble fraction to anionic exchange chromatography and then hydrophobic interaction chromatography, and recovering a MUCl glycoprotein (extracellular domain) containing sample.

Preferably the protease is trypsin.

Preferably the aqueous suspension of MFGM is obtained by homogenising a milk derived cream fraction to buttermilk, separating MFGM from the buttermilk and optionally resuspending the MFGM if necessary. Preferably the MFGM is separated from the buttermilk by filtration and/or centrifugation. Preferably, the digestion of MFGM with protease (such as trypsin) is conducted for between about 30 minutes and about 8 hours, at a temperature of between about 35⁰C and about 4O⁰C and at a pH of between about 6.5 and about 8.5. Preferably protease (eg. trypsin) is inactivated using benzamidine and EDTA. In a preferred embodiment the soluble protein fraction from said digestion is recovered by filtration and/or centrifugation.

According to another embodiment of the invention there is provided a process for purification of MUCl glycoprotein (extracellular domain) from milk whey that has been exposed to agitation, storage and/or temperature fluctuation, or from butter milk or cheese whey, comprising exposing milk whey, butter milk or cheese whey to anionic exchange chromatography and then hydrophobic interaction chromatography, and recovering a MUCl glycoprotein (extracellular domain) containing sample.

In another preferred embodiment a dialysis step is conducted prior to one or both of the chromatography steps. Preferably DEAE Sepharose is used as the anion exchange resin and preferably elution from the anion exchange resin is achieved by applying a 0-1 M NaCl gradient, wherein MUCl glycoprotein containing fractions are eluted at 200-400 mM NaCl. Preferably Phenyl Sepharose is used as the hydrophobic interaction resin and preferably the MUCl glycoprotein containing fraction is eluted in the break-through fraction.

According to a further embodiment of the present invention there is provided a method of treating or preventing bacterial infection in a mammal, which comprises administering to the mammal an effective amount of the glycoprotein or active fragment or derivative thereof according to the invention.

According to a still further embodiment of the present invention there is provided use of the glycoprotein or active fragment or derivative thereof according to the invention in preparation of a medicament for the treatment or prevention of bacterial infection in a mammal.

Preferably the mammal is a human.

According to a still further embodiment of the present invention there is provided a method of preventing or inhibiting bacterial growth at a locus, which comprises exposing the locus to an effective amount of the glycoprotein or active fragment or derivative thereof according to the invention. For example, the locus may be a contact lens, denture, mouth guard, food or drink package or component thereof or a food or drink preparation or processing area.

According to another embodiment of the present invention there is provided a method of determining the MUCl variable number of tandem repeats (VNTR) repeat number for a mammal, which comprises amplifying a nucleotide sequence encompassing the MUCl VNTR region from a DNA sample from the mammal and determining the VNTR repeat number from the amplified sequence.

According to a further embodiment of the present invention there is provided a method of determining the MUC 1 variable number of tandem repeats (VNTR) repeat number for a mammal, which comprises exposing a DNA sample from the mammal to PCR amplification using a forward primer complimentary to a MUCl sequence located 5' to the VNTR region and a reverse primer complimentary to a MUCl sequence located 3' to the VNTR region, and sequencing the amplified sequence. Preferably the mammal is bovine and in one preferred embodiment the forward primer comprises the nucleotide sequence 5'- CATAAACCCCCGCAGAACTA-S¹ and the reverse primer comprises the nucleotide sequence S'-TAATATGGCTGGCAGCAGTG-S'.

According to a still further embodiment of the present invention there is provided a method of increasing the VNTR repeat number of MUC 1 in milk produced by a herd of cows which comprises determining the MUCl VNTR number of individual cows in the herd and selecting cows to contribute to the milk based upon MUCl VNTR repeat number for each individual cow being at least 10. Preferably the MUCl VNTR number for each individual cow is at least 14, more preferably at least 16 and most preferably at least 20.

According to a further embodiment of the present invention there is provided a method of up-regulating MUCl glycoprotein production in the milk of a mammal which comprises exposing mammary gland cells of the mammal to cell walls, cell wall components or other cellular components from Gram-negative or Gram-positive bacteria. Preferably the mammal is bovine.

According to a further embodiment of the present invention there is provided a method of measuring the quantity of MUC 1 glycoprotein in the milk of a mammal which comprises exposing a sample of the milk to a competitive ELISA assay or immunomagnetic separation utilising an antibody to MUCl or a fragment thereof. Preferably the MUCl antibody is raised against the MUCl extracellular region.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be described further and by way of example only, with reference to the figures, wherein: Fig 1 is a schematic representation of the human MUCl protein as part of the MFGM (ref 5);

Fig 2 is a photograph of an SDS-PAGE gel of MFGM and MUCl fractions, where bovine MFGM is in lane 2, trypsin released protein from MFGM is in lane 3 and phenyl sepharose-purified MUCl protein stained with silver is in lane 4 and stained with Alcian blue is in lane 5 (the arrow indicates the position of MUCl);

Fig 3 is a schematic representation of the domain architecture of MUCl proteins which outlines that three isoforms of MUCl exist that are derived from the same gene by alternative splicing and in one instance (for MUCl) proteolytic processing (the splicing variants are: MUCl, MUCl/sec, and MUC1/Y);

Fig 4 is a diagrammatic representation of the MUCl protein and the relative positions of PCR primers based on the corresponding genomic sequence, where the primers denote the regions of the genomic DNA (corresponding to and contained within exon 2) that were amplified, which correspond with the polymorphic region (VNTRs) of MUCl;

Fig 5 is a schematic illustration of MUCl allelic variants which outlines the number of repeat units, the amino acid sequences and potential glycosylation sites of the different repeat units as well as the structure of repeat units within each allelic variant, for the MUCl alleles A-H;

Fig 6 is a photograph of an agarose gel showing the PCR products obtained for 11 different MUCl genotypes, where each vertical lane represents a genotype and where samples from homozygote animals have one band and samples from heterozygote animals have two bands (e.g. A and B for genotype AB) and each allele is identified by a letter;

Fig 7 shows bar graphs of inhibition of bacterial binding to Caco-2 adenocarcinoma cells by (A) MFGM and (B) trypsin-released MFGM proteins, where each assay was performed twice using triplicate wells and where error bars represent the standard error of the mean across each assay;

Fig 8 shows plots of percentage binding of FITC-labelled E. coli (A), S. typhimurium (B), S. aureus (C) and B. subtilis (D) to Caco-2 cells following pre-incubation of bacteria with serial dilutions of bovine MUCl, where triplicate wells were analysed in duplicate assays and where error bars represent the standard error of the mean;

Fig 9 shows direct binding of bacteria to different concentrations of immobilised MUCl. The graph plots the log of MUCl concentration (ng/ml) against intensity (au) for biotinylated E. coli (A), S. typhimurium (B), S. aureus (C) and B. subtilis (D), where each assay is performed twice in triplicate and where error bars represent the standard error of the mean (SEM);

Fig 10 shows the generic bovine MUCl amino acid sequence where italicised type indicates signal sequence, bold type indicates extracellular domain, indicates the VNTR region (sequence for each allelic variant VNTR is shown in SEQ ID NOS. 8 - 14), underlined type indicates transmembrane domain and normal type ^' indicates intracellular domain. The amino acid sequence for seven allelic variants of MUCl are shown in SEQ ID NOS. 1 - 7 (which correspond to the VNTR variants of SEQ ID NOS. 8 - 14, respectively).

Fig 11 shows a photograph of an SDS-PAGE gel demonstrating release of MUCl from milk fat globule membranes by a range of proteases. The SDS-PAGE was stained with silver and Alcian blue. The latter agent preferentially stains MUCl as a poorly focused high molecular weight blue band. Lane 1, size standards. Lane 2, milk fat globule membrane protein (100 μg) without addition of any protease for 24 h at 2O⁰C (control). Lanes 3-5, proteins released from milk fat globule membrane protein (100 μg) incubated with trypsin, endoproteinase Lys C or chymotrypsin (each 1 μg) for 24 h at 2O⁰C, respectively. All three proteases preferentially released MUCl from milk fat globule membranes without destroying the integrity of the released MUCl. Fig 12 shows SDS-PAGE analysis of the stability of MUCl after its release from milk fat globule membranes. The SDS-PAGE was stained with silver and Alcian blue. The latter agent preferentially stains MUCl as a poorly focused high molecular weight blue band. MUCl was released from milk fat globule membranes by the action of trypsin and then subjected to digestion with alcalase (1% wt/wt for 1 h). The 'pre' sample shows the starting material while the ' 1 hr' sample shows the sample after one hour of incubation with alcalase at 2O⁰C. Alcalase completely digested MUCl and therefore cannot be used in its preparation.

Fig 13 shows SDS-PAGE isolation of two different homozygous genotypic variants of the MUCl protein (alleles A (16 VNTR repeats) and C (11 VNTR repeats)). The two variants were tested for their relative abilities to inhibit the binding of E. coli bacteria to CACO2 cells grown in cell culture. The AA genotype showed 31% greater inhibitory activity compared with the CC genotype. Thus, it is concluded that the greater the number of VNTRs in the MUCl protein, the greater is the inhibitory activity.

Fig 14 shows SDS-PAGE profiles of purified recombinant bovine MUCl proteins. H and F represent the two different recombinant proteins produced. The left hand panel shows the recombinant MUCl proteins after purification using Ni-NTA affinity chromatography. The protein was stained with Coomassie Blue. The right hand panel shows the recombinant MUCl proteins purified Jacalin affinity chromatography and stained with silver and Alcian blue.

Fig 15 shows a bar graph of mole percentage of total monosaccharide for natural Mucl (pool of allelic variants A (16 VNTR), B (14 VNTR), and C (1 IVNTR); 'nat Mucl') and recombinant Mucl allelic variants H (23 repeats) ('rec Mucl -H') and F (7 VNTR) ('rec Mucl-F'). DESCRIPTION OF THE SEQUENCE LISTINGS

The invention will be further described, also by way of example only, with reference to the sequence listings, wherein:

SEQ ID No. 1 shows the amino acid sequence of a first allelic variant of bovine MUCl.

SEQ ID No. 2 shows the amino acid sequence of a second allelic variant of bovine MUCl.

SEQ ID No. 3 shows the amino acid sequence of a third allelic variant of bovine MUCl.

SEQ ID No. 4 shows the amino acid sequence of a fourth allelic variant of bovine MUCl.

SEQ ID No. 5 shows the amino acid sequence of a fifth allelic variant of bovine MUCl.

SEQ ID No. 6 shows the amino acid sequence of a sixth allelic variant of bovine MUCl.

SEQ ID No. 7 shows the amino acid sequence of a seventh allelic variant of bovine MUCl.

SEQ ID No. 8 shows the amino acid sequence of a first allelic variant of the bovine MUCl VNTR region.

SEQ ID No. 9 shows the amino acid sequence of a second allelic variant of the bovine MUCl VNTR region.

SEQ ID No. 10 shows the amino acid sequence of a third allelic variant of the bovine MUCl VNTR region.

SEQ ID No. 11 shows the amino acid sequence of a fourth allelic variant of the bovine MUCl VNTR region. SEQ ID No. 12 shows the amino acid sequence of a fifth allelic variant of the bovine MUCl VNTR region.

SEQ ID No. 13 shows the amino acid sequence of a sixth allelic variant of the bovine MUCl VNTR region.

SEQ ID No. 14 shows the amino acid sequence of a seventh allelic variant of the bovine MUCl VNTR region.

SEQ ID No. 15 shows the nucleotide sequence of the forward primer used to amplify the VNTR region of bovine MUC 1.

SEQ ID No. 16 shows the nucleotide sequence of the reverse primer used to amplify the VNTR region of bovine MUCl.

SEQ ID NO. 17 shows the amino acid sequence for MUCl allelic variant H (23 VNTR units).

SEQ ID NO. 18 shows the amino acid sequence for MUCl allelic variant F (7 VNTR units.)

SEQ ID No. 19 shows the nucleotide sequence of the forward primer used for cloning of vector constructs for expression of MUCl allelic variants.

SEQ ID No. 20 shows the nucleotide sequence of the reverse primer used for cloning of vector constructs for expression of MUCl allelic variants.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

As can be seen from Figure 1 (taken from reference 5), human MUCl consists of a long, highly glycosylated extracellular projection, a transmembrane domain embedded in the lipid bilayer of the MFGM, and an intracellular segment that is connected to the actin cytoskeleton. MUCl presents a large extended conformation in comparison to other surface molecules such as receptors and membrane proteins. This scheme does not show the full extracellular projection of human MUCl which, on the basis of the VNTR and the negative charges carried by the glycan moiety, is predicted to range from 0.2-0.5 μm in size (from (5)).

Figure 2 shows that MUCl is present in MFGM and can be purified from this milk fraction. The MUCl glycoprotein stains poorly with Coomassie blue or silver in SDS- PAGE but can be more easily visualised using Alcian blue as a high molecular weight poorly focussed blue band.

MUC 1 is highly polymorphic because of extensive allelic variations and qualitative and quantitative differences in its glycosylation between tissues, individuals and species and because of proteolytic processing (14). Structurally, the MUCl protein is organised into distinct domains (see Fig. 3, Fig. 4 and Fig 10). The first domain is an amino-terminal signal sequence, which directs the protein to the extracellular surface and is then cleaved off from the mature protein. The second domain is a transmembrane region that is responsible for the binding of this protein to the lipid bilayer in the milk fat globule membrane. The third and most conspicuous domain encodes a variable number of non- identical tandem repeat units (VNTRs) of approximately 20 amino acids each. These repeats are typically characterised by a prevalence of proline, serine and threonine residues. Importantly, the amino acid sequence of the repeat units varies significantly between species, e.g. bovine and human MUCl share only about 35% sequence identity in the VNTR repeat domains while the cytoplasmic regions are about 85% identical. Due to these major sequence differences the functions of the extracellular repeat domains cannot be inferred from the function of these repeats in other species. This extracellular repeat domain is extensively glycosylated, and is flanked by degenerate repeat sequences and a short N-terminal region. The allelic VNTR (variable number of tandem repeats) polymorphism in MUCl is substantial in most mammalian species, particularly in humans where the repeat numbers range from 21 to 125 (15). However, the murine gene does not display this genetic variability. Bovine MUCl has been less extensively studied but appears to have a lower degree of allelic variation than human MUCl. Two separate research groups have each identified five protein variants of bovine MUCl based on SDS- PAGE mobility (16, 17). Based on recent genetic sequence information for bovine MUCl (9) a more recent study has more reliably identified four allelic variants, with repeat numbers ranging from 10 to 24 (18). However, the genetic survey conducted by the present inventors of several Australian dairy breeds has demonstrated seven allelic variants containing between 7 and 23 repeats.

Located towards the C-terminus of the VNTR region is an extended fourth domain incorporating an SEA module that contains five potential N-linked glycosylation sites and a major processing site. The processing site is the target for an endogenous protease whose specific activity results in the shedding of MUCl from the surface of cells. There is also an intracellular domain (fifth domain) that may be involved in signal transduction and protein-protein interactions.

The human MUCl gene consists of seven exons; exon 1 encodes the leader (signal) peptide, exon 2 the central domain containing the VNTR repeats, and exons 6 and 7 respectively, the transmembrane sequence and the cytoplasmic tail. A number of different MUCl isoforms generated by alternative splicing have also been described for human and mouse MUCl. Thus, MUCl can be expressed as four distinct protein iso-forms: membrane-anchored; soluble (proteolytic cleavage of the membrane-bound form); secreted MUCl /SEC (no transmembrane domain); and the splicing variant MUC 1/Y, which does not contain a tandem repeat domain (Fig. 3).

Human and bovine milk MUCl contain approximately 50% carbohydrate by mass and this is the major architectural feature of the protein (19). Moreover, the inventors have demonstrated that the oligosaccharides are important for the binding of bacteria to MUCl and thus oligosaccharide quantity and specific structures are important determinants of the overall bacterial sequestering activity of MUCl.

The inventors have determined the monosaccharide content of purified MUCl obtained by protease treatment of MFGM obtained from pooled milk samples from a large number of cows with different numbers of VNTRs. That analysis revealed the following average mole percent composition: Galactose 32.8± 3.9; N-acetyl Galactose 23.5± 1.4; Glucose 0.6± 0.2; N-acetyl glucosamine 5.5± 0.6; Fucose 0.3 ±0.1; Mannose 5.1± 1.7; N-acetyl Neuraminic acid (sialic acid) 32.2± 1.6.

Since the size and composition of MUCl varies with the number of genetically determined tandem repeats (YNTRs), the content of oligosaccharides can be significantly different from sample to sample of milk obtained from different cows or herds. Snow et al. (19) showed a high content of sialic acid and N-acetylgalactosamine and a significant amount of galactose, mannose and N-acetylglucosamine in MUCl from bovine milk. Species- specific differences exist between human and bovine MUCl. For example, while reactivity with the lectins, PNA (Peanut agglutinin) and WGA (wheat germ agglutinin), has been reported for both human and bovine MUCl, the lectin Con A (Concanavalin A) has been reported to bind only to bovine MUCl (20). This is consistent with the absence of detectable (or perhaps accessible) mannose in human MUCl. Further, the glycosylation pattern of MUCl is believed to change during the bovine lactation cycle.

The remarkable and very large variation in the structure of MUCl indicates that there is substantial variation in the extent to which these molecules protrude from the cell surface. The present inventors have determined that variations in size and carbohydrate profile of MUCl are major influences on its anti-bacterial activity.

In a broad aspect the invention is directed to isolated, purified or recombinantly produced anti-bactierially active bovine MUCl glycoprotein or active fragments or derivatives thereof. The invention is intended to extend to active fragments and derivatives of bovine MUCl, to the extent that such fragments and derivatives are functionally equivalent, in that they retain anti-bacterial activity. Preferably, a given quantity of the fragment or derivative is at least 10%, preferably at least 30%, more preferably at least 50, 60, 80, 90, 95 or 99% as effective as an equivalent amount of a native bovine MUCl from which the fragment or derivative is derived. Determination of the relative efficacy of the fragment or derivative can readily be carried out by utilising a prescribed amount of the fragment or derivative in an assay of anti-bacterial activity (such as described below in Example 3), in comparison to the same amount of native bovine MUCl (having the same number of VNTRs).

Derivatives are intended to encompass proteins having amino acid sequence differing from the protein from which they are derived by virtue of the addition, deletion or substitution of one or more amino acids to result in an amino acid sequence that is preferably at least 60%, more preferably at least 80%, particularly preferably at least 85, 90, 95, 98, 99 or 99.9% identical to the amino acid sequence of the original protein. The derivatives specifically include polymorphic variants and derivatives of fragments as discussed below.

By reference to "fragments" it is intended to encompass fragments of a protein that are of at least 5, preferably at least 10, more preferably at least 20 and most preferably at least 40, 60, 80 or 100 amino acids in length and which are functionally equivalent to the protein of which they are a fragment. Preferred fragments of the invention include those of SEQ ID nos. 8-14.

Reference herein to the proteins being "bovine" is intended to convey that the proteins (more specifically glycoproteins) are either directly derived from a bovine source (eg. gastrointestinal tract, female reproductive tract, pulmonary tissue or mammary tissue and most preferably from milk) or are active fragments or derivatives (as discussed above) that could be derived therefrom, even if actually obtained by recombinant or synthetic means, or indeed if sourced from an animal of another species.

The proteins (including peptide fragments and derivatives) of the invention are "anti- bacterially active" in that they exhibit some form of bacterial cytotoxicity, inhibition of bacterial proliferation or enhancement of the natural immune response to thereby at least reduce the bacterial load. Standard assays, such as those described in Example 3, are available to test for anti-bacterial activity. The anti-bacterial activity of the proteins of the invention can of course be tested in vitro, but in practice the proteins preferably exhibit anti-bacterial activity that is effective in vivo in a human or animal system.

The proteins of the invention can therefore be incorporated into a wide variety of products, such as food and drink products, animal feed, pharmaceutical and veterinary products, nutraceutical or health supplement products, medical plastics, contact lenses, contact lens cleaning solutions, cleaning solutions for dentures or mouthguards, cosmetics, skin lotion, mouth wash or spray or saliva substitute for dry mouth syndrome, lozenges, veterinary supplements and cleaning/disinfecting products, for example. In each case the proteins of the invention can be formulated with other agents such as excipients, other active agents and food and drink ingredients conventional with products of that type. In the case of food (including drink) and feed products, compatible food or feed components include, for example, nutritious components such as those of fruit, vegetable or animal derivation and including grains, seed, pulses, meat, milk (from cattle or other mammals) or materials derived from any of these as well as sugar or other sweetening agents, emulsifiers, thickeners, flavouring agents, colouring agents, flour, yeast or other leavening or rising agents, salts, pH adjusting agents and water as well as other conventional ingredients. Food products that may incorporate the proteins include, for example, baked goods such as breads, biscuits and cakes, processed foods such as pasta, sauces, jams and spreads, processed meats, cereals, confectionary, desserts, pre-made meals, drinks (including sport drinks/fruit drinks) and foods coated with the proteins. In preferred embodiments the proteins are included in milk derived products such as modified milk, flavoured milk, milk based drinks, condensed milk, yogurt, cheese, cream, butter or other dairy based spreads, junket, custard, baked goods, desserts, ice-confections, milk powder and bulk milk derived material whether in liquid or dried form, each of which has had the protein added or is enriched with the protein such that it is included at higher levels than normal.

Preferably care will be taken to ensure processing of products comprising the proteins does not involve conditions and exposure to other ingredients that adversely affect activity of the proteins to any significant extent. For example, it is preferred that the proteins are not exposed to extremes of pH or long periods of high temperature that can result in protein denaturation, as would be well understood by persons skilled in the art.

The present invention also relates to pharmaceutical compositions comprising the proteins, optionally in conjunction with other active agents, and combined with one of more conventional pharmaceutically acceptable additives. The pharmaceutically acceptable additives may be in the form of carriers, diluents, adjuvants and/or excipients and they include all conventional solvents, dispersion agents, fillers, solid carriers, coating agents, antifungal or antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and slow or controlled release matrices. The proteins may be presented in the form of a kit of components adapted for allowing concurrent, separate or sequential administration. Each carrier, diluent, adjuvant and/or excipient must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and physiologically tolerated by the subject. The compositions may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the protein with the carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the proteins with liquid carriers, diluents, adjuvants and/or excipients or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the protein; as a powder or granules; as a solution or a suspension in an aqueous phase or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. inert diluent, preservative disintegrant (eg. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made my moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the conjugate therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating or may be encapsulated, to provide release in parts of the gut other than the stomach and/or to provide protection from extreme conditions encountered in the gastrointestinal tract.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended subject; and aqueous and non-aqueous sterile suspensions which may include suspended agents and thickening agents. The compositions may be presented in a unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Compositions suitable for topical administration to the skin, ie transdermal administration, may comprise the active agents dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gels, creams, pastes, ointments and the like. Suitable carriers may include mineral oil, propylene glycol, waxes, polyoxyethylene and long chain alcohols. Transdermal devices, such as patches may also be used and may comprise a microporous membrane made from suitable material such as cellulose nitrate/acetate, propylene and polycarbonates. The patches may also contain suitable skin adhesive and backing materials.

The proteins may also be presented within medical devices such as implants, contact lenses or other forms of medical plastic, which comprise the protein and a polymer device wherein the polymer is biocompatible and non-toxic. Suitable polymers include hydrogels, silicones, polyethylenes and biodegradable polymers.

The proteins of the invention can be administered in a sustained (ie controlled) or slow release form. A sustained release preparation is one in which the protein is slowly released within the body of the subject once administered and maintains the desired concentration over a minimum period of time. The preparation of sustained release formulations is well understood by persons skilled in the art. Dosage forms include oral forms, implants and transdermal forms. For slow release administration, the proteins can be suspended as slow release particles or within liposomes, for example.

It should be understood that in addition to the ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art, having regard to the type of composition in question. For example, agents suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents, disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.

Other details of pharmaceutically acceptable carriers, diluents and excipients and methods of preparing pharmaceutical compositions and formulations are provided in Remmington's

Pharmaceutical Sciences 18^th Edition, 1990, Mack Publishing Co., Easton, Pennsylvania, USA, the disclosure of which is included herein in its entirety by way of reference.

The proteins for use in the invention may also be presented for use in veterinary compositions. These may be prepared by any suitable means known in the art. Examples of such compositions include those adapted for:

(a) oral administration, eg drenches including aqueous and non-aqueous solutions or suspensions, tablets, boluses, powders, granules, pellets for admixture with feedstuffs, pastes for application to the tongue;

(b) parenteral administration, eg subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension;

(c) topical application, eg creams, ointments, gels, lotions, etc.

Throughout this specification the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms apply equally to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to both naturally and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. "Amino acid analogues" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is a carbon that is bound to a hydrogen, a carboxyl group, an amino group and an

R group, e.g., homoserine, norleucine, methionine sulfoxide and methionine methyl sulphonate. Such analogues have modified R groups (e.g. norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure different from the general chemical structure of an amino acid, but retain a function similar to that of a naturally occurring amino acid. Proteins, peptides and polypeptides of the invention may be isolated or purified from naturally occurring sources or may be synthetically produced by routine chemical or molecular biological techniques, as well understood in the art. By the terms "isolated" and "purified" it is intended that the proteins are extracted from a naturally occurring source and have been removed from at least some components with which they are associated in nature, such that they are preferably concentrated relative to their natural state. Preferably the isolated or purified proteins are at least 10 percent pure, more preferably at least 20, 30, 50, 70, 80, 90, 95, 98, 99 or 99.9 percent pure when considered by weight relative to other components such as proteins, nucleic acids, carbohydrates, fats and the like. In a preferred embodiment of the invention the proteins are 100% pure, and may for example be in an aqueous pH buffered and isotonic form or in the form of a lyophilisate.

The proteins or peptides of the invention include glycoproteins, that is, proteins to which one or more sugar moieties are bound. Such glycoproteins include both those linked via

OH groups of the protein chain (O-linked glycoproteins) and those linked via amide nitrogens on the protein chain (N-linked glycoproteins). Further details of routine techniques for protein and peptide production and purification are provided in Sambrook &

Russell, Molecular Cloning: A laboratory manual, 3rd Edition, 2001, Cold Spring Harbour Laboratory Press, New York, the disclosures of which are included herein in their entirety by way of reference.

The proteins according to the invention are preferably characterised by significant levels of O-linked glycosylation, with lower levels of N-linked glycosylation. For example, proteins of the invention may be 0-link glycosylated at between about 51 to about 300 sites, preferably between 51 to 200 sites or most preferably between 51 to 161 sites. For example, the proteins may be glycosylated at at least 70, 90, 110, 130 or 150 sites.

It is to be understood that reference herein to VNTRs is intended to include regions comprising imperfect tandem repeating units, which may vary for example by the substitution, deletion or addition of one or more amino acids, as long as there is a general pattern of repetition across the domain in question. Preferably, the VNTR region of bovine MUCl glycoproteins according to the invention is characterised by repeating units of between about 15 and about 25 amino acids in length, more preferably about 18 to 22 and most preferably about 20. Preferably also the VNTR region is characterised by comprising between about 7 to about 23 repeats (that is the "repeat number" is between about 7 and about 23), preferably at least 10, more preferably at least 12, 14, 16, 18, 20 or 22 repeating units. Particularly in recombinantly or synthetically produced proteins the repeat number of the VNTR domain can be in excess of 23, such as 24 to 200, preferably 24 to 150, 24 to 100, 24 to 75, 24 to 50 or 24 to 30, for example.

Suitable routes of administration for the proteins of the invention or for pharmaceutical, nutraceutical and veterinary compositions comprising them include oral, rectal, nasal, inhalation of aerosols or particulates, topical (including buccal and sublingual), transdermal, vaginal, intravesical and parenteral (including subcutaneous, intramuscular, intravenous, intrasternal, intrathecal, epidural and intradermal). Preferably, administration of the proteins will be via the oral route. However it will be appreciated that the preferred route will vary with the condition and age of the subject, and the judgement of the physician or veterinarian.

An important aspect of the invention relates to a process for purification of MUCl glycoprotein, or more particularly its extracellular domain, from milk fat globule membranes. The process generally comprises the steps of:

(b) inactivating the protease and recovering a soluble fraction obtained from the digestion:

(c) exposing the soluble fraction to anionic chromatography and then hydrophobic interaction chromatography, and recovering a MUCl glycoprotein (extracellular domain) containing sample. Preferably the protease is trypsin, although other acceptable proteases include any that release the extracellular glycosylated domain of MUCl, preferably intact, such as endoproteinase lys C, chymotrypsin, thermolysin and other proteases with similar cleavage specificity to trypsin.

MUCl glycoprotein (extracellular domain) may also be purified from milk whey that has been exposed to agitation, storage and/or temperature fluctuation, or from butter milk or cheese whey, by exposing milk whey, butter milk or cheese whey to anionic chromatography and then hydrophobic interaction chromatography, and recovering a MUCl glycoprotein (extracellular domain) containing sample.

The butter milk or cheese whey can, for example, be obtained as by-products of butter and cheese manufacture.

Preferably the aqueous suspension of MFGM is obtained by homogenising a milk derived cream fraction to buttermilk, separating MFGM from the buttermilk and optionally resuspending the MFGM, if necessary. Preferably the MFGM is separated from the buttermilk by filtration and/or centrifugation and preferably the digestion of MFGM with trypsin is conducted for between about 30 minutes and about 8 hours, at a temperature of between about 35⁰C and about 4O⁰C and at a pH of between about 6.5 and about 8.5. Preferably trypsin is inactivated using benzamidine and EDTA. In a preferred embodiment the soluble MUCl fraction from the digestion is recovered by filtration and/or centrifugation.

In another preferred embodiment a dialysis step is conducted prior to one or both of the chromatography steps. Preferably DEAE Sepharose is used as the anion exchange resin, although it is possible to use any conventional anion exchange resin. These can be weak or strong anion exchange resins which are defined as an insoluble organic polymer containing cation groups that attract and hold anions present in a surrounding solution in exchange for anions previously held. Examples of other suitable resins include Q-Sepharose, DEAE Cellulose, quaternary amine QAE Sephadex.

Preferably elution from the anion exchange resin is achieved by applying a 0-1 M NaCl gradient, wherein MUCl glycoprotein containing fractions are eluted at 200-400 mM NaCl. Persons skilled in the art will of course understand that other conditions may equally be adopted. Preferably Phenyl Sepharose is used as the hydrophobic interaction resin, although other hydrophobic interaction resins such as those containing a range of hydrophobic functional groups such as alkanes and aromatics may also be used eg HiTrap HIC, ProPac HIC-10. Preferably the MUCl glycoprotein containing fraction is eluted in the break-through (unbound) fraction.

Examples of bacterial infections that may be treated or prevented according to the invention include, but are not limited to, pathogenic gram-negative infections caused for example by E.coli, salmonella, shigella, Campylobacter, helicobacter, cholera, Vibrio choleras, and Vibrio parahaemalyticius, Serratia marcescens, the Pasteurella- Haemophilus group, Proteus and Providencia, pathogenic gram-positive infections caused for example by Staphylococcus and Streptococcus and Bacillus spp. (based upon Figures 7-9 data) enteric infections in animals such as swine dysentery (Brachyspira) in pigs .

The proteins according to the invention are also useful in treatment/prevention of inflammation in chronic inflammatory diseases of the gut such as IBD, UC and Crohn's disease, in the reduction in ulcerations in mouths of chemotherapy patients, and in wound healing such as in hospitalised patients, in skin burns victims where pseudomonas can be a problem and as an agent against propionobacterium in acne. In a particularly preferred embodiment of the invention the proteins can be incorporated in traveller's anti-biotic formulations to treat/protect against food and water bound bacterial infections commonly encountered by travellers. Preferably the mammal is a human, but in the context of this invention the term "mammal" is intended to encompass both humans and other mammals. Examples of other mammals include laboratory animals including rats, mice, simians and guinea pigs, domestic animals including cats, dogs, rabbits, agricultural animals including cattle, sheep, goats, horses and pigs and captive wild animals such as lions, tigers, elephants and the like.

As used herein, an "effective amount" refers to an amount of the protein or active fragment or derivative thereof that provides the desired anti-bacterial treatment or preventative effect. For example, dosing may occur at intervals of minutes, hours, days, weeks or months. Suitable dosage amounts and regimes can be determined by the attending physician or veterinarian, and will depend upon the age, weight, sex and general health of the patient as well as the purpose of the treatment. For example the proteins may be administered as a single dose, by infusion over a suitable time period or as multiple doses at separate times, preferably at regular intervals. For example, the proteins can be administered in amounts of between about lOμg/kg to about 500mg/kg, preferably between about lOOμg/kg to about 100mg/kg. Naturally the administration amounts can be varied if administration is conducted more or less frequently, such as by continuous infusion, by regular dose every few minutes or by administration every 10, 20, 30 or 40 minutes or every 1, 2, 3, 4, 6, 8, 10, 12, 16 or 24 hours, for example.

According to a still further embodiment of the present invention there is provided a method of preventing or inhibiting bacterial growth at a locus, which comprises exposing the locus to an effective amount of the glycoprotein or active fragment or derivative thereof according to the invention. For example, the locus may be a contact lens, denture, mouth guard, food or drink package or component thereof or a food or drink preparation or processing area. In particular this aspect of the invention is useful for preventing, or at least slowing the rate of formation, of bacterial bio-films that are often associated with packaged foods. By applying the proteins of the invention to the foods themselves prior to packaging and/or to the packaging it is possible to increase the shelf-life of the product.

Another use of the proteins of the invention is as mouth moisturiser or lubricant, which may, for example, be useful for singers, actors, teachers and others who use their voice for an extended period of time.

According to another embodiment of the present invention there is provided a method of determining the MUC 1 variable number of tandem repeats (VNTR) repeat number for a mammal, which comprises amplifying a nucleotide sequence encompassing the MUCl VNTR region from a DNA sample from the mammal and determining the VNTR repeat number from the amplified sequence.

According to a further embodiment of the present invention there is provided a method of determining the MUCl variable number of tandem repeats (VNTR) repeat number for a mammal, which comprises exposing a DNA sample from the mammal to PCR amplification using a forward primer complimentary to a MUCl sequence located 5¹ to the VNTR region and a reverse primer complimentary to a MUCl sequence located 3' to the VNTR region, and sequencing the amplified sequence. Preferably the mammal is bovine and in one preferred embodiment the forward primer comprises the nucleotide sequence 5'- C AT AAACCCCCGC AGAACTA-3¹ and the reverse primer comprises the nucleotide sequence 5'-TAATATGGCTGGCAGCAGTG-S'.

According to a further embodiment of the present invention there is provided a method of up-regulating MUCl glycoprotein production in the milk of a mammal which comprises exposing mammary tissue or mammary epithelial cells of the mammal to cell walls or cell wall components from Gram-negative or Gram-positive bacteria (preferably Gram- negative bacteria). Preferably the mammal is bovine. A method is also provided to measure the quantity of MUCl in milk or fractions derived from milk, which can be achieved using an immuno-assay approach. For example, MUCl isolated from MFGM can be used as an immunogen in chickens. After three injections antibody can be harvested from eggs. The antibody can be immuno-affinity purified on a column of immobilized MUCl. The antibody can then be labelled with biotin. Unlabelled and biotin labelled anti-MUCl antibodies are then used in an antigen capture ELISA assay, in which unlabelled antibody to MUCl is immobilised on the ELISA plate and used to capture MUCl in milk. The biotin labelled MUCl in conjunction with streptavidin peroxidase is used for detection.

A rapid immunoassay-based field kit based on the above mentioned immuno-assay approach can be prepared, which will, for example, allow farmers to conduct herd screening to determine by examining fresh milk the ability of individual cows to produce MUCl and/or to produce milk containing MUCl with a high VNTR number.

The invention will now be described further with reference to the following non-limiting examples:

EXAMPLES

Example 1:

Isolation and purification of MUCl from Milk Fat Globule Membranes (MFGM) MFGM is isolated from fresh pooled, unpasteurised full cream dairy milk as described by Pallesen, et al. (9) but with the following modifications. Briefly, full cream milk is centrifuged at 2,000xg for 15 min at 10 ⁰C. The cream fraction is then collected, washed (x3) in Washing Buffer (5O mM Tris, pH 7.5, 15O mM NaCl [TBS] and 1 mM EDTA, 1 mM Benzamidine [Sigma Chemical Co.]) and centrifuged at 2,000xg for 15 min at 1O⁰C after each wash. The cream is subsequently resuspended in two volumes of Washing Buffer and homogenized to butter using an Ultra-turrax tissue homogenizer (IKA works, Wilmington, NC) at 4⁰C. The expressed buttermilk is collected by filtration through cheese cloth and centrifuged at 100,000xg for 30 min at 4⁰C. The MFGM pellet is collected, washed in TBS and re-centrifuged at 100,000xg for 30 min at 4⁰C before storing at -8O⁰C.

Trypsin digestion of MFGM

MUCl is highly glycosylated and this property has been exploited in a novel approach to the isolation and purification of this protein. This procedure exploits the resistance of the extracellular domain of MUCl to proteolytic cleavage due to its extensive N-linked and O- linked glycosylation. Crude MUCl preparation is prepared by resuspending, MFGM (160 mg) in 20 ml of Protease (Trypsin) Digestion Buffer (100 mM Tris pH 8.0) containing 8 mg of bovine pancreatic trypsin (Roche) and incubated for 4 h at 37⁰C. This treatment releases the large, glycosylated extracellular domain of MUCl from the MFGM. Benzamidine hydrochloride and EDTA (Sigma) are then added to a final concentration of 1 mM to inactivate the protease. The digest is then centrifuged at 100,000xg for 30 min at 4 ⁰C and the resultant supernatant (soluble fraction) containing the released MUCl is collected and filtered (0.45μm) to remove residual lipid.

Chromatography Purified MUCl is isolated from the MFGM tryptic digest using a combination of anion exchange chromatography followed by hydrophobic interaction chromatography. Anion exchange chromatography

The supernatant derived from the tryptic digestion of MFGM is dialysed against 20 mM Tris pH 7.0, 1 mM benzamidine hydrochloride and EDTA. After dialysis the sample is filtered and fractionated on a 10 ml DEAE Sepharose (Amersham Biosciences) column. MUCl is recovered from the column by applying a 0—1 M NaCl gradient wherein MUCl- rich fractions elute between 200 mM-400 mM NaCl.

Phenyl Sepharose chromatography MUCl -rich fractions from the DEAE Sepharose chromatography are then pooled, concentrated to 5.0 ml and dialysed against 50 mM sodium phosphate, pH 7.0 containing 0.8 M ammonium sulphate. The dialysate is then applied to a 10 ml Phenyl Sepharose (low substitution, Amersham Biosciences) column and purified MUCl (extracellular domain) is then eluted in the break-through fraction and is termed "trypsin-released MUCl".

SDS-P olyacrylamide Gel Electrophoresis

The proteins present in MFGM and those released from MFGM by tryptic digestion are subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 6-18% gradient gels and subsequently stained with Silver (10), Coomassie Blue R250 (11) or Alcian blue (12- 13). 10 and 25 μg of total and reduced protein are loaded per lane for the MFGM and proteins released from the MFGM by trypsin digestion, respectively. All samples are electrophoresed under reducing conditions. MUCl stains very poorly with Silver and Coomassie Blue but is clearly visible as a diffuse high molecular weight blue band using Alcian Blue (Figure 2).

Amino-terminal amino acid sequencing and peptide mass mapping Proteins released from the MFGM by proteolytic (trypsin) digestion are blotted to polyvinylidine fluoride (PVDF) membrane (Problot Applied Biosystems) and the amino- terminal amino acid sequence of selected bands determined using a Procise Protein Sequencer (Applied Biosystems). The presence of MUCl in MFGM and the proteins released from the MFGM by proteolytic (tryptic) treatment is also confirmed by tryptic peptide mass mapping and amino acid sequencing using matrix-assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry or MS/MS and Edman sequencing approaches (Proteomics Analyzer 4700, Applies Biosystems).

Direct amino-terminal amino acid sequencing of purified MUCl confirmed the N-terminal sequence of the isolated protein (NVPTLTTSD). In addition, three internal peptides were identified from the tryptic digest of MUCl (YVPPGSTK, SIWGLILQIYKQR and YVPPGSTK). However, the majority of the MUCl protein is unaffected by the analytical tryptic treatment, presumably due to its extensive glycosylation.

Example 2: Characterisation of Bovine MUCl allelic variants

Allelic polymorphism in the VNTR (variable number of tandem repeats) region of bovine MUCl can be detected by PCR amplification using primers that target sequences outside the VNTR domain, as shown in Figure 4.

This PCR approach has been used to survey MUCl VNTR polymorphism in DNA samples from a diverse range of cattle breeds, including Holstein, Jersey, Guernsey, Ayrshire, Brown Swiss, Illawarra, Australian Red Breed, Australian Friesian Sahiwal and Brahman. DNA can be extracted from bovine sources using methods well known by persons skilled in the art, such as those disclosed in Sambrook & Russell, Molecular Cloning: A laboratory manual, 3rd Edition, 2001, Cold Spring Harbour Laboratory Press, New York, the disclosures of which are included herein in their entirety by way of reference. PCR Method

Primers used to amplify the VNTR region of bovine MUCl: The forward primer has the nucleotide sequence 5'-CATAAACCCCCGCAGAACTA-S', The reverse primer has the nucleotide sequence 5'-TAATATGGCTGGCAGCAGTG-S'.

PCR is carried out using Ix PCR buffer, 200 μM dNTPs, 500 nM of each primer, 50 ng genomic DNA and 0.6 U HotStar™ Taq DNA polymerase (Qiagen). The PCR cycling includes an initial denaturation at 94⁰C for 15 min, followed by 10 cycles of denaturation at 94⁰C for 30 s, touch-down annealing for 45 s at a starting temperature of 7O⁰C with a decrease of 0.5⁰C per cycle, and extension at 72⁰C for 1.5 min. Another set of 25 cycles with denaturation at 94°C for 30 s, annealing for at 65⁰C for 45 s, and extension at 72°C for 1.5 min is carried out, followed by a final extension step at 72°C for 10 min. The PCR products are visualised and separated by agarose gel electrophoretic analysis, excised from the gel, purified and sequenced.

MUC 1 polymorphic alleles in cattle

DNA sequencing of the polymorphic alleles of bovine MUCl has demonstrated a fundamental repeat structure of 60 bp (coding for 20 amino acids), which shows marked variation in copy number. The number of VNTRs observed ranges from 7-23 repeats in a total of seven allelic variants. The alleles (as graphically depicted in Fig. 5) have been designated A (16 repeats), B (14 repeats), C (11 repeats), D (9 repeats), F (7 repeats), G (12 repeats), and H (23 repeats). There is also an intra-repeat polymorphism that has the potential to change glycosylation from O-linked to N-linked glycosylation at some specific regions of the repeat. The inventors understand this has effects on the physical properties of the protein and potentially its ability to bind bacteria.

The most predominant alleles detected are A, B and C, and the majority of the animals tested are either homozygous for one of these three alleles (e.g. AA), or heterozygous (eg. AB, AC and BC). Figure 5 shows the repeat sequences and structure of each allelic variant, and Figure 6 shows the range of genotypes detected. The marked differences in size between the smaller and larger alleles (e.g. 7 repeats versus 23 repeats) significantly affect the anti-bacterial activity of the bovine MUCl protein. Without wishing to be bound by theory the inventors believe this is due to the extent of O- linked glycosylation (which is in proportion to the number of VNTRs) and also to the different types of glycosylation resulting from allelic differences that introduce an N- linked glycosylation site in some alleles. For example, the longest allelic variant detected (H) has 161 putative O-linked glycosylation sites and 8 putative N-linked glycosylation sites in the VNTR region (assuming that all putative glycosylation sites are occupied). The corresponding numbers for the shortest allelic variant detected (F) are 51 and 2, respectively. These differences are correlated to the ability of each MUCl protein variant to sequester pathogenic bacteria, with increased levels of O-linked glycosylation correlated to improved anti-bacterial activity.

Example 3: Anti- bacterial activity of bovine MUCl Using human intestinal Caco-2 cells, the inventors' experimental data show that MUCl purified [termed MUCl glycoprotein (extracellular domain)] from bovine milk fat globule membranes can bind gastrointestinal pathogens and inhibit their adherence to human gut epithelial cells. Intestinal mucin-pathogen interactions represent a primary step in causing electrolyte and fluid secretion leading to gastroenteritis. Interruption of the adherence of enteropathogens to human intestinal mucins can prevent or improve intestinal infections. The distinct bovine glycosylated MUCl forms; membrane-anchored, soluble (proteolytic cleavage of the membrane-bound form), secreted MUCl /SEC (no transmembrane domain), and especially the O-glycosylated variants of MUCl are suitable candidates for food-derived therapeutic approaches to intestinal bacterial infections and for other applications requiring anti-bacterial activity.

The present inventors have also shown that MUCl both prevents bacterial binding and displaces bacteria bound to bovine mammary epithelial cells, indicating that this glycoprotein can be used to treat mastitis in cattle and humans. In vitro model of MFGM inhibition of bacterial binding to human intestinal Caco-2 cell line

Two bovine milk fractions rich in the membrane bound form of MUCl, namely the milk fat globular membrane (MFGM) and trypsin-released proteins cleaved from the surface of the MFGM (MFGM_TRY) (a fraction rich in MUCl, Figure 2), have been tested for their ability to inhibit microbial binding to a human intestinal epithelial cell line, Caco-2. The testing employs a Caco-2 adenocarcinoma cell line as an in-vitro model of the human lower intestinal tract and representative strains of intestinal pathogens including E. coli, coagulase positive S. aureus, pathogenic B. subtilis and a non-invasive aroA mutant of S. typhimurium.

Inhibition of bacterial binding to Caco-2 cells is quantitated using a variation of the microplate assay described by Simon et al (28). This assay is performed twice with each dilution analysed in triplicate. Caco-2 cells are seeded in 96 well, black-sided tissue culture plates (Greiner-bio) with 100 μl of supplemented A-DMEM. Media is refreshed daily and the cells allowed to grow to 100% confluency. To fluorescently label bacteria, 1 x 10⁹ cfu in 1 ml PBS are incubated with 100 μg of fluorescein-5-isothiocyanate (FITC; Molecular Probes) for 30 min at room temperature (7). Excess probe is removed with three washes (5 min, 3000^χg) in 1 ml of PBS followed by final dilution to 1 * 10⁸ cfu/ml in tissue culture media. A 25 μl aliquot of FITC labelled bacteria is pre-incubated with one volume of the potential inhibitor for 20 min at room temperature with gentle shaking. Unbound bacteria are removed using three gentle washes with PBS. The wells are monitored between each wash to ensure there is no detachment of the monolayer resulting from cell injury. The fluorescence intensity within each well is measured on a Fusion™ universal microplate analyser (Perkin-Elmer Life and Analytical Sciences) with excitation and emission filters set at 490 nm and 530 nm, respectively. Binding of bacteria is determined using the following formula:

% binding = 100 X (Iexperimental " Inegative)/(Ipositive " Inegative) Equation 1 where I_exp_erim_ental is the fluorescence intensity measured in wells incubated with FITC labelled bacteria, test agent and Caco-2 cells, In_ega_tiv_e is the fluorescence intensity in wells incubated with unlabelled bacteria and Caco-2 cells (accounting for autofluorescence of samples), I_poSi_ti_ve is the fluorescence intensity in wells incubated with FITC labelled bacteria and Caco-2 cells in the absence of a test agent.

Figure 7 shows strong inhibition of Gram-negative bacteria (E. coli and S. typhimuήum) binding to the Caco-2 cell line by both milk fractions, and a lesser inhibition of the Gram- positive bacteria S. aureus and B. subtilis. Thus, milk protein fractions rich in MUCl strongly inhibit the binding of some pathogenic bacterial species to a human intestinal epithelial cells grown in culture.

Inhibition of bacterial binding to Caco-2 by purified bovine MUCl glycoprotein (extracellular domain)

Inhibition assays are conducted using serial dilutions of bovine MUCl purified by the chromatographic methods described in Example 1 above. Binding of Gram-negative bacteria to Caco-2 cells is reduced dramatically by MUCl in a dose dependant manner (Figure 8, A and B). At the highest MUCl concentration (1 μg/ml) binding of E. coli to the Caco-2 cells is abolished and that of S. typhimurium is reduced by greater than 80%. Inhibition of the Gram-positive bacteria is less pronounced however. At the highest MUCl concentration adherence of S. aureus and B. subtilis are reduced by 43% and 26%, respectively (Figure 8, C and D).

These results demonstrate that bovine MUCl purified by the procedures in Example 1 inhibits binding of both Gram-negative and Gram-positive bacteria to human intestinal epithelial cells (human Caco-2 cells). The inhibition of binding of the Gram-negative E.coli and aroA mutant of Salmonella typhimurium is more pronounced.

Solid-phase binding of pathogenic bacteria to immobilised bovine MUCl The ability of purified bovine MUCl to effectively interact with the bacterial cell surface and bind to bacteria has been examined using a solid phase (slot blot) assay. Binding of bacteria to MUCl is performed using a variation of the method previously described by Takahashi et al (29). Samples of MUCl are serially diluted (100 ng/ml - 100 μg/ml) and adsorbed onto Westran S^® PVDF membrane (Schleicher & Schuell) using a Bio-Dot^® SF microfiltration apparatus (BioRad). The membrane is blocked for 1 h at room temperature with PBS-0.1% Tween20. Bacteria are labelled with Sulpho-NHS-LC-biotin and overlayed onto the membrane overnight at a final concentration of 2.5 x 10⁷ cfu/ml. Non-adherent bacteria are removed by five washes with PBS-0.1% Tween 20. Adherent bacteria are detected using conjugated streptavidin-horse radish peroxidase and standard chemiluminescence techniques (Pierce). Figure 9 shows a dose dependant binding of all bacteria tested to increasing concentrations of immobilised MUCl. Maximum binding is seen with 10 μl of 100 μg/ml concentration of MUCl (1 μg). However, the Gram negative bacteria show much greater binding to lower quantities of MUCl compared with the Gram positive bacteria. This result is consistent with the greater ability of MUCl to inhibit the binding of Gram negative bacteria to human intestinal Caco-2 cells.

Inhibition of bacterial binding to Caco-2 cells by purified MUCl pretreated with neuraminidase

Bovine MUCl is a heavily glycosylated protein with sialic acid being the predominant terminal residues on the majority of the O-linked oligosaccharides (31). A 96 well inhibition assay was performed using neuraminidase treated MUCl (purified by the procedure of Example 1) (NMUCl) and untreated MUCl purified by the procedure of Example 1 as the control to examine the role of sialic acid in the inhibition of bacterial binding to human Caco-2 cells by MUCl. Neuraminidase from C. perfringens is well characterised in its ability to specifically hydrolyse terminal glycosidic linkages of sialic acid residues on glycoproteins. Sialic acid removal from bovine MUCl is confirmed by a shift in SDS-PAGE migration of the protein and loss of Alcian Blue staining (data not shown). As shown in Table 1, neuraminidase treatment dramatically decreases the ability of MUCl to inhibit bacterial adhesion indicating a major role played by sialic acid residues in binding intestinal pathogens. Table 1. Loss of inhibition of bacterial binding to Caco-2 cells by MUCl following neuraminidase treatment.

*Values represent percentage loss of inhibition of bacterial binding compared to control samples.

Example 4: Abilities of proteases to release MUCl from milk fat globule membranes and susceptibility of MUCl to digestion by various proteases.

Materials and Methods

Milk fat globule membranes were separately incubated with a number of individual proteases under a variety of conditions optimum for protolysis. The specific conditions are outlined below. This assay measured the ability of a protease to release MUCl from milk fat globule membranes whilst maintaining the structural integrity of the extracellular domain of MUC 1.

Bovine MUCl released from milk fat globule membranes by the action of trypsin (described previously) was incubated with a number of individual proteases, under a variety of conditions optimum for protolysis. The specific conditions are outlined below. This assay measured the stability of trypsin released MUCl to a range of proteases.

Trypsin, Chymotrypsin and Proteinase K

Milk fat globule membranes or MUCl released from milk fat globule membranes was incubated with the respective protease, at a protein to protease ratio of 25:1 (wt/wt), in 100 mM Tris-HCl pH 8.5, at 37°C for 24 hours. Proteolysis of MUCl was detected by SDS- PAGE, gels were initially stained with Silver and then counterstained with Alcian blue to visualize the MUCl. Alcalase

Milk fat globule membranes or MUCl was incubated with Alcalase at a protein to protease ratio of 100:1 (wt/wt), in 100 mM Tris-HCl pH 8.5, at 56°C for 1 hour. Proteolysis of MUCl was detected by SDS-PAGE, gels were initially stained with Silver and then counterstained with Alcian blue to visualize the MUCl .

Pepsin

Milk fat globule membranes or MUC 1 was incubated with Pepsin at a protein to protease ratio of 50:1 (wt/wt), in 200 mM HCl, 200 mM KCl pH 2.0, at 37⁰C for 24 hour. Proteolysis of MUCl was detected by SDS-PAGE, gels were initially stained with Silver and then counterstained with Alcian blue to visualize the MUCl .

Thermolysin Milk fat globule membranes or MUCl was incubated with thermolysin at a protein to protease ratio of 5:1 (wt/wt), in 100 mM Tris-HCl pH 7.5, at 37⁰C for 24 h. Proteolysis of MUCl was detected by SDS-PAGE, gels were initially stained with Silver and then counterstained with Alcian blue to visualize the MUC 1.

Bromelain and Multifect P-3000

Milk fat globule membranes or MUCl was incubated with the respective protease, at a protein to protease ratio of 100:2 (wt/wt), in 100 mM Tris-HCl pH 8.5, at 56⁰C for 4 h. Proteolysis of MUCl was detected by SDS-PAGE, gels were initially stained with Silver and then counterstained with Alcian blue to visualize the MUCl.

Results

The SDS-PAGE of Fig 11 demonstrates release of MUCl from milk fat globule membranes by a range of proteases. The SDS-PAGE was stained with silver and Alcian blue. The latter agent preferentially stains MUCl as a poorly focused high molecular weight blue band. Lane 1 shows size standards. Lane 2 is milk fat globule membrane protein (100 μg) without addition of any protease for 24 h at 2O⁰C (control). Lanes 3-5 are proteins released from milk fat globule membrane protein (100 μg) incubated with trypsin, endoproteinase Lys C or chymotrypsin (each 1 μg) for 24 h at 2O⁰C, respectively. All three proteases preferentially released MUCl from milk fat globule membranes without destroying the integrity of the released MUCl.

Figure 11 demonstrates that trypsin, endoproteinase Lys C or chymotrypsin can be used to release MUCl from milk fat globule membranes, a key step in the isolation and purification of MUCl from milk. Table 2 summarizes MUCl release and stability results generated for several different proteases.

Figure 12 shows an SDS-PAGE analysis of the stability of MUCl after its release from milk fat globule membranes by the action of trypsin. The SDS-PAGE was stained with silver and Alcian blue. The latter agent preferentially stains MUCl as a poorly focused high molecular weight blue band. MUCl was released from milk fat globule membranes by the action of trypsin and then subjected to digestion with alcalase (1% wt/wt for 1 h).

The 'pre' sample shows the starting material while the ' 1 hr' sample shows the sample after one hour of incubation with alcalase at 2O⁰C. Alcalase completely digested MUCl and is therefore not preferred for use in its preparation.

MUCl is not stable to the action of some proteases and therefore these proteases are not preferred for releasing MUCl from milk fat globule membranes or for use in other stages of its purification. Table 2. Action of proteases in the release of MUCl from milk fat globule membranes and effect on MUCl stability

Source of proteases (catalogue number)

Trypsin Roche (10109819001) Chymotrypsin Sigma (C-3142) Endoproteinase Lyc-C Sigma (P-3428) Protex 6L Enzyme Solutions Pepsin Sigma (P-6887) Thermolysin Sigma (T-7902) Proteinase K Sigma (P-2308) Bromelain Enzyme Solutions Multifect P-3000 Enzyme Solutions

+, released or stable; -, not released or not stable to the action of the protease.

Example 5: Longer alleles of the MUCl protein have greater ability to inhibit the binding of E. coli bacteria to human intestinal epithelial cells (CACO2).

Material and Methods

Bacterial strain and culture A non-pathogenic strain of E. coli (O-, H48) was used. Bacteria were grown in Brain Heart Infusion (BHI, Oxoid) medium. BHI broth was inoculated with a single bacterial colony forming unit (cfu) taken from a BHI agar plate and incubated overnight at 37°C under anaerobic conditions. A log phase culture was then prepared by inoculating 10 ml of BHI broth with a 1 *10⁹ cfu aliquot of the overnight culture followed by further incubation for 3 h. Cultures were used within two passages.

Caco-2 cell culture

All cell culture solutions were purchased from Invitrogen, Australia. Caco-2 adenocarcinoma cells were cultured at 37°C in humidified air with 5% CO₂ in Advanced Dulbecco's modified Eagles Medium (A-DMEM; 4.5 g/1 glucose) supplemented with 2% foetal bovine serum, 2 mM L-glutamine and 10 mM HEPES buffer. Assays were performed between passages 69 and 79. Cells were seeded at a density of 6x10⁴ cells/ cm² and monolayers used within 48 hours of reaching confluency. Assays were performed using bacterial cultures in log phase growth and at a multiplicity of infection (MOI) of 100.

Inhibition of E. coli binding to Caco-2 cells by MUCl

The MUCl variants AA and CC and a BSA control (all in triplicate 25 μl aliquots and 10- fold serially diluted in PBS from 100 μg/ml - 10 ng/ml) were pre-incubated with E. coli cells (25 μl aliquots of I xIO⁸ cfu/ml) at 37°C for 20 minutes. The solutions were then added to Caco-2 cells seeded in a 96-well, black-sided tissue culture plate (Greiner-bio) in 100 μl medium. E. coli in PBS only was used as a positive control. After a 1 hour incubation, non-adherent bacteria were removed from the Caco-2 cells with three gentle washes of the cells in PBS. The Caco-2 cells and adhering bacteria were detached from the plate with trypsin, diluted 1 : 10,000 in PBS, and plated in triplicate onto agar growth plates and incubated at 37°C for 48 h. The number of bacteria adhering to Caco-2 cells in the presence of the MUCl variants was determined by counting cfu on the agar plates (adjusted for dilution factor). The inhibitory activity of MUCl was defined as the mean percentage reduction in bound bacteria compared to the control. Results

Figure 13 shows the isolation of two different homozygous genotypic variants of the MUCl protein (alleles A (16 VNTR repeats) and C (11 VNTR repeats. Although these were not the most extreme VNTR variants of the protein, they represent the two genotypes that are predominant in the Australian production herd. The two variants were tested for their relative abilities to inhibit the binding of E. coli bacteria to CACO2 cells grown in cell culture. The AA genotype showed 31% greater inhibitory activity compared with the CC genotype. Thus, it is concluded that the greater the number of VNTRs in the MUCl protein, the greater is the inhibitory activity.

Example 6: Production of recombinant forms of MUCl

Materials and Methods

Chinese Hamster Ovary (CHO) cell culture

All cell culture solutions were purchased from Invitrogen, Australia. Chinese Hamster Ovary (CHO) Kl cells (ATCC CCL-61) were cultivated at 37°C in humidified air with 5% CO₂ in F- 12 Nutrient Mixture (Ham) containing 1 mM L-glutamine and supplemented with non-essential amino acids (Ix) and 5% foetal bovine serum. Transfections were performed between passages 11 and 25.

Design of expression constructs for production of recombinant MUCl allelic variants

PCR primers were designed to amplify the coding sequence of exon 2 from the MUCl gene. This exon encodes all of the variable number of tandem repeats (VNTR) encompassing most of the extracellular domain of MUCl. The amino acid sequences encoded by the amplified DNA sequences for MUCl allelic variants H (SEQ ID NO. 17) and F (SEQ ID NO. 18) are provide in Fig. 10 and in the sequence listings. In Fig. 10 ITALIC font indicates the VNTR sequence.

Cloning of vector constructs for expression of MUCl allelic variants Primers were purchased from Sigma-Proligo. The sequence of the forward primer was 5'- TACAAGCTTCAATGTCCCTACCCT-3' (SEQ ID NO. 19), corresponding to that encoding the amino acid sequence NVPT and incorporating a Hindlll restriction site at the 5' end. The sequence of the reverse primer was 5'-GACTCGAGCAGACAACTGCTGAG- 3' (SEQ ID NO. 20), corresponding to the amino acid sequence QQLS and incorporating an Xhol restriction site at the 5' end. Genomic DNA from animals with particular MUCl allelic variants was used as PCR templates.

PCR was carried out in 25 μl reactions containing Ix PCR buffer, 200 μM dNTPs, 500 nM of each primer, 20-50 ng genomic DNA and 0.3 U HotStar™ Taq DNA polymerase

(Qiagen). The PCR cycling included an initial denaturation at 94⁰C for 15 min, followed by 10 cycles of denaturation at 94⁰C for 30 s, touch-down annealing for 45 s at a starting temperature of 62°C with a decrease of I⁰C per cycle, and extension at 72⁰C for 1.5 min.

Another set of 25 cycles with denaturation at 94⁰C for 30 s, annealing at 52⁰C for 45 s, and extension at 72°C for 1.5 min was carried out, followed by a final extension step at 72⁰C for 10 min. The amplicons were separated by agarose gel electrophoresis and visualized with SYBR Safe DNA gel stain (Invitrogen). The PCR products corresponding to MUCl allelic variants H and F were excised from the gel, cloned into pSecTag2A vector

(Invitrogen) using the restriction sites incorporated in the primers, and sequenced using BigDye® Terminator v3.1 (Applied Biosystems). Transfection grade plasmid DNA was generated using EndoFree Plasmid Maxi Kit (Qiagen), according to the manufacturer's instructions.

Expression of MUCl allelic variants in CHO cells and protein purification CHO cells were transiently transfected with the constructs encoding MUCl variants H and

F as described above using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Briefly, 210 μg plasmid DNA and 525 μl Lipofectamine

2000 were individually mixed with 13.7 ml Opti-MEM medium and incubated separately for 5 minutes, then mixed together and co-incubated for 20 minutes at room temperature. The transfection mixture was added to three Tl 75 flasks (9 ml to each flask) containing

60-80% confluent CHO cells in 16 ml Opti-MEM. After 5-6 hours incubation at 37°C, the transfection mixture was removed and fresh Opti-MEM was added to the cells (100 ml per flask). The medium was collected and changed after three days incubation, and the cells were incubated for a further three days.

The medium collected during transfection was filtered and dialysed into 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0, and then concentrated to 20 ml. The solution was incubated with 3 ml of Ni-NTA agarose for 30 minutes. The Ni-NTA agarose was then washed three times in 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0. Bound protein was eluted in 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0, concentrated to 1 ml, and visualized by SDS-PAGE analysis.

Characterisation of recombinant MUCl proteins

The recombinant MUCl proteins were purified by Ni-NTA affinity chromatography and characterized by SDS-PAGE migration patterns; staining with silver and Alcian Blue after SDS-PAGE; and binding to Jacalin lectin. The latter lectin immobilized on Sepharose 4B can be used to independently purify the recombinant proteins. The lectin is specific for O- linked oligosaccharides which are prevalent on MUCl.

Native MUCl, purified as described elsewhere, and recombinant MUCl variants H and F, produced and purified as described above, were dialyzed against 50 mM Tris-HCl pH 7.5,

6 M urea and 10 mM dithiothreitol. The proteins were then further purified by gel permeation chromatography on a Tosohaas TSK-GEL G3000SW column using High

Performance Liquid Chromatography (HPLC). MUCl eluted in the void volume of the column. The MUCl proteins were buffer exchanged into water and lyophilized. Monosaccharide composition analysis was performed by the Glycotechnology Core

Resource at the University of California, San Diego (http://glvcotech.ucsd.edu/).

Briefly, the MUCl samples were treated with 2 M trifluoroacetic acid at 100°C for 4 hours. After drying the hydrolyzate, samples were dissolved in water and analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a CarboPac™ PAl column (Dionex) and eluted with a low concentration of sodium hydroxide (Dionex). Monosaccharide standards (Man, Gal, GIc, GIcNAc, GaINAc, and Fuc) were treated in parallel and used for calibration of the HPAEC-PAD response. For sialic acid determination, the samples were dissolved in 2 M acetic acid and heated to 80°C for 3 hours. The released sialic acid was collected by ultrafiltration through a 3 kDa nominal molecular weight cut-off filter and derivatized with 1,2- diamino-4,5-methylene dioxybenzene. The fluorescent sialic acid derivatives were analyzed by reverse-phase HPLC with on-line fluorescence detection. Identification and quantitation were based on known standards (Neu5Gc, Neu5Ac) run in parallel. The relative amounts of monosaccharides (including sialic acid) on native and recombinant MUCl were calculated and are presented as mole percent of the total monosaccharide content.

Results

Two recombinant bovine MUCl proteins were expressed in CHO (Chinese Hamster Ovary) cells, which are known for their ability to add O-linked and N-linked oligosaccharides including sialic acid to proteins. A commercial expression vector (pSecTag2; Invitrogen) was used according to the manufacturer's instructions with appropriate DNA corresponding to that encoding various artificial forms of bovine MUC 1. The DNA encoding the MUCl spanned regions from the beginning of the mature protein to a potential natural cleavage site C-terminal to the variable VNTR domain (GSVV) in all allelic variants The recombinant MUCl proteins were specifically designed for secretion from the CHO cells. The two recombinant proteins expressed represented versions with 23 (Allele H) and 7 (Allele F) VNTR units. The constructs also contained hexa-his tags to facilitate detection and purification and the corresponding proteins.

The recombinant bovine MUCl proteins preferentially stained with Alcian blue and were purified by Jacalin affinity chromatography (Figure 14). In addition their apparent size on SDS-PAGE was much greater than that predicted directly from the encoded amino acids. Thus, it is concluded that recombinant MUCl proteins expressed and secreted by CHO cells are extensively glycosylated. Figure 15 shows an oligosaccharide composition analysis of these recombinant proteins and a comparison with natural bovine MUCl isolated from milk. The latter contains a natural mixture of alleles A, B and C. The results indicate strongly similarities in the oligosaccharide compositions of the recombinant MUCl proteins and natural bovine MUC 1. Moreover, all proteins have a predominance of Gal, GaINAc and Neu, which are characteristic of extensively 0-linked glycoproteins. It is concluded that recombinant forms of MUCl have an oligosaccharide composition and structure similar to natural bovine MUC 1 and hence activities of the former proteins are likely to be the same as for the natural MUCl.

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Claims

1. An isolated, purified or recombinantly produced anti-bactierially active bovine MUCl glycoprotein or active fragment or derivative thereof.

2. The glycoprotein or active fragment or derivative thereof according to claim 1 comprising a variable number of tandem repeats (VNTR) region with a repeat number of between about 7 and about 23.

3. The glycoprotein or active fragment or derivative thereof according to either claim 1 or claim 2 wherein the repeat number is at least 10.

4. The glycoprotein or active fragment or derivative thereof according to either claim 1 or claim 2 wherein the repeat number is at least 14.

5. The glycoprotein or active fragment or derivative thereof according to either claim 1 or claim 2 wherein the repeat number is at least 16.

6. The glycoprotein or active fragment or derivative thereof according to either claim 1 or claim 2 wherein the repeat number is at least 20.

7. The glycoprotein or active fragment or derivative thereof according to claim 1 comprising an amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 14.

8. The glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 7 which is O-link glycosylated at between about 51 to about 161 sites.

9. The glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 8 which is O-link glycosylated at at least about 70 sites.

10. The glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 8 which is O-link glycosylated at at least about 90 sites.

11. The glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 8 which is O-link glycosylated at at least about 110 sites.

12. The glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 8 which is O-link glycosylated at at least about 130 sites.

13. The glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 8 which is O-link glycosylated at at least about 150 sites.

14. An anti-bacterially active food or feed comprising the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13 and one or more food or feed compatible components.

15. An anti-bacterially active pharmaceutical composition comprising the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13 and one or more pharmaceutically acceptable additives.

16. An anti-bacterially active veterinary composition comprising the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13 and one or more veterinary acceptable additives.

17. A bovine milk or milk derived product enriched with the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13.

18. The enriched milk derived product according to claim 17 which is a modified milk, a flavoured milk, a milk based drink, an infant formula, a geriatric food/formula, a yogurt, a cheese, a cream, an ice-confection, a butter, a junket, a custard, a baked good, a dessert or a milk powder.

19. A process for purification of MUCl glycoprotein (extracellular domain) from milk fat globule membranes (MFGM), comprising the steps of:

(a) exposing an aqueous suspension of MFGM to digestion with a protease at an appropriate temperature for a suitable period;

(b) inactivating said protease and recovering a soluble fraction obtained from said digestion; (c) exposing said soluble fraction to anionic exchange chromatography and then hydrophobic interaction chromatography, and recovering a MUCl glycoprotein (extracellular domain) containing sample.

20. The process according to claim 19 wherein the aqueous suspension of MFGM is obtained by homogenising a milk derived cream fraction to buttermilk, separating MFGM from the buttermilk and optionally resuspending the MFGM if necessary.

21. The process according to claim 20 wherein MFGM is separated from the buttermilk by filtration and/or centrifugation.

22. The process according to any one of claims 19 to 21 wherein digestion of MFGM with protease is conducted for between about 30 minutes and about 8 hours, at a temperature of between about 35⁰C and about 4O⁰C and at a pH of between about 6.5 and about 8.5.

23. The process according to any one of claims 19 to 22 wherein protease is inactivated using benzamidine and EDTA.

24. The process according to any one of claims 19 to 23 wherein the protease is trypsin.

25. The process according to any one of claims 19 to 24 wherein the soluble protein fraction from said digestion is recovered by filtration and/or centrifugation.

26. A process for purification of MUCl glycoprotein (extracellular domain) from milk whey that has been exposed to agitation, storage and/or temperature fluctuation, or from butter milk or cheese whey, comprising exposing milk whey, butter milk or cheese whey to anionic chromatography and then hydrophobic interaction chromatography, and recovering a MUCl glycoprotein (extracellular domain) containing sample.

27. The process according to any one of claims 19 to 26 wherein a dialysis step is conducted prior to one or both of the chromatography steps.

28. The process according to any one of claims 19 to 27 wherein DEAE Sepharose is used as the anion exchange resin.

29. The process according to claim 28 wherein elution from the anion exchange resin is achieved by applying a 0-1 M NaCl gradient, wherein MUCl glycoprotein containing fractions are eluted at 200-400 mM NaCl.

30. The process according to any one of claims 19 to 29 wherein Phenyl Sepharose is used as the hydrophobic interaction resin.

31. The process according to claim 30 wherein the MUCl glycoprotein containing fraction is eluted in a break-through or unbound fraction.

32. A method of treating or preventing bacterial infection in a mammal, which comprises administering to said mammal an effective amount of the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13.

33. Use of the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13 in preparation of a medicament for the treatment or prevention of bacterial infection in a mammal.

34. The method according to claim 32 or the use according to claim 33, wherein the mammal is a human.

35. A method of preventing or inhibiting bacterial growth at a locus which comprises exposing the locus to an effective amount of the glycoprotein or active fragment or derivative thereof according to any one of claims 1 to 13.

36. The method according to claim 35 wherein the locus is a contact lens, denture, mouth guard, food or drink package or component thereof or a food or drink preparation or processing area.

37. A method of determining the MUCl variable number of tandem repeats (VNTR) repeat number for a mammal, which comprises amplifying a nucleotide sequence encompassing the MUCl VNTR region from a DNA sample from the mammal and determining the VNTR repeat number from the amplified sequence.

38. A method of determining the MUCl variable number of tandem repeats (VNTR) repeat number for a mammal, which comprises exposing a DNA sample from the mammal to PCR amplification using a forward primer complimentary to a MUCl sequence located 5' to the VNTR region and a reverse primer complimentary to a MUCl sequence located 3' to the VNTR region, and sequencing the amplified sequence.

39. The method according to claim 38 wherein the mammal is bovine.

40. The method according to claim 39 wherein the forward primer comprises the nucleotide sequence 5'-CATAAACCCCCGCAGAACTA-3^l and wherein the reverse primer comprises the nucleotide sequence 5'- TAATATGGCTGGCAGCAGTG-S".

41. A method of increasing the VNTR repeat number of MUCl in milk produced by a herd of cows, which comprises determining the MUCl VNTR repeat number of individual cows in the herd and selecting cows to contribute to the milk based upon MUCl VNTR repeat number for each individual cow being at least 10.

42. The method according to claim 41 wherein the MUCl VNTR repeat number for each individual cow is at least 14.

43. The method according to claim 41 wherein the MUCl VNTR repeat number for each individual cow is at least 16.

44. The method according to claim 41 wherein the MUCl VNTR repeat number for each individual cow is at least 20.

45. A method of up-regulating MUCl glycoprotein production in the milk of a mammal which comprises exposing mammary gland cells of the mammal to cell walls, cell wall components or other cellular components from Gram-negative or Gram-positive bacteria.

46. The method according to claim 45 wherein the mammal is bovine.

47. The method according to claim 45 wherein the bacteria is Gram-negative bacteria.

48. A method of measuring the quantity of MUCl glycoprotein in the milk of a mammal which comprises exposing a sample of the milk to a competitive ELISA assay or immunomagnetic separation utilising an antibody to MUCl or a fragment thereof.

49. The method according to claim 47 wherein the MUCl antibody or fragment thereof is raised against the MUCl extracellular region.