WO1995002692A1 - Modified alpha-lactalbumin - Google Patents
Modified alpha-lactalbumin Download PDFInfo
- Publication number
- WO1995002692A1 WO1995002692A1 PCT/GB1994/001514 GB9401514W WO9502692A1 WO 1995002692 A1 WO1995002692 A1 WO 1995002692A1 GB 9401514 W GB9401514 W GB 9401514W WO 9502692 A1 WO9502692 A1 WO 9502692A1
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- WO
- WIPO (PCT)
- Prior art keywords
- lactalbumin
- modified
- phe
- human
- protein
- Prior art date
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C07—ORGANIC CHEMISTRY
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- This invention relates to modified proteins useful in the diet of patients with hyperphenylalaninemia.
- the frequency of PKU varies worldwide, with a rate of 1 in 5,000 in Ireland to about 1 in 100,000 in Japan. A conservative average estimate worldwide for incidence of PKU is 50 cases per million live births. In the US the frequency of PKU is about 1 in 10,000 of all screened newborns.
- phenylalanine content is then added as natural supplements to the amino acid formula base diet. Since these foods contain varying levels of phenylalanine, what is chosen and how much is consumed gives more or less flexibility and palatability to the overall diet.
- the level of phenylalanine allowed is only that which is required to meet growth and/or maintenance needs.
- the maximum level of tolerance of phenylalanine intake throughout childhood, for example, is 500mg/day, with an acceptable range of 200-500mg/day.
- such products should taste good, smell good, and allow maximum flexibility of food choices and menu planning when using them.
- the ideal product or formula should be highly palatable and low, preferably free, in phenylalanine: these are key qualities encouraging compliance and allowing the maximum intake of low protein phenylalanine-containing foods and a more normal diet.
- a high biological value food which is low or totally free of phenylalanine would be an extremely valuable dietary component because a greater variety and volume of natural foods containing phenylalanine would be allowed.
- DNA sequences encoding a variety of medically and nutritionally important proteins have been cloned in recent years. These include insulin, plasminogen activator, c. 1 -antitrypsin, blood factors VIII and IX, lysozyme, ⁇ t-lactalbumin and lactoferrin. From these DNA sequences the protein sequences can be deduced and verified by peptide mapping and Edman degradation.
- Protein X-ray crystallography and multidimensional NMR spectroscopy has enabled the three-dimensional structure of many proteins to be determined. This has been coupled with very powerful computers and advanced programs, which can model how changes in the amino acid residues may affect the structure and function of the proteins (Nilsson et al , Curr. Opin . in Struc. Biol . 2 569-575 (1992); Presta, L.G., Curr. Opin. in Struc. Biol . 2 593- 596 (1992); Cech, T.R., Curr. Opin . in Struc. Biol . 2 605-609 (1992) ; Pickersgill and Goodenough, Trends in Food Sci . and Tech . 5 122-126 (1991)) .
- strategies can be developed to isolate the DNA coding for that protein, and to generate a mutant DNA sequence which encodes the desired amino acid changes.
- Such strategies involve the subcloning of that region(s) of the gene which contains the amino acids which are to be changed. The sub-cloned fragments are then subjected to site directed mutagenesis. This technique can be used to target precisely the location and type of modification desired. At its most accurate, a single base in the DNA sequence can be changed to any of the other three bases, by means of oligonucleotide primers, which serve to mutagenise the DNA sequence.
- the DNA may then encode a totally different amino acid (McPherson, M.J., Directed Mutagenesis, Oxford Univ. Press, NY (1991); Carter, P., Biochem. J. 237 1-7 (1986); Nickoloff and Deng, Anal. Biochem. 200 81-88 (1992) ) .
- protein characteristics such as activity, specificity and stability are controlled by the relative positions of amino acids in the protein and by the physical and chemical properties of the amino acid.
- the structure of the protein is closely related to its function. Generally, it is important when making amino acid, and thus conformational, changes to a protein, not to alter its function, and to make sure it is processed properly and, if necessary, secreted from the cell.
- Physiological concerns depend on the nature of the chosen protein and whether it is an enzyme, a structural, regulatory, transport or food protein.
- One potential problem is that the mutation(s) affect structural and functional properties of a protein, such that the host cell or its function is compromised in some way.
- the protein needs to be as chemically well balanced as possible. Therefore, before any thought is given to possible mutations, the native protein must be analysed to assess its nutritional potential, in terms of amino acid content, digestibility and allergenicity. The ease of extraction will to some extent, be dependent on the method of production. This will dictate what contaminating substances are present and hence what method of extraction is used.
- the present invention is based, at its broadest, on the realisation that all these various and sometimes conflicting considerations can be adequately addressed by the choice of ⁇ -lactalbumin as the candidate for modification.
- a modified ⁇ -lactalbumin comprising no phenylalanine residues or fewer phenylalanine residues than wild type ⁇ -lactalbumin, other than a variant of human ⁇ -lactalbumin in which Phe-80 is substituted by lysine.
- a-Lactalbumin is the major whey protein in human milk, being present at levels of -2.5g/l (29% of total protein - Heine et al , J. Nutr. 121 277-283 (1991)). It is a globular, calcium binding protein, with a relative molecular mass of 14,200, and is found in almost all mammalian milks.
- ⁇ -Lactalbumin has a high nutritional value with essential amino acids accounting for 63% of the total.
- bovine Vilotte et al , Biochimie 69 609-620 (1987) and human ⁇ - lactalbumins (Hall et al , Biochem. J. 242 735-742 (1987) contain 123 amino acid residues, with only four phenylalanine, and four tyrosine residues per monomer
- ⁇ -Lactalbumin has formed part of our diet for millennia in the form of human, cow, sheep, goat and camel milks.
- the function of ⁇ -lactalbumin is twofold. Its main role is as one component of the lactose synthase system.
- ⁇ -Lactalbumin acts as a specifier protein, by modifying the substrate specificity of UDP / 3l,4- galactosyl transferase (E.C. 2.4.1.22), the second component of the enzyme system. Secondly, ⁇ -lactalbumin has a role as a source of nutrition.
- ⁇ -Lactalbumin is a relatively small secretory protein which has only four phenylalanine residues. As it is a milk protein it is ideally suited for expression in the mammary gland of a transgenic mammal which, as will be discussed below, is the expression system of choice, ⁇ - Lactalbumin is well suited to meeting the structural, physiological, nutritional and harvestability considerations discussed above. Structurally, the four phenylalanine residues in the human and bovine mature proteins are at positions 3, 31, 53 and 80, and 9, 31, 53 and 80 respectively. A protein engineering study of these two proteins shows that almost any residues could in principle be accommodated in place of phenylalanine, on the basis of size and space. However, positions Phe-31 and 80 are involved with the lactose synthase reaction and calcium binding loop respectively, and so more care had to be taken when considering suitable substitutions for these positions.
- ⁇ -lactalbumins can function with galactosyl transferases from other species (Khatra et al , Eur. J. Biochem. 44 537-560 (1974) ; Powell and Brew, Biochem. J. 142 203-209 (1974)).
- Phenylalanine-reduced, or phenylalanine-free, ⁇ - lactalbumin product will for preference be produced in the mammary glands of lactating transgenic animals, in which the native protein may still be present. This may make purification difficult unless the amino acid substitutions by themselves alter the protein's characteristics.
- a particular advantage of modifying ⁇ -lactalbumin and expressing it in the milk of a dairy animal is that purification away from the native ⁇ -lactalbumin may not be essential if the expression of the dairy animal's native ⁇ -lactalbumin is very low relative to expression of the modified ⁇ -lactalbumin, or if expression of the native or wild-type form is prevented or suppressed; this latter effect may be achieved by making the dairy animal transgenic through the use of embryonic stem cells. If purification is needed and conservative changes are necessary to maintain the protein's structure, then one possibility is to attach an additional amino acid sequence, such as a tail to the C-terminus, (Smith et al , Gene 32 321-327 (1984)) to facilitate purification.
- an additional amino acid sequence such as a tail to the C-terminus
- the additional amino acid sequence would have one or more characteristics which lead to easier separability of the protein; for example, the additional amino acid sequence may enable the protein to be separated on the basis of charge.
- a poly-Arg, or poly-Arg/Lys, tail would alter the charge sufficiently to allow more efficient and cheaper extraction.
- the modified ⁇ -lactalbumin should for preference be at least partially purified away from such proteins as was native ⁇ -lactalbumin.
- the invention is particularly useful in improving the palatability of, and hence compliance for, the protein content in the diet of PKU sufferers. This is accomplished by choosing a suitable protein, namely ⁇ - lactalbumin, which fulfils the criteria of having a high nutritional value, and of being able to address all of the considerations discussed above.
- the DNA encoding this protein can then be cloned, mutated to remove at least some, but preferably all, of the phenylalanine residues, and expressed in a system which can produce a quantity large enough to supply the world market.
- the ⁇ -lactalbumin which is used as the base for the modifications can be of any suitable animal origin. Humans and dairy animals such as cows, sheep, goats and camels are preferred. ⁇ -Lactalbumin haying either a human or a bovine origin is likely to be optimal. Both have similar nutritional value and humans have been consuming them for millennia.
- Bovine or human ⁇ -lactalbumin would be particularly appropriate to use as the starting point for the mutational modifications as man is accustomed to eating both proteins; and they are very similar, having 72% homology at the amino acid level.
- Some, or all of the phenylalanine residues will be replaced with one or several amino acids depending on structural and/or nutritional considerations.
- the preferred amino acids from which the choice can be made are shown in Table 2.
- a combination of any of these amino acids, in any of the four phenylalanine positions, may be substituted. It is preferred to make substitutions at all these positions, but failing that at three or two of them, although the invention also covers substitutions at one position.
- Tyr 3 -Leu 31 -Tyr 53 -Tyr 80 human ⁇ - lactalbumin andTyr 9 -Leu 31 -Tyr 53 -Tyr 80 bovine ⁇ -lactalbumin are preferred (or substitution mutants having fewer than those four substitutions) .
- Tyr 3/9 -Tyr 31 -Tyr 53 -Tyr 80 human and bovine ⁇ -lactalbumin are the mutants of choice.
- a further way of determining the optimal substitution(s) is to look at the amino acids occupying corresponding positions in ⁇ -lactalbumins of other species and in lysozymes of vertebrates, since the ⁇ -lactalbumins are lysozymes from vertebrate species have, where examined on publicly accessible databases, very similar structures and are believed to have derived from the same ancestral gene; on this basis, Tyr 3 -Tyr 31 - Leu 53 -Leu 80 human ⁇ -lactalbumin (or substitution mutants having fewer than those four substitutions) are preferred.
- phenylalanine residues could be substituted. One or more of them may be preferred for structural purposes, and may therefore be conserved. In the absence of any structural considerations, at least one, two, three or four of the natural phenylalanine residues may be substituted, in increasing order of preference. It is known that a natural variant of human ⁇ -lactalbumin exists which has its Phe-80 substituted by lysine (Maynard, F., J " . Dairy Res . 59 425-429 (1992)); this variant per se is not part of the invention, although it may be used in the formulas and methods of the invention to be discussed below.
- ⁇ - lactalbumin can be made; for example, a poly-Arg or Arg/Lys tail may be added at the C-terminus of the protein. This will alter the charge of the protein and enable it to be purified relatively cheaply on a large scale. It could also have the advantage of improving the nutritional quality of the protein.
- Modified ⁇ -lactalbumin of the invention may be produced by recombinant DNA technology, using a collection of techniques that have come to be known as protein engineering.
- the co-ordinates for baboon ⁇ -lactalbumin are available from the Brookhaven database (Acharya et al , J. Mol . Biol . 208 99-127 (1989)). Those for the human protein and the cow protein are not presently available, but the three dimensional structure of the baboon protein is thought to provide an acceptable template for modelling the human and bovine wild type and mutant ⁇ -lactalbumins.
- the first stage is to assess the state of each of the four Phe residues in the native presumed three- dimensional structure. These are: Phe-3 (human) or Phe-9 (bovine) , which is on the surface; Phe-31, which is exposed on the surface; Phe-53, which is in an internal cavity; and Phe-80, which is largely buried. Conveniently, the four residues are fairly well separated such that it is reasonable to assume that, provided changes introduced are largely conservative, little interaction is likely between the sites.
- Model building shows that essentially any residue could be accommodated at Phe-3/9 and 31, since both are on the surface.
- the buried Phe residues can both be accommodated with little extra movement of the surrounding protein.
- Leu and Met can be put in, but polar or charged groups are likely to disrupt the structure and so are less preferred.
- SYBYL from Tripos Associates may be useful in assessing any other modifications which it may be desirable to make.
- Posi tion 3 Surface residue which can accommodate most residues: Tyr, Leu, Arg, Met, Trp in order of increasing relative energy, the most favoured being
- Posi tion 9 Surface residue which can accommodate most residues. Most ⁇ -lactalbumins have a Ser at this position: Tyr, Ser, lie.
- Posi tion 31 Exposed on the surface, so any residue will do. However, this region is important for the lactose synthase reaction and a hydrophobic interaction may be important. Therefore, Leu and possibly lie, then Trp, would be the most preferred here if retention of the activity is important.
- Posi tion 53 Largely buried. Tyr, Leu, Met, Trp is the order in increasing energy.
- Position 80 Totally buried. Tyr, Met, Trp, Leu is the order here. This is the calcium binding loop region and so changes here might be important. Tyr is the nearest isomorph.
- modified ⁇ -lactalbumins in accordance with the invention may be made by any process, including chemical synthesis, the process of choice will generally involve the use of recombinant DNA technology in which the protein is expressed from a corresponding nucleic acid sequence.
- a process for the preparation of a modified ⁇ - lactalbumin as described above comprising coupling together successive amino acids, and/or ligating oligo- and/or poly-peptides.
- coupling together successive amino acids will preferably be ribosomally mediated.
- nucleic acid useful in such expression itself forms part of the invention.
- nucleic acid useful in such expression itself forms part of the invention.
- nucleic acid encoding a modified ⁇ -lactalbumin comprising no phenylalanine residues or fewer phenylalanine residues than wild type ⁇ -lactalbumin.
- nucleic acid sequences will encode any particular protein within the invention. All such sequences are encompassed within the invention, although in practical terms the preferred nucleic acid sequences will be those: (a) whose codons correspond where possible to those of the wild type gene;
- Recombinant DNA encoding modified ⁇ -lactalbumin in accordance with the invention is not restricted either to cDNAs or to otherwise natural genomic sequences encoding the modified protein, although these sequences may be preferred.
- the "minigene" approach of WO-A-9005188, in which some, but not all, of the introns present in the natural gene are present in the construct introduced into the host may be used.
- Recombinant DNA in accordance with this aspect of the invention will often be in the form of a vector.
- the invention is not limited to any particular type of vector, which may for example be a plasmid, cosmid or phage.
- Vectors containing sufficient regulatory sequences are not limited to any particular type of vector, which may for example be a plasmid, cosmid or phage.
- Vectors not including regulatory sequences operatively coupled may be useful as cloning vectors .
- Recombinant DNA in accordance with the invention may also be in the form of a construct suitable for use in preparing a transgenic animal, for example by microinjection or by homologous recombination.
- Nucleic acid of the invention may be prepared, in a fourth aspect of the invention, by coupling together successive nucleotides and/or ligating oligo- and/or polynucleotides, as is well within the knowledge of those skilled in the art.
- a host containing recombinant DNA as described above may be a cloning host, in which case it will often be a bacterium such as Escherichia coli , or it may be an expression host.
- DNA encoding a modified ⁇ -lactalbumin will be operatively coupled to sufficient regulatory sequences (including a promoter) for expression and the host will permit expression to take place either constitutively or under regulated conditions.
- a wide range of expression hosts may be useful in the invention, including bacteria
- yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha and Pischia pastoris
- insect cells such as Spodoptera frugiperda
- mammalian cell lines such as BHK and CHO cell lines
- the transformed host may properly be said to be transgenic; for example a cow or bull carrying DNA encoding modified human ⁇ -lactalbumin is transgenic for the human gene.
- Not all host animals in accordance with the invention are transgenic, though, as it is well within the possibilities of the invention, and it may be preferred in certain circumstances, to provide a host animal of a given species (for example cattle) carrying DNA encoding modified ⁇ -lactalbumin of the same species (in the example, bovine ⁇ -lactalbumin) .
- Such "quasi-transgenic" animals may be prepared by routine adaptations of the techniques which have been developed and will continue to be developed for the preparation of transgenic animals, or by the techniques of gene therapy, which are being developed for the replacement or supplementing of one version of a gene with another.
- the mammary gland expression system has the advantages of high expression levels, low cost, correct processing and accessibility.
- Bovine and human ⁇ -lactalbumin have been produced in lactating transgenic animals by several workers (Vilotte et al , Eur. J. Biochem. 186 43-48
- the ⁇ -lactalbumin promoter can direct the expression of heterologous genes (Stinnakre et al , FEBS Letters 284(1) 19-22 (1991)) to the lactating mammary gland (WO-A-8801648) .
- transgenic animals including transgenic mammals expressing heterologous protein in the milk of adult females
- transgenic mammals such as sheep which express pharmaceutically valuable proteins including factor IX and ⁇ - ⁇ antitrypsin.
- the methodology set out in all the above and other publications may be adapted to produce transgenic or quasi-transgenic animals in accordance with the present invention. It is preferred to use known dairy mammals, such as cows, sheep, goats or even camels or pigs, as hosts in the present invention, but the choice of host will be dictated primarily by convenience and not by any requirement of the invention itself.
- Transgenic animals or other hosts according to the invention may be prepared by any convenient methodology. Accordingly, the invention is not limited to any particular method for their preparation. Transgenic animals, for example, may be prepared by microinjection, as described in WO-A-8800239 and WO-A-9005188 above, or they may be prepared by embryonic stem cell technology such as is described in WO-A-9003432.
- Expression of the wild type ⁇ -lactalbumin gene of the host may be prevented or at least attenuated, so as to simplify purification.
- a foodstuff suitable for sufferers of hyperphenylalaninemia comprising at least one modified ⁇ -lactalbumin comprising no phenylalanine residues or fewer phenylalanine residues than wild type ⁇ -lactalbumin.
- Preferred features of the modified ⁇ -lactalbumin(s) are as described above.
- the foodstuff may, and often will, contain other components.
- the modified protein alone or in combination with L-amino acids and/or other low or phenylalanine-free proteins or peptides, would be appropriate for use by infants, children and adults with PKU.
- a formula with low or phenylalanine-free ⁇ -lactalbumin as the protein base would look and taste more like normal whole protein infant formulas and would, as a result, be better tolerated than the current special dietary formulas of protein hydrolysate or amino acid mixtures.
- a special dietary food or supplement for infants and very young children containing modified ⁇ -lactalbumin in accordance with the invention may contain other ingredients, such as a fat source, a carbohydrate source, and appropriate levels of vitamins, minerals and other nutritional factors.
- An additional nitrogen source besides the modified ⁇ -lactalbumin can also be any L- amino acid (such as L-tyrosine) , which would be added to improve the overall essential amino acid quality of the product to make it nutritionally adequate.
- the total phenylalanine content of the final product should generally be less than 80 mg, preferably less than 25 mg per 100 g powder equivalent, or phenylalanine free.
- any low-Phe or Phe-free peptide such as casein- derived peptide (CDP) (derived from the cheese making process when the enzyme chymosin (rennet) splits the milk protein, ⁇ -casein, and releases CDP into the whey fraction)
- CDP casein- derived peptide
- L-tyrosine and non- essential amino acids may also be added in amounts up to and/or higher than that found in human milk and normal children's diets to ensure nutritional adequacy.
- This special dietary food or supplement for infants and young children containing modified ⁇ -lactalbumin may be a ready-to-feed liquid, or in the form of a powder or concentrated liquid adapted to provide a ready-to-feed form by the additional of water and stirring.
- Each 100 ml (60-75 kcal/ml) of the ready-to-feed liquid formula or reconstituted powder will generally contain from 1.2 to 3.0 g, preferably about 1.3 to 1.5 g, of protein; from 2.2 to 4.0 g, preferably about 3.6 g, of a fat or mixture of fats; and from 6 to 9 g of carbohydrate.
- the carbohydrate source lactose is generally preferred for most infants, but corn syrup solids, sucrose or other sugars can also be used in the special dietary food for infants and young children with PKU.
- special dietary food containing modified ⁇ - lactalbumin as low-Phe or Phe-free protein source would generally contain minerals to provide nutritionally acceptable quantities of calcium, phosphorus, potassium, sodium, chloride, magnesium, iron, copper, zinc, manganese, molybdenum, selenium, chromium, fluorine and iodine plus adequate quantities of vitamins such as vitamins A, D, E, B 1# B 2 , B 6 , B 12 , C, nicotinamide, pantothenic acid, folic acid, vitamin K, biotin and choline.
- Other nutritional factors such as carotene, taurine, inositol, carnitine and nucleotides may also be added.
- the diets of older children, adolescents and pregnant women in particular with PKU might be comprised of a low -Phe or Phe-free formula plus more than 50% of total calories from low phenylalanine foods.
- Such a formula can be specifically designed for each age group, ie, with recommended daily intakes in mind, or one common formula can be developed with specific usage directions and dilution differences for each category.
- Such a formula for those other than infants and very young children would include (approximately) the following RDI levels of nutrients in about 500-1000 kcal or 500-1000 ml, as shown in Table 5.
- a special dietary supplement For children and adults, including pregnant women, the formulation goal of a special dietary supplement would be that the formula is palatable and a high quality component to the remainder of the diet.
- Such a formula should contain all essential amino acids (other than phenylalanine) , and those vitamins and minerals in particular which are not typically present in high amounts in low protein foods, such as fruits and vegetables.
- Such nutrients, besides protein, are calcium, iron, folic acid, magnesium and trace minerals.
- a typical formula for children, adults and pregnant women with PKU would contain but not be limited to the following preferred ranges of nutrients, as shown in Table 6.
- Low-phenylalanine or phenylalanine-free ⁇ -lactalbumin would also be highly appropriate for inclusion in milk drinks for older children, pregnant women, adolescents and adults with PAH deficiencies.
- modified ⁇ -lactalbumin or dietary food supplement containing modified ⁇ -lactalbumin could be used in foods, such as breads, ice cream and frostings, as sources of functional protein; they would still be free of phenylalanine, or at least of reduced phenylalanine content.
- the milk of transgenic or quasi-transgenic host animals as described above may be suitable as foodstuffs in accordance with the invention with or without further modification, particularly if expression of the host's wild type ⁇ -lactalbumin gene has been attenuated or prevented. Further processing of the modified protein could involve removal of the amino acid tail for regulatory or other purposes, for example, prior to its incorporation into a formula.
- a method of feeding a sufferer of hyperphenylalaninemia comprising administering to a sufferer of hyperphenylalaninemia one or more foodstuffs containing modified ⁇ -lactalbumin comprising fewer phenylalanine residues than wild type ⁇ - lactalbumin.
- modified ⁇ -lactalbumin is as described in relation to the first aspect of the invention above.
- the amount and timing of the administration may be left to the discretion of the sufferer, but will generally be within the guidance of a physician, nutritionist or other medical adviser.
- Figure 3 is discussed in Example 1 and shows SDS-PAGE analysis of skimmed milk from human ⁇ -lactalbumin transgenic mice run against non transgenic mouse milk;
- Figure 4 is discussed in Example 1 and shows a Western blot of the milk from human ⁇ -lactalbumin transgenic mice run against human ⁇ -lactalbumin standard;
- Figure 5 is discussed in Example 2 and shows the sequence of bovine ⁇ -lactalbumin PCR primers
- Figure 6 is discussed in Example 2 and shows position of bovine ⁇ -lactalbumin PCR primers and products
- Figure 7 is discussed in Example 3 and shows the sequence of bovine ⁇ -lactalbumin PCR primers used for mutagenesis
- Figure 8 is discussed in Example 4 and 5 and shows PCR strategies for Phe substitutions and cloning strategy for transgene constructs PKU1 to 4;
- Figure 9 is discussed in Example 4 and shows Western analysis of milk from PKU1 to 4 transgenic mice run against control mouse milk and bovine ⁇ -lactalbumin standard;
- Figure 10 is discussed in Example 4 and shows quantitation by Western blot analysis of milk from two PKU1 transgenic mouse lines run against dilutions of bovine ⁇ -lactalbumin standard;
- Figure 11 is discussed in Example 5 and shows PCR strategies for Phe substitutions and cloning strategy for transgene constructs PKU5 and PKU1H;
- Figure 12 is discussed in Example 6 and shows cloning strategy for bovine ⁇ -lactalbumin and PKU5 to 10 constructs for expression in COS cells;
- Figure 13 is discussed in Example 6 and shows an autoradiograph of immunoprecipitated material from the supernatants of COS cells transfected with pC-BOVA and pC-PKU5 to 10 constructs.
- pHA-1 consists of the human a-lactalbumin coding region, -1.8kb of 5' flank and 3kb of 3' flank derived from 1 clone 5b.1 cloned as a 7kb EcoRI/Sall fragment into pUC18.
- pOBHA ovine S-lactoglobulin, human ⁇ -lactalbumin
- pHA-1 containing the human ⁇ -lactalbumin gene and flanking regions
- pOBHA containing the human ⁇ -lactalbumin gene driven by the ovine .-lactoglobulin promoter
- mice The expression levels of human ⁇ -lactalbumin in the milk from these mice ranged from undetectable to -3mg/ml. Table 7 gives a summary of the relative amount of the transgenic protein. Skimmed milk from these animals was analysed by SDS-PAGE stained with Coomasie blue, isoelectric focusing, Western blots visualised with a commercial anti-human ⁇ -lactalbumin antibody (Sigma) and chromatofocusing. The results from these analyses showed that the transgenic protein was of the correct size, pi and antigenicity when compared to a human ⁇ -lactalbumin standard (Sigma) .
- Figure 3 shows an SDS-PAGE analysis of skimmed transgenic mouse milk run against a non-transgenic control mouse milk.
- Figure 4 shows a Western blot of these milks run against a human ⁇ -lactalbumin standard.
- Table 7 shows the relative levels of human ⁇ -lactalbumin in transgenic mouse milk as estimated by comparison with protein standards on Coomassie gels and Western blots.
- Bovine ⁇ -lactalbumin There are three known variants of bovine ⁇ -lactalbumin, of which the B form is the most common.
- the A variant from Bos (Bos) nomadicus f . d. indicus differs from the B variant at residue 10: Glu in A is substituted for Arg in B.
- the sequence difference for the C variant from Bos (Bibos) javanicus has not been established (McKenzie & White, Advances in Protein Chemistry 41 173-315 (1991) .
- the bovine ⁇ -lactalbumin gene (encoding the B form) was cloned from genomic DNA using the PCR primers indicated in Figure 5. The primers have been given the following SEQ ID NOs:
- the source of DNA in all the PCR reactions was blood from a Holstein-Friesian cow.
- Ba-7 in combination with primer Ba-8 is 2.05kb. This BamHI/_-C ⁇ RI fragment was cloned into Bluescript (pBA-P2) .
- the entire bovine ⁇ -lactalbumin gene including 300bp of 3'-flanking region was amplified using primer Ba-9 in combination with primer Ba-2.
- primers include Bam ⁇ l restriction enzyme recognition sites, which allowed direct subcloning of the amplified 3kb fragment into the BamHI site of pUC18, giving rise to construct pBA-G3.
- the construct pBA ( Figure 6) was injected into mouse embryos and has given rise to 9 transgenic animals. Milk analysis was carried out as described in Example 1. Analysis by SDS-PAGE gel stained with Coomasie blue and comparison with standard amounts of ⁇ -lactalbumin showed expression levels varying from non-detectable to ⁇ 0.5-lmg/ml.
- the four phenylalanine (Phe) residues in bovine ⁇ - lactalbumin occur within the first 3 exons of the gene.
- a set of seven oligos (PKU-PCR primers 1, 2, 2L, 3, 4, 7 and 8) was designed for Phe substitutions based on protein modelling, nutritional aspects, and on amino acid variants present in either native ⁇ -lactalbumin or lysozyme genes from different species.
- the sequence of the primers (complementary to either the coding or non-coding strand) , the predicted change of amino acid and the mutagenised site of each are shown in Figure 7.
- These oligos act as both PCR and mutagenic primers.
- PKU primer 1 was used in combination with 2 or 2L and 7 in combination with 8, giving rise to a 435bp amplified fragment (A) containing the first two Phe substitutions.
- PKU primer 3 and 4 or 9 and 10 would produce a PCR product (B) of 601bp containing the second two Phe substitutions (see Figure 8) .
- the PCR products were subcloned for sequencing using the sites EcoRI / BairSil and Bam ⁇ l/Xbal respectively.
- the Pvul site which had been engineered into PKU primers 2, 2L, 3, 7, and 8, created a unique restriction site between amino acid 31 and amino acid 53, without changing the encoded amino acids in that region.
- primers PKU-5 and 6 were used to amplify up the 3' end of the bovine ⁇ -lactalbumin gene giving rise to the 0.44kb PCR product C.
- Primer PKU 6 contained the coding region for six Arg residues, immediately following the last natural Leu amino acid in the bovine ⁇ -lactalbumin sequence and created unique Sail and SnaBI sites.
- the unique Sall/SnaBI sites can be digested, and linkers inserted, to create any length of poly-Arg or poly-Arg/Lys tail that is required.
- pBARG-H was constructed from five DNA fragments:
- PKU-1 was constructed from six DNA fragments:
- PKU-2 was constructed in the same way as PKU-1 with the exception of fragment (5) , which was derived from pBARG-H and contained the poly-Arg tail.
- PKU-3 was constructed in the same way as PKU-1 with the exception of fragment (2) (PCR product A) , which had been amplified using PKU-primer 1 in combination with 2L.
- PKU-4 was constructed in the same way as PKU-3 with the exception of fragment (5) , which was derived from pBARG-H and contained the poly-Arg tail.
- the amino acid substitutions resulting from the mutagenesis above are shown for plasmids PKU-1 to PKU-4 in Table 8.
- the 9.3kb BamHI fragment derived from ⁇ clone 4a ( Figure 1) contains 3' flanking region of the human ⁇ -lactalbumin gene and was included in all PKU constructs as a suitable target for preimplantation screening in transgenic embryos.
- the 9.3kb fragment was inserted into the unique Bglll site present in all the above constructs (see Figure 8) .
- DNA for microinjection was digested away from the vector sequences by cutting with BamHI. DNA was prepared for microinjection and transgenic mice generated as published (Hogan et al . , "Manipulating the Mouse Embryo:
- the protein is further analysed with a combination of established techniques such as ELISA, pi, sequencing and circular dichroism. These methods are intended to give information about the size and charge of the protein, and some idea of the folding.
- the bovine ⁇ -lactalbumin mRNA present in the extracted RNA was cloned by the method of RT-PCR and sequenced. The results proved that the mRNA was transcribed from the PKU1-4 constructs and that the translated protein should therefore be phenylalanine free.
- EXAMPLE 5 Expression of Mutagenised Bovine ⁇ -lactalbumin under the Control of the Human ⁇ -lactalbumin Promoter in vivo Data from Examples 1, 2 and 4 indicated that expression of the human ⁇ -lactalbumin transgene is considerably higher than that of the native bovine ⁇ -lactalbumin transgene, reflecting the difference in expression levels of the endogenous bovine and human genes. As this might contribute to differences in the 5' control region, the 5'region of the bovine ⁇ -lactalbumin transcriptional start site was substituted with sequences from the human ⁇ -lactalbumin gene.
- the final construct included 6 DNA fragments:
- PKU-IH was constructed in the same way as PKU-5 with the exception of fragment (3) , which was derived from PKU-1 (Example 4) .
- Table 10
- the two constructs PKU-IH and PKU-5 have been injected into mouse embryos . So far transgenic animals were derived for the PKU-5 construct . These animals are set up for breeding to allow milk analysis .
- Figure 13 shows that transient expression in COS cells leads to secretion of native and mutated bovine ⁇ -lactalbumin.
- Amino acid substitution Phe 9 to Ser (pC-PKUlO) gave expression levels equivalent to wild type bovine ⁇ -lactalbumin.
- Substitution Phe 30 to Tyr in combination with substitution Phe 53 to Leu (pC-PKU6)
- substitution Phe 80 to Tyr (pC-PKU8) had reduced expression levels.
- Expression of pC-PKU5, 7, and 9 was below the level of detection in this assay system. This system will allow the definition of amino acid substitutions for maximum expression in transgenic animals.
- EXAMPLE 7 How to use Milk Containing Modified Protein For children with phenylketonuria, a special dietary supplement free of phenylalanine is required. Since dietary compliance is critical during the early years, any therapeutic formula which tastes good and enhances dietary compliance is preferred.
- a typical daily menu for children with phenylketonuria would contain up to 3 servings/day of a special supplement containing modified ⁇ -lactalbumin and other nutrients in Table 3 described previously.
- the current recommendation is that the restricted phenylalanine diet, adjusted regularly to meet age, growth and maintenance requirements, be continued for life.
- EXAMPLE 8 Preparation/Isolation of Modified Protein from Milk For most if not all cases of special formula preparation it will be desirable to separate and purify regular ⁇ - lactalbumin from modified ⁇ -lactalbumin protein. For children, adolescents and pregnant women, for example, formulas zero or the least amount of Phe would allow for greatest dietary flexibility and make adherence to lifelong dietary management of PKU most likely.
- EXAMPLE 9 Formulation of Modified Protein From this invention it will be possible to formulate several special dietary formulas for the treatment of hyperphenylalaninemia. These formulas are unique from all current formulas because a whole protein - modified ⁇ -lactalbumin - is the predominant protein (N) and Phe- free amino acid source, whereas, in current formulas, the N source is exclusively in the form of L-amino acids or extensively hydrolysed casein protein.
- MOLECULE TYPE cDNA
Abstract
Description
Claims
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP7504407A JPH09500273A (en) | 1993-07-16 | 1994-07-13 | Modified α-lactalbumin |
EP94920557A EP0711344A1 (en) | 1993-07-16 | 1994-07-13 | Modified alpha-lactalbumin |
NZ268406A NZ268406A (en) | 1993-07-16 | 1994-07-13 | Alpha-lactalbumin with fewer phenylalanine residues |
AU71306/94A AU698597B2 (en) | 1993-07-16 | 1994-07-13 | Modified alpha-lactalbumin |
AU28962/95A AU700224B2 (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
CA002193513A CA2193513A1 (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
PCT/GB1995/001651 WO1996002640A1 (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
NZ289197A NZ289197A (en) | 1994-07-13 | 1995-07-12 | Alpha lactalbumin gene constructs and transgenic cattle |
EP95924467A EP0765390A1 (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
JP8504802A JPH10502816A (en) | 1994-07-13 | 1995-07-12 | α-lactalbumin gene construct |
CN95194129A CN1157635A (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
IL11456895A IL114568A0 (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
ZA955850A ZA955850B (en) | 1994-07-13 | 1995-07-13 | "Alpha-lactalbumin gene constructs" |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9314802.1 | 1993-07-16 | ||
GB939314802A GB9314802D0 (en) | 1993-07-16 | 1993-07-16 | Modified proteins |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995002692A1 true WO1995002692A1 (en) | 1995-01-26 |
Family
ID=10738971
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1994/001514 WO1995002692A1 (en) | 1993-07-16 | 1994-07-13 | Modified alpha-lactalbumin |
Country Status (10)
Country | Link |
---|---|
EP (1) | EP0711344A1 (en) |
JP (1) | JPH09500273A (en) |
CN (1) | CN1127528A (en) |
AU (1) | AU698597B2 (en) |
CA (1) | CA2167155A1 (en) |
GB (1) | GB9314802D0 (en) |
IL (1) | IL110312A0 (en) |
NZ (1) | NZ268406A (en) |
WO (1) | WO1995002692A1 (en) |
ZA (1) | ZA945217B (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995018224A1 (en) * | 1993-12-29 | 1995-07-06 | Gene Pharming Europe Bv | Recombinant production of modified proteins lacking certain amino acids |
WO1996002640A1 (en) * | 1994-07-13 | 1996-02-01 | Ppl Therapeutics (Scotland) Ltd. | Alpha-lactalbumin gene constructs |
US5852224A (en) * | 1994-12-15 | 1998-12-22 | Ppl Therapeutics (Scotland) Limited | α-lactalbumin gene constructs |
WO2010119088A3 (en) * | 2009-04-15 | 2011-06-30 | Bodo Melnik | Milk and milk-based products modified to exhibit a reduced insulinemic index and/or reduced mitogenic activity |
WO2016046234A3 (en) * | 2014-09-22 | 2016-08-11 | Nexttobe Ab | Recombinant phe-free proteins for use in the treatment of phenylketonuria |
EP3290436A1 (en) * | 2016-09-01 | 2018-03-07 | metaX Institut für Diätetik GmbH | Phenylalanine-free protein for the treatment of pku |
US11076615B2 (en) | 2014-08-21 | 2021-08-03 | Perfect Day, Inc. | Compositions comprising a casein and methods of producing the same |
WO2022182814A1 (en) * | 2021-02-24 | 2022-09-01 | Milk Care Co., Inc. | Infant formulas containing human breast milk proteins |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105325556A (en) * | 2015-10-29 | 2016-02-17 | 北京银河美科技有限公司 | Liquid milk product containing heat-sensitive alpha-lactalbumin and preparation method |
CN113785972B (en) * | 2021-09-17 | 2023-10-24 | 江苏冬泽特医食品有限公司 | Nutritional powder for rare disease phenylketonuria, preparation method and application |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0014362A1 (en) * | 1979-01-26 | 1980-08-20 | Societe Des Produits Nestle S.A. | Method of debittering a protein hydrolysate and debittered hydrolysate obtained |
-
1993
- 1993-07-16 GB GB939314802A patent/GB9314802D0/en active Pending
-
1994
- 1994-07-13 JP JP7504407A patent/JPH09500273A/en active Pending
- 1994-07-13 CA CA002167155A patent/CA2167155A1/en not_active Abandoned
- 1994-07-13 WO PCT/GB1994/001514 patent/WO1995002692A1/en not_active Application Discontinuation
- 1994-07-13 NZ NZ268406A patent/NZ268406A/en unknown
- 1994-07-13 AU AU71306/94A patent/AU698597B2/en not_active Ceased
- 1994-07-13 CN CN94192790A patent/CN1127528A/en active Pending
- 1994-07-13 EP EP94920557A patent/EP0711344A1/en not_active Withdrawn
- 1994-07-14 IL IL11031294A patent/IL110312A0/en unknown
- 1994-07-15 ZA ZA945217A patent/ZA945217B/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0014362A1 (en) * | 1979-01-26 | 1980-08-20 | Societe Des Produits Nestle S.A. | Method of debittering a protein hydrolysate and debittered hydrolysate obtained |
Non-Patent Citations (4)
Title |
---|
HALL L. ET AL.: "Organization and sequence of the human alpha-lactalbumin gene", THE BIOCHEMICAL JOURNAL, vol. 242, no. 3, March 1987 (1987-03-01), pages 735 - 742 * |
MAYNARD F.: "Identification of a new molecular form of human alpha-lactalbumin", JOURNAL OF DAIRY RESEARCH, vol. 59, no. 3, August 1992 (1992-08-01), pages 425 - 429 * |
SHIN-ICHI HOCHI ET AL.: "Secretion of bovine alpha-lactalbumin into the milk of transgenic rats", MOLECULAR REPRODUCTION AND DEVELOPMENT, vol. 33, October 1992 (1992-10-01), pages 160 - 164 * |
VILOTTE J.-L. ET AL.: "Complete nucleotide sequence of bovine alpha-lactalbumin gene: comparision with its rat counterpart", BIOCHIMIE, vol. 69, no. 6/7, 1987, pages 609 - 620 * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995018224A1 (en) * | 1993-12-29 | 1995-07-06 | Gene Pharming Europe Bv | Recombinant production of modified proteins lacking certain amino acids |
WO1996002640A1 (en) * | 1994-07-13 | 1996-02-01 | Ppl Therapeutics (Scotland) Ltd. | Alpha-lactalbumin gene constructs |
US5852224A (en) * | 1994-12-15 | 1998-12-22 | Ppl Therapeutics (Scotland) Limited | α-lactalbumin gene constructs |
WO2010119088A3 (en) * | 2009-04-15 | 2011-06-30 | Bodo Melnik | Milk and milk-based products modified to exhibit a reduced insulinemic index and/or reduced mitogenic activity |
US11076615B2 (en) | 2014-08-21 | 2021-08-03 | Perfect Day, Inc. | Compositions comprising a casein and methods of producing the same |
US11457649B2 (en) | 2014-08-21 | 2022-10-04 | Perfect Day, Inc. | Compositions comprising a casein and methods of producing the same |
WO2016046234A3 (en) * | 2014-09-22 | 2016-08-11 | Nexttobe Ab | Recombinant phe-free proteins for use in the treatment of phenylketonuria |
US10174354B2 (en) | 2014-09-22 | 2019-01-08 | Nexttobe Ab | Recombinant Phe-free proteins for use in the treatment of phenylketonuria |
WO2018041920A1 (en) * | 2016-09-01 | 2018-03-08 | Metax Institut Für Diätetik Gmbh | Phenylalanine-free protein for the treatment of pku |
AU2017317652B2 (en) * | 2016-09-01 | 2020-11-19 | Metax Institut Für Diätetik Gmbh | Phenylalanine-free protein for the treatment of PKU |
US10640538B2 (en) | 2016-09-01 | 2020-05-05 | Metax Institut Für Diätetik Gmbh | Phenylalanine-free protein for the treatment of PKU |
RU2764796C2 (en) * | 2016-09-01 | 2022-01-21 | Метакс Институт Фюр Диететик Гмбх | Phenylalanine-free protein for treatment of pku |
EP3290436A1 (en) * | 2016-09-01 | 2018-03-07 | metaX Institut für Diätetik GmbH | Phenylalanine-free protein for the treatment of pku |
WO2022182814A1 (en) * | 2021-02-24 | 2022-09-01 | Milk Care Co., Inc. | Infant formulas containing human breast milk proteins |
Also Published As
Publication number | Publication date |
---|---|
GB9314802D0 (en) | 1993-08-25 |
AU698597B2 (en) | 1998-11-05 |
JPH09500273A (en) | 1997-01-14 |
CN1127528A (en) | 1996-07-24 |
IL110312A0 (en) | 1994-10-21 |
EP0711344A1 (en) | 1996-05-15 |
AU7130694A (en) | 1995-02-13 |
CA2167155A1 (en) | 1995-01-26 |
ZA945217B (en) | 1996-01-15 |
NZ268406A (en) | 1998-03-25 |
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