JPH09500273A - Modified α-lactalbumin - Google Patents

Abstract

  (57) [Summary] Modified α-lactalbumin, eg of bovine or human origin, containing less phenylalanine residues than wild-type α-lactalbumin is used as a dietary ingredient for patients with hyperphenylalanineemia. Preferably all phenylalanine residues are replaced. The modified α-lactalbumin is expressed in the mammary gland of a non-human host animal so that it can be accumulated in milk and, if necessary, can be separated from milk.

Description

Detailed Description of the Invention                     Modified α-lactalbumin   The present invention provides a therapeutic diet for patients with hyperphenylalaninemia. To a modified protein useful for   Human hyperphenylalaninemia due to genetic causes is phenylalanine At the hydroxylase (PAH; EC 1.99.1.2) locus, At the locus encoding DHPR (dihydropteridine reductase) And BH_Four(Tetrahydrobiopterin) This is the result of mutations at the two loci. Suddenly at a locus other than PAH The mutation rate was less than 3% of all hyperphenylalaninemia patients, and Kafka It is considered to be close to 1% of Zu patients. High phenyla due to mutation of PAH Severe PAH deficiency (typical phenylketonuria) in more than half of patients with raninemia Disease or "PKU"). This liver enzyme turns phenylalanine into It catalyzes the conversion to syn.   As shown in the above figure, deficiency of PAH results in phenylara in tissues and blood Nin and its organic acid metabolites accumulate, which cannot be investigated. If left untreated, it causes severe and irreversible mental retardation in infants and young children.   The frequency of PKU varies around the world, but from 1 / 5,000 in Ireland to a day It is up to about 1 / 100,000 of a book. Of the worldwide inner circle where PKU develops The average estimate is 50 per million live born children. A In the United States, the frequency of PKU is about 1/10 of all newborns screened, 000.   When an infected baby is found, firstly to lower the blood level of phenylalanine It is important to start your diet. Generally, the level of phenylalanine in the blood Less than 1000 μmol / liter (16.5 mg / dl), preferably 600 μmol Treatment is terminated by maintaining less than 1 liter / liter. Recent neurological and behavioral studies Behavioral evidence points out that regarding lower future treatment endpoints: : Phenylalanine value as close to normal as possible (<120 μmol / l , Or <1.98 mg / dl).   Theoretically, ideal biochemical control of PKU would require frequent blood monitoring and And phenylalanine are achieved by adhering to a restricted diet [Trams (T rahms), "Nutritional Care in Metabolic Disorder (Nutritional Care in Metabolic Disorders) ", Food, Nutrition   In And Diet Therapy (Food Nutition and Diet Terapy), 8th Edition (1992), Mahan & Arli n), W. W. Bee. Sanders Corporation (W.B. Sanders Co.) , Philadelphia, Pennsylvania; Kitagawa et al., Enzyme (E 38, pp. 321-327 (1987); and Owada et al. Kuta Pediator. Japan (Acta Paediatr.jpn.) 3 0, pages 405-409], but in practice this ideal is not always achieved. Details In fact, nutritional therapy is based on the use of prescriptions with reduced phenylalanine content. Be planned. 9 commonly used prescriptions for PKU infants and children Hydrolyzed protein free of phenylalanine at a rate of 5 to 100% Or a mixture of free amino acids with little or no added phenylalanine It is one of the compounds. Tyrosine has been shown to be a factor in the treatment of phenylketonuria patients. As there continues to be the view that rosin levels can be kept below the normal mean, Typically, it is a supplemented amino acid.   Late as well as early to prevent mental retardation in infected infants and children , I.e., in adults and especially in pregnant women with hyperphenylalaninemia, It is important to follow a diet. Almost all children of pregnant women who are not treated And suffer from complications such as mental retardation and microcephaly. Also, a small number are congenital Some people suffer from various heart diseases or have a low birth weight. Most places These results are very unfortunate because the synthetic infant is not hyperphenylalaninemia per se. It is. IQ gradually decreases, learning is difficult, and there is a problem in behavior. However, although the lifespan is extended, the control of phenylalanine by diet is almost or Older children, underage with hyperphenylalaninemia who have never been Reported in adults. Therefore, PKU is a lifelong and descendant (transge nerational) problems and require continuous and lifelong dietary control. It is especially clear from recent reports [Scriver et al. The Metabolic Basis of Inherited Diseases ic Basis of Inherited Disease), Scriber, C. R. (Scriver, C.R. ), Beaudet, A., Sly, W., Valet , Dee (Valle, D.), (Author), 7th edition (7th edit.) McGraw Hill, Lee In press, Chapter 27; Joint Feo ー / W Etch O / You N Ex-part Consultation , Energy and Protein Liquids (Joint FAO / WHO / UNU Exp ert Consultation, Energy and Protein Requirements), WHO   Tech. Lept. Series number. 724 (WHO Tech. Rept. Ser. No. 724) WHO, Geneva, Switzerland (1985); Levy etch. El. (Levy H.L.), Treatment of Genetics Kudizisesu (Treatment of genetic diseases), author, Earl. J. De R.j. Desnick, Churchill-Livingsto n), New York (1991)].   The feeding strategy of PKU patients has three aspects. First, it supplies all essential amino acids except phenylalanine to help infants and small children Allows the child to grow and develop moderately or, in the case of adults, be maintained You. Vitamins and minerals usually have the prescribed prescribed forms, Supplied with amino acid components. For infants, the standard amino acid profile Content of phenylalanine is lower than that of human breast milk. Of breast milk from human breasts, unless present or absent at all Must agree [Nayman et al., 32, p. 1279-12. 89 (1979) and Joint FA / W Etch / You N Excipart Consultation, Energy and Pro TEIN REQUIREMENTS (joint FAO / WHO / UNU Expert Consultation, Energy a nd Protein Requirements), W etch technology. Lept. Series number. 724 (WHO Tech. Rept. Ser. No. 724), WHO, Geneva, Switzerland ( 1985)]. For children and adults, the minimum except phenylalanine Formulated amino acid profiles are FEO / DUBLI for the above groups The necessary conditions for patterns suggested by FAO / WHO / UNU Must match.   Second, foods with a defined phenylalanine content are further formulated with amino acids. It is added as a supplement to the basic diet. These foods vary It contains a quantity of phenylalanine, so what to choose and what Depending on how much you consume, your overall diet may have some flexibility or palatability (palatab). ility) occurs. The acceptable amount of phenylalanine is needed to meet growth Only the amount needed to maintain and / or maintain. For example, through childhood The maximum acceptable level of ingestion of phenylalanine is 500 mg / day with an acceptable range of 2 It is from 00 to 500 mg / day.   The determination of the complete phenylalanine requirement in humans is shown in Table 1 below. Like Young & Pellett [Am J Clini   Neutr (Am.j.Clin.Nutr.) Vol. 45, p. 1323 (1987)) and The Committee on Nutrition of the American Academy   Of Pediatrics (the Commitee on Nutrition of The American Aca demy of Pediatrics (Pediatrics Volume 57, p. 783 (1 976)]:   The third component of the diet for PKU is deficient in essential amino acids and phenylalanine. This is to ensure proper calories after being filled. This is due to low protein, high carbohydrate foods such as fruits, vegetables, fats and oils. Achieved.   Common therapeutic formulations such as those described above provide essential amino acids and It is suitable for limiting the uptake of nin, and for proper biochemical control. Vitaminally effective but nutritionally relevant and / or bioavailable , Depending on minerals and amino acids [Acosta et al., J. Inher. Metab. Dis. Vol. 5, p. 107 (1982) Year)]. In addition, they have poor taste and odor and have a high osmotic load, so As a result, it is difficult to follow. Need for lifelong dietary follow-up and regulatory control Sex is more acceptable in food products with or without small amounts of phenylalanine. Stresses the need for   Ideally, such foods taste good, smell good, and, when used, Must have maximum flexibility in meal selection and menu planning No. Such an ideal product or formulation is It has a high degree of palatability and contains a small amount of phenylalanine, preferably no phenylalanine Must: Such products are easy to follow and have low protein phenylene A key quality that maximizes the intake of foods containing Lanin and the more common diet . Raw with low phenylalanine content or no phenylalanine Foods of higher physical value are more diverse and have a larger capacity, including phenylalanine. It is a very expensive dietary ingredient as it allows for natural foods.   Techniques currently exist to address the above issues in new and more efficient ways. specific Site-directed mutagenesis, protein engineering ngineering) and recombinant DNA technology in combination. A known protein is transformed into a phenyi by mutating the codons of the associated DNA It can be converted to reduce or eliminate lualanine residues.   In recent years, our understanding of recombinant DNA and gene technology, and protein engineering has been very high. Spread. With this knowledge, mutations can be made based on known sequences, structures and functions. It has become possible to select specific proteins for use. Moreover, such a tan The DNA encoding the protein is cloned from an appropriate source to obtain the desired contents. Mutated to analyze the sequence and confirm changes in the sequence, and then select the appropriate vector for expression. Can be cloned.   DNA sequences encoding various medically and nutritionally important proteins are the most Closely cloned. Such proteins include insulin and plasma. Nogen activator, α₁-Antitrypsin, blood factors VIII and IX, Examples include zozyme, α-lactalbumin and lactoferrin. These D From NA sequence, protein sequence is prepared by peptide map creation and Edman degradation. Can be guessed and confirmed Wear.   X-ray crystallography and multidimensional NMR of proteins Spectroscopy (multidimensional NMR spectroscopy) It became possible to determine the three-dimensional structure. This and a very powerful computer By combining this with advanced programs, changes in amino acid residues can be You can create a model of how it affects the structure and function of the protein. Nilsson et al., Careent Opinion Instructural   Biology (Curr. Opin. In Struc. Biol.) Volume 2, pages 569-575 (19) 1992); Presta, El. Gee. (Presta, L.G.), Career Opinion Volume 2 of Instructural Biology (Curr. Opin. In Struc. Biol.) P. 593-596 (1992); Czech Republic, T. R. (Cech, T.R.), Ki Target Opinion in Structural Biology (Curr. Opin.i n Struc. Biol. 2, p. 605-609 (1992); Picke's Gill (Picke) rsgill) and Goodenough, Torrens in Food Science   And Technology (Trends in Food Sci. And Tech.) Vol. 5, pp. 122-1 26 (1991)].   Which proteins are modified and what the modifications are Since it has been established, the DNA encoding the protein can be isolated and Strategies for obtaining mutated DNA sequences encoding changes can be developed . One such strategy is the gene region containing the amino acid to be changed. Sub-cloning of the region. In addition, this subcloned fragment A specific site is subjected to mutagenesis. By using this technology, The position and type of modification can be precisely targeted. In the most accurate case, DN An oligonucleotide that plays a role in mutagenizing a DNA sequence from one base in the A sequence. Oh It can be changed to any of the other three bases by the tide primer. in this way The resulting DNA encodes totally different amino acids [McPher Son, M. J. (Mcpherson, M.j.), Directed Mutagen Directed Mutagenesis, Oxford University Press (O xford Univ. Press), New York (1991); Carter P. ), Biochem. J. 237, pp. 1-7 (1986); Nickoloff and Deng, Anal Biochem. 2 00, pp. 81-88 (1992)].   Must be considered when choosing a suitable protein for phenylalanine substitution There are some points that do not. First, the protein that you are trying to use as a food Must be available in large quantities. Therefore, high levels of expression should not be achieved. It is preferable that the product is easy to obtain and easy to purify. Yes. Secondly, protein modeling is becoming more and more complicated, The structural and functional consequences of the changes in the must be inferrable. The larger the polypeptide, the more amino acids it contains and thus its larger size. This usually increases the number of phenylalanine residues, which is proportional to More mutagenesis is required to replace these residues. Pre-poly Smaller peptides are more preferable in operation. In principle, Can select secretory proteins. However, by this choice the expression system Will be decided.   When considering which protein to modify, the structure, physiological and And nutritional relevance, and the ease of extracting proteins from the cell-synthesizing host Various factors such as etc. must be considered.   As far as structure is concerned, protein properties such as activity, specificity and stability Depends on the relative position of amino acids in proteins and on the physical properties of amino acids Controlled by physical and chemical properties. The structure of a protein is closely related to its function. I am involved. If it is necessary for the cells to proceed properly without changing their functions, Secretion usually produces amino acids, ie the conformation of the protein Is important when changing.   Physiological relevance depends on the nature and enzymes of selected proteins, structurally regulated transport proteins. Protein (structural regulatory protein) or food protein (food protei It depends on whether it is n). The host cell or its function is compromised in some way. Mutations, such as compromise, affect the structural and functional properties of proteins. Giving is one of the major problems.   Nutritionally, proteins are as chemically balanced as possible There is a need. Therefore, before thinking about possible mutations, Regarding amino acid content, digestibility and allergenicity (allergen icity) must be analyzed to assess nutritional excellence I won't.   The ease of extraction depends to some extent on the manufacturing method. Any pollution by this It is determined if the substance is present and what extraction method to use.   The invention, in its broadest scope, is all of the above various and possibly spoilers. Appropriately shielded thoughts by choosing α-lactalbumin as a candidate for modification It is based on the realization of handling.   According to the first concept of the present invention, the 80th Phe (Phe-80) is replaced with lysine. Containing a phenylalanine residue other than a variant of human α-lactalbumin. Or less phenylalanine residues than wild-type α-lactalbumin Providing modified α-lactalbumin containing I do.   α-lactalbumin is the major whey protein in human milk, About 2.5 g / liter [29% of total protein-Heine et al. Existence at the level of J. Nutr. 121, p. 277-283 (1991)]. Exist. α-Lactalbumin is a spherical calcium with a relative molecular weight of 14,200. A sium-binding protein found in milk of almost all mammals .   α-lactalbumin has a high nutritional value, 63% of which is an essential amino acid. Is what you do. Beef [Vilotte et al., Biochemie 6] 9, p. 609-620 (1987)] and humans [Hall et al., Biol. Chem. J. (Biochem. J.) Vol. 242, pages 735-742 (1987)]. -Lactalbumin both have 123 amino acid residues and have one monomer Contains 4 phenylalanine residues and 4 tyrosine residues per. α-Luck Tobumin is in the form of human, bovine, ovine, goat and camel milk for millennia. Has formed part of the meal. The function of α-lactalbumin has two aspects. Its main role is as a component of the lactose synthase system. This enzyme Lactose is synthesized in the milk-secreting mammary gland. α-lactalbumin is UDP β-1,4-galactosyl transferase, the second component of the enzyme system (UDP β-1,4-galactosyl transferase) (EC 2.4.1.22) ) Substrate specificity by modifying the specifier protein (speci acts as a fier protein). Second, α-lactalbumin serves as a nutrient source. Have a role.   The sequences of 9 α-lactalbumin have been completely determined [Macken. Gee (Mackenzie) and White (Adv. In  Prot. Chem.) 41, pp. 173-315 (199). (1 year); Vilotte et al., Gene, 112, pp.251-255 ( 1992)], the amino acid composition of 13 α-lactalbumin has been published. [Mackenzie and White, Advent Rotochem (Adv. In Prot. Chem.) 41, pp. 173-315 (1991)] . The crystal structure of baboon α-lactalbumin at 1.7 Å is Acharya et al. [Journal of Molecular Biology (J. Mol. iol.) 208, pp. 99-127 (1989)]. Recently, crystals of human α-lactalbumin at 1.7 Å The structure was published [Acharya et al., Journal of Molecular   Biology (J. Mol. Biol.) 221, 571-581 (1991)]. .   α-lactalbumin and c-type lysozyme are separated from a common precursor That is generally accepted [Hall and Campbell, Essays in Biochem., Vol. 22, pages 1-2 6 (1986) The Biochemical Soc.] .   α-lactalbumin is relatively small with 4 phenylalanine residues It is a secreted protein. Since this is milk protein, as described below, Ideal for expression in the mammary gland of transgenic mammals, the expression system of choice Is preferred. α-lactalbumin is structural, physiological and nutritional And very well suited to meet the importance of harvestability.   In terms of structure, four phenylaras in human and bovine mature proteins Nin residues are 3, 31, 53 and 80th, and 9, 31, 53 and 80th Located in each. These two protein tags Based on protein engineering research, almost every residue can be On the basis of intervening, it is shown to adapt instead of phenylalanine. However Et al., 31 and 80 Phe is lactose synthase reaction and calcium binding Note that when considering the appropriate permutations for these positions, they are associated with each loop. I have to pay more.   α-lactoactbumin along with galactosyltransferase from other species Known to work [Khatra et al., European Journal   Of Biochemistry (Eur. J. Biochem.), Vol. 44, pages 537-560 ( 1974); Powell and Brew, Biochem J. chem. J.), 142, pp. 203-209 (1974)].   Nutritionally, the following three factors should be considered when selecting an appropriate protein for modification. The concept of should be considered: amino acid content, digestibility and antigenicity. Food like this Nutritional quality and levels of essential amino acids as things feed infants and even adults Must be as high as possible. Some milk proteins generally contain essential amino acids. Noic acid is 51.4% of casein, 52.5% of total milk protein, and bovine whey tongue. It accounts for 58.8% of quality. However, in α-lactalbumin it is essential Amino acids account for 63.2% of the total [Heine et al. (J. Nutr.), 121, p. 277-283 (1991)]. More importantly Α-lactalbumin has a high content of lysine and cysteine, especially tryptophan. High content (5.9% of all amino acids). Regarding the measurement of true digestibility in vivo However, commonly used studies have shown that α-lacto The digestibility of albumin is as high as that of casein, and β-lactalbumin (BL It is suggested to be better than G). Bovine whey protein One BLG is human milk Is not found, and is considered to be a predominant human antigen that is susceptible to infection. Bovine α-lactalbumin is very similar to human α-lactalbumin and these Is believed to minimize the antigenicity of bovine α-lactalbumin. This seems correct when compared to other milk proteins [Goldman, et al. Goldman, A.S., Pediatrics 32, 425-Page 425 443 (1963); Strobel, S., Hume Newt. Le: Hum. Nutr ,: Appli. Nutr. 40A (1), pages 45-5 4 (1986)].   Α-la with reduced or no phenylalanine content The lactalbumin products secrete milk (l actating) It is preferably produced in the mammary gland of a transformed animal. This is a Make purification difficult unless the property of the protein is changed by the substitution of the mino acid . However, expression of native α-lactalbumin in dairy animals Is very low compared to the expression of modified α-lactalbumin, or When the expression of the natural or wild-type form is prevented or suppressed, the Purification of α-lactalbumin is not essential. The modified and modified α-lactalbumin expresses in milk of milking animals. Of particular advantage; the latter effect transforms milking animals by using embryonic stem cells It is obtained by Requires purification to maintain protein structure When a conservative change is needed, one possibility is To further facilitate purification, additional amino acid sequences, such as a tail, are attached to the C-terminus. Sometimes [Smith et al., Gene, 32, pp. 321-327 (1 984)]. The additional amino acid sequence makes it easier to separate the proteins. One It has more properties; for example, when the added amino acid sequence changes Enables separation of proteins. Poly-Arg, or poly-Arg / Lys Tail changes enough charge to allow more efficient and cheaper extraction .   Considering the phenylalanine content of other milk proteins such as casein, In some cases, modified α-lactalbumin is a type of natural α-lactalbumin. It is considered preferable that the protein is at least partially purified.   The present invention provides a palatabilit of protein content in the diet of PKU patients. y), and thus is particularly effective in improving compliance. This is expensive Appropriate to meet the criteria of having nutritional value and being able to apply all of the above considerations. Achieved by selecting the protein of interest, α-lactalbumin . In addition, the DNA encoding the above protein is cloned, mutated, Removing at least some, and preferably all, phenylalanine residues, and In a system that can produce enough to supply the world markets Can be expressed.   The α-lactalbumin used as a base for modification may be from a suitable animal source . Milking animals such as humans and cows, sheep, goats and camels are preferred. There is a human It is considered that α-lactalbumin of bovine origin is the most suitable. both With equivalent nutritional value, humans have consumed them for millennia.   Bovine or human alpha-lactalbumin, human eats both proteins It is particularly suitable for use as a starting point for modification by Furthermore, they are very similar, with 72% homology at the amino acid level. I do. Some or all phenylara The nin residue may contain one or more nin residues depending on structural and / or nutritional considerations. It is replaced with an amino acid. The amino acids preferably selected are shown in Table 2. These Any of the four phenylalanines in combination with any of the amino acids of the mino acids May be replaced with the position. Although making substitutions at one position is also included in the invention, Substitute at all positions without substitution at 3 or 2 of these Is preferred.   Select amino acid substitutions only based on energy minimization and structural considerations And Tyr_Three-Leu₃₁-Tyr₅₃-Tyr₈₀Human α-lactalbumin and Tyr₉-LeU₃₁ -Tyr₅₃-Tyr₈₀Bovine α-lactalbumin (or less than these four Substitution variants with substitutions are preferred. Taking into account nutritional needs as well,  Tyr_3/9-Tyr₃₁-Tyr₅₃-Tyr₈₀Of human and bovine α-lactalbumin (or Substitution variants with less than these four substitutions) are the variants selected. Most Other methods of determining suitable substitutions are vertebrate α-lactalbumin and lysozyme. When searching the publicly accepted database, the structure is very similar. Of the same ancestor gene, it is considered that the α-lactoal Finding the corresponding positions occupied by amino acids in bumin and in vertebrate lysozyme Based on this, Tyr_Three-Tyr₃₁ -Leu₅₃-Leu₈₀Human α-lactalbumin (or less than these four Substitution variants with substitutions are preferred.   Some or all phenylalanine residues are replaced. One of these Alternatively, more residues may be conserved as they are preferred for structural purposes. Structure If construction is not considered, at least one, two, three or four natural The nylalanine residue is replaced, with the number of substitutions preferably increasing. Human α-lactoa in which the 80th phenylalanine (Phe-80) was replaced with lysine It is known that there are naturally occurring variants of rubumin [Meinaldo, F .. ( Maynard, F.), J. Dairy Res., Volume 59, p. 425. .. 429 (1992)]; this mutant itself is the compound of the present invention described below. However, it is not part of the present invention.   The α-lactalbumin may be further modified to aid in purification; For example, the tail of poly-Arg or poly-Arg / Lys is added to the C-terminal of the protein. Added. This changes the charge of the protein and makes it possible to purify it in large quantities at relatively low cost. It becomes possible to do. It also says to improve the nutritional quality of the protein It also has advantages.   The modified α-lactalbumin of the present invention is not known as protein engineering. Produced by recombinant DNA technology, collectively using emerging technology.   The equivalent of baboon α-lactalbumin is a Brookhaven database. Available from the Brookhaven database [Acharya et al. Le of Molecular Biology, Vol. 208, p. 99-1 27 (1989)]. The equivalent of human and bovine proteins is Although not currently available, the three-dimensional structure of the baboon protein is not found in human and bovine wild-type and And mutant α-lactoal It is believed to be an acceptable template for making a Bumin template.   The effect of substituting amino acids is first considered at the level of the type of amino acid. did Therefore, aromatic is replaced with aromatic, electric charge is replaced with electric charge, and shape is replaced with shape etc. You.   The first step is for each 4-phenylalanine in the native putative 3D structure. To assess the status of the residues. These include: On the surface Yes, the third phenylalanine (Phe-3) (human) or the ninth phenylalanine Nin (Phe-9) (bovine); the 31st phenylalanine (Phe-3) exposed on the surface 1); 53rd phenylalanine (Phe-53) in the internal cavity And very buried, phenylalanine at position 80 (Phe-80); Simply The four residues are fairly well separated, so the changes introduced are very similar (conservative) and it is reasonable to think that almost no interaction occurs between sites. ing.   By building the model, Phe-3 / 9 and 31 are both on the surface, so , Necessarily show that any residue may adapt to Phe-3 / 9 and 31 It is. Both of the buried phenylalanine residues are found in most of the surrounding proteins. You can adapt without moving it. In addition, leucine and methionine may be added. However, polar or charged groups are thought to destroy the structure and are less preferred. Not good.   α-lactalbumin or some guidance not specific to the purpose under development The literature [Bordo and Argos (Journal of Molecular   Biology (J. Mol. Biol.) 217, p. 721-729 (1991)) , Lesk and Boswell (Cal Opinist Structual Buy Curr. Opin. Struct. Biol. Vol. 2, p. 242 (1992)), Sinfu (S ingh) and Thornton (T hornton) ("Atlas of Protein Sidechain Interlac (Atlas of Protein Side-Chain Interactions) ", Volumes 1 and 2, Aia IRL Press, Oxford, England (1992) ))]; And additionally, computer programs (eg, Lipos Associates Program Civil (SYB YL^TM)) Is useful for assessing other modifications that it is desirable to make.   The following remarks summarize the location:       3rd position: surface residue that can accommodate the most residues: Tyr, Leu, Arg, Met, and Trp are sequentially increased in relative energy, and Tyr is the highest. Is also preferred.       9th position: a surface residue capable of accommodating the most residues. Most α-la Octalbumin has a Ser at this position: Tyr, Ser, Ile.       31st position: Since it is exposed on the surface, any residue can adapt. . However, this region is important for the lactose synthase reaction and Interaction is important. Therefore, when retention of activity is important, Leu and And probably Ile, then Trp are most preferred here.       53rd position: very embedded. Tyr, Leu, Met, T The energy increases in the order of rp.       80th position: Entirely embedded. Tyr, Met, Trp, L eu is the order. This is a calcium-binding loop region, so change here Is important. Tyr is the closest isomorph.       C-terminal: Very exposed because it is on the surface. Of multiple residues       The addition has no significant effect on the structure, but improves the separation properties.   These suggested substitutions were made in human, bovine or other α-lactalbumin. They may be used in combination.   In principle, modified α-lactalbumin according to the present invention should include chemical synthesis. Although it may be produced by a method such as The use of recombinant DNA technology to express proteins from corresponding nucleic acid sequences. .   According to a second concept of the invention, coupling of consecutive amino acids, and And / or ligating oligo- and / or poly-peptides And a method for preparing the modified α-lactalbumin as described above. Preferred Preferably, as predicted above, the coupling of consecutive amino acids is a ribosome. It is preferably mediated by a some. Many expression methods are common to those skilled in the art today Known as general knowledge. In particular, Sambrook et al., Molecular Ra Cloning (Molecular Cloning) 2nd Edition (2nd ed.), Cold Spring Cold Harbor Harbor Laboratory Press ) (1989).   The nucleic acids used for such expression itself form part of the invention. The present invention According to the third concept, phenylalanine residue-free or wild-type α-lacto Modified α-Lactor with fewer phenylalanine residues than albumin Provided are isolated or recombinant nucleic acids that encode rubumin.   Given the degeneracy of the genetic code, many different nucleic acid sequences are included in the invention. Encodes a special protein. In practice the preferred nucleic acid sequences are: However, all such sequences are included in the invention:       (A) The codon matches the codon of the wild-type gene if possible Do;       (B) That codon follows the codon preference of the expression host Selected; or       (C) The codon is selected on the basis of a compromise between criteria (a) and (b) .   Recombinant DNA encoding the modified α-lactalbumin according to the invention is The following sequences are preferred, but the cDNA or this modified protein is It is not limited to other natural genomic sequences to be cloned. Existing in natural genes, Not all, but some introns are present in the construct that are taken up by the host The study of "minigenes" in WO-A-90051188 may be used.   Recombinant DNA according to the inventive concept often has the form of a vector. The present invention , But not limited to specific types of vectors, such as plasmids, cosmids, Or phage. Sufficient regulatory sequences (such as promoters) have been modified By genetically engineering the sequence encoding α-lactalbumin (operatively) The ligated vector is used as an expression vector. Regulatory sequences for genetic manipulation Therefore, the unligated vector is used as a cloning vector.   The recombinant DNA according to the present invention may also be used, for example, in microinjection. (jection) or by homologous recombination It may have the form of the construct that is preferably used.   In the fourth concept of the invention, the nucleic acid of the invention is well within the knowledge of a person skilled in the art. But coupling and / or oligo of consecutive nucleotides And / or prepared by ligating poly-nucleotides. It is.   According to a fifth concept of the present invention, there is provided a host having the above recombinant DNA. You. This host is a cloning host, in which case it is often Escherichia coli (Esc). herichia coli) or an expression host. In the expression host, DNA encoding the modified α-lactalbumin was generated by genetic engineering. It is ligated to a sufficient current regulatory sequence (promoter, etc.) to make the host constitutively or under controlled conditions. A wide variety of expression hosts are used in the present invention. Can be used, specifically, bacteria [especially E. coli], yeast [Saccharomyces   Saccharomyces cerevisiae, Hansenula polymorpha (Hanse nula polymorpha) and Pischia pastoris], insect cells [Spodoptera frugiperda, etc.] and mammalian cells Systems [BHK and CHO cell lines, etc.]. However, so far The most preferred expression host is an animal, especially a germline modified α-lactoalbu Non-human mammals (placental) having a placenta with DNA encoding min  non-human mammals) of which adult females have modified α-lactoalbu Express modified α-lactalbumin in the mammary gland so that min accumulates in milk It is something that can be done. The modified α-lactalbumin differs from the host animal When it is from a different species, the transformed host is a transformed host. (Transgenic); for example, modified human α-lactalbumin Cows or Bulls with Encoding DNA Transformed for Human Genes Was done. All host animals according to the invention, although well within the scope of the invention Are not transformed and, in some cases, the species of interest (eg livestock). Bovine modified α-lactalbumin (eg bovine α-lactalbumin) It is preferred to obtain a homologous host animal carrying the DNA encoding This Such quasi-transgenic animals have been and will be developed. Animal By constantly using techniques that will continue to be developed for production, Or replace one type of gene with another type of gene or replace one type of gene with another By gene therapy technology developed for the purpose of adding , Produced.   Mammary expression system, high expression level, cheap cost, correct processing And has the advantage of easy availability. Bovine and human α-lactalbumin are Produced by various researchers in lactating transgenic animals [Vilot otte) et al., European Journal of Biochemistry (Eur. J, Bio chem.), 186, pp. 43-48 (1989); Hochi et al., Morlep. Rod. And Devel., Vol. 33, pp. 160-164. (1992); Soulier et al., FBS Letters (FEBS) Letters), 297 (1, 2), pages 13-18 (1992)], high-level tan. It has been shown to produce protein. The promoter of α-lactalbumin is milk Expression of a heterologous gene for the secretory mammary gland (WO-A-8801648) [Stinnakre et al., FEB Letters (FEB S Letters), 284 (1), pages 19-22 (1991)].   Other traits, such as transformed mammals expressing heterologous proteins in the milk of adult females Converted animals have been reported in the literature. For example, WO-A-8800239 and WO- A-90051188 is a factor IX and α₁-Pharmacologically such as antitrypsin Describes the production of transformed mammals such as sheep expressing valuable proteins. ing. By using the methods listed above and in all other publications Can produce transformed or sub-transformed animals according to the invention. Cow, sheep, ya Known milking animals such as barley or camel or pigs as hosts in the present invention Use However, the selection of the host is simple and not by the requirements of the present invention. Therefore, it can be decided first.   Transformed animals or other hosts according to the present invention are made by conventional methods. Therefore, the present invention is not limited to a particular manufacturing method. For example, a transformed animal , WO-A-8800239 and WO-A-9005188, described above. Made by microinjection, Also, embryonic stem cell technology as described in WO-A-9003432. (embryonic stem cell technology).   Expression of the host wild-type α-lactalbumin gene is used to simplify purification. Is inhibited or at least suppressed.   According to a sixth concept of the present invention, no phenylalanine residue or wild type At least one containing less phenylalanine residues than α-lactalbumin of Consisting of two modified α-lactalbumin Providing suitable foodstuffs to patients with enylalaninemia.   The preferable embodiment of the modified α-lactalbumin is as described above.   The food product may, or will often, include other ingredients. Qualified Proteins can be L-amino acids and / or other phenylalanines alone or Protein or Peptide Low in Phosphorus Content or Free of Phenylalanine In combination with, it is preferred to be used for infants, children and adults with PKU. Good. For infants and very young children, a protein base With a low phenylalanine content or without phenylalanine The formulation with octoalbumin has the appearance and appearance of a normal whole protein infant formula. The taste is close As a result, the general special nutrition of protein hydrolysates or amino acid mixtures It will be more tolerable than the raw mix.   Infants and very young children containing modified α-lactalbumin according to the invention Special dietary supplements or supplements for children include fat sources, charcoal water. Other constituents such as oxidant sources, and appropriate amounts of vitamins, minerals, and other nutritional factors It may include a child. In addition, L-amino acids (L-tyrosine, etc.) are not modified. In addition to the α-lactalbumin prepared above, it may be added as a nitrogen source. It is added to improve the quality of the overall essential amino acids of the product, which makes it biologically relevant. In all cases, the total phenylalanine content of the final product is usually 100 g powder. Less than 80 mg, preferably less than 25 mg, or phenyl It does not contain lualanine. In addition, [enzyme chymosin (rennet) The quality of cheese when decomposing α-casein which is a protein and releasing CDP into the whey fraction Includes phenylalanine such as casein-derived peptide (CDP) derived from the production process Peptides with low abundance or no phenylalanine are also available with Preferred as a nitrogen source added to improve the overall quality of the acid profile Yes. L-tyrosine and non-essential amino acids are also used to ensure nutritional suitability. Up to and / or in the amount found in human milk and the diet of normal children You may add in the amount which exceeded.   The above special regimen for infants and young children containing modified α-lactalbumin Food or curing supplements should be prepared with a ready-to-feed liquid or water It is used so that it can be made into a ready-to-feed form by stirring It may have the form of a powder or a concentrated liquid that is 100 ml (60-75 kcal / ml) easy-to-feed liquid formulation or reconstituted powder The end is usually 1.2 to 3. 0 g, preferably about 1.3-1.5 g protein; 2.2-4.0 g, preferred Or about 3.6 g of fat or fat mixture; and 6-9 g of carbohydrates You.   The source of carbohydrates, lactose, is usually preferred in most babies, but corn white Corn syrop solid, sucrose or other sugars also affect PKU May be used as a special diet for infants and young children.   In addition, it has a low phenylalanine content or does not contain phenylalanine. A special dietary diet containing modified α-lactalbumin as a protein source is Appropriate amount of vitamins A, D, E, B₁, B₂, B₆, B₁₂, C, Nicotine In addition to vitamins such as glucose, pantothenic acid, folic acid, vitamin K, biotin and choline. Nutritionally acceptable amounts of calcium, phosphorus, potassium, sodium, chloride Objects, magnesium, iron, copper, zinc, manganese, molybdenum, selenium, chromium, fluorine Contains minerals so that fluorine and iodine can be obtained. Carotene, taurine, boar Other trophic factors such as tall, carnitine and nucleotides may also be added.   The nutrients listed above and their acceptable chemical forms are included in the special dietary diet. A large amount is a user of Federal Regulations. S . U.S. Code (Parts 181-184) and the 1980 U.S. Code. S . Infant Formula Act, US Federation Food and Drug Cosmetics Act, Section 2, Chapter IV, Section 192 Public Law 96-359 (the U.S. Federal Food, Drug and Cosmetic) Act, Section 2, Chapter IV, Section 192 Public Law 96-359), 1980 9 26th, October 7, 1985 amendment.   Not exactly the same, but a similar recommendation is EC Commission Dilek Tiv on Infant and Follow-On Formula, Offy The Shar Journal of the European Communities, No. L 1 75/35, 1991 (EC Commission Directive on infant Formula and Fol low-on Formula, Official Journal of the European Communities, No. L 175 / 35, 1991), and The Codex Alimentarius Commission, Recommendation Dead International Standards For Foods For Infa And the Children (the Codex Alimentarius Commission, Recommende d International Standards for Foods for Infants and Children), Join TOF AO / W HOT STANDARDS PROGRAM, CIE C / RS, 72/74, 1967 (Joint FAO / WHO Food Standards Pr ogramme, CAC / RS, 72/74, 1967). Minimum value recommended in Table 3 And the maximum and preferred values.   Therefore, in infants and very young children, the ideal special supplement is A phenylalanine content of less than 80 mg per 100 g of the formulation or solids. On the outside, it should resemble breast milk from a human breast. Infant, small Continued diet with low phenylalanine content in infants and adolescents Kicking is inevitable. Table 4 below shows the daily requirements:   Diets for other children, adolescents and pregnant women, especially those with PKU, include phenylala Phenylalanine in formulations with low nin content or no phenylalanine Consists of more than 50% calories of low content foods. Such a distribution It is recommended that the compound be taken daily for each age group, i.e. in mind. Because it is designed, or has one common formulation in each category specific usage direction And may be developed with a dilution difference. People other than these infants and very young children Formulations for about 50-1000 kcal or 50, as shown in Table 5. Contains nutrients with RDI levels below (approximately) 0-1000 ml.   Pregnant women have good tasting formulations and are high quality ingredients for the rest of the diet For children and adults, this is the end point for the formulation of special curative supplements. like this The formulation should include all essential amino acids (except phenylalanine), especially fruits and vegetables. Of the above vitamins and minerals that are not present in high amounts in low protein foods Must be included. Other such nutrients in protein include: There are calcium, iron, folic acid, magnesium and trace minerals.   Specific formulations for children, adults and pregnant women with PKU are limited to Although not included, it includes the following preferred ranges of nutrients shown in Table 6:   Α-Lact with low phenylalanine content or no phenylalanine Albumin is also used in older children, pregnant women, and adult children with PAH deficiency. Highly preferred for inclusion in milk drinks for years and adults.   Or such modified α-lactalbumin or modified α-la A dietary food supplement containing octoalbumin is a functional food supplement. Used in foods such as bread, ice cream and sugar coatings as a source of puffiness; They are also free of phenylalanine, or at least phenylalanine. Content is low.   The milk of transformed or sub-transformed host animals as described above may be When the expression of the α-lactalbumin gene is suppressed or inhibited, It is suitable as a foodstuff according to the invention with or without further modification. Qualified By further processing of the protein, eg added to the formulation Prior to this, the amino acid tail is removed for regulatory or other purposes.   According to a seventh concept of the invention, less flux than wild-type α-lactalbumin. One containing modified α-lactalbumin containing a phenylalanine residue, or Higher levels consisting of administering more foodstuffs to patients with hyperphenylalaninemia A method for supplying a patient with phenylalanineemia is provided.   Again, the preferred embodiment of modified α-lactalbumin is the invention As described above in connection with the first concept of.   Preferred aspects of the food product are as described above in relation to the second concept of the invention. . More generally, aspects of the inventive concepts may be modified (mut atis mutandis), similar to other concepts.   The amount and timing of administration is at the discretion of the patient, but in general, Follow the instructions of your doctor, nutritionist or other medical adviser.   The present invention is explained in detail by the following examples, which are examples of the present invention. There is no restriction. The examples refer to the following figures:   FIG. 1 illustrates Example 1 and relates to human α-lactalbumin. Figure 3 shows a restriction map of two overlapping genomic lambda clones;   FIG. 2 illustrates Example 1 and shows that human α-lactalbumin tras FIG. 3 shows a transgene construct;   FIG. 3 illustrates Example 1, humans versus untransformed mouse milk. SDS-PAG of milk from α-lactalbumin transformed mice Shows E analysis;   FIG. 4 illustrates Example 1 and is a human α-lactalbumin standard. Quality of Human α-Lactalbumin Transformed Mouse Milk for Quality Demonstrate stun blotting;   FIG. 5 illustrates Example 2 and bovine α-lactalbumin PCR. Shows the sequence of the primer;   FIG. 6 illustrates Example 2 and bovine α-lactalbumin PCR. Shows the location of the primer and product;   Figure 7 illustrates Example 3 and uses bovine α- used for mutagenesis. 1 shows the sequence of a lactalbumin PCR primer;   FIG. 8 illustrates Examples 4 and 5 and illustrates the substitution of phenylalanine. PCR strategy and transgene of PKU1 to 4 FIG. 6 shows the cloning strategy for the construct;   FIG. 9 illustrates Example 4 in which control mouse milk and cows are used. 1 to 4 for α-lactalbumin standard of Showing Western blotting analysis of milk run of transgenic mouse Is;   FIG. 10 illustrates Example 4, a bovine α-lactalbumin standard. Two PKU1 transgenic mouse lines of milk run against dilutions of material Quantification by Eastern blotting analysis is shown;   FIG. 11 illustrates Example 5 and relates to the substitution of phenylalanine. PCR strategy and transgene constructs for PKU5 and 1H Shows the relevant cloning strategy;   FIG. 12 illustrates Example 6 and shows bovine α-lactalbumin and Cloning of 10 constructs from PKU5 for expression in COS cells It shows the strategy;   FIG. 13 illustrates Example 6 and shows pC-BOVA and pC-PKU. Immunoprecipitated material (imm from the supernatant of COS cells infected with constructs 5 to 10 unoprecipitate material).Example 1-Cloning of human α-lactalbumin in transgenic mice And expression Cloning of human α-lactalbumin gene   The DNA sequence of human α-lactalbumin is known [Hall et al., Va. Iochem J. (Biochem. J.) Vol. 242, pp. 735-742 (1987)] . PCR primers were designed using human sequences, and 2 from human genomic DNA We cloned two small fragments, one of which was the 5'end of the gene And the other was the 3'end. Substituting these into the pUC18 vector After cloning, the commercially available (Stratagen (Stratagen e) Made) λ Genomera Ibrary was screened. two containing the α-lactalbumin gene Recombinant bacteriophage, 4a and 5b. 1, the established method [Sambrook (Sambrook) et al., Molecular Cloning, Second Edition (2nd e d.), Cold Spring Harbor La boratory) (1989)]. By creating a restriction map, Both clones contain the complete coding sequence for human α-lactalbumin. It was shown that the 5'and 3'sequences contained rows but differed in abundance (Figure 1).   Clone 5b. Sequence analysis of 1 exon and partial sequence analysis of clone 4a Showed that these clones matched the published sequence. Transgene construct (Figure 2)   pHA-1 is a human α-lactalbumin coding region, pUC18. 5 clones cloned in as a 7 kb EcoRI / SalI fragment b. 1'derived about 1.8 kb 5'flank and 3 kb 3'flank It is composed of (3'flank).   pOBHA (sheep β-lactalbumin, human α-lactalbumin) Was constructed from the following four DNA fragments:       (1) 4.2 kb containing sheep β-lactalbumin promoter SalI / EcoRV fragment (WO-A-9005188);       (2) used as an 8 bp BclI linker and a blunt BglI fragment 74 bp corresponding to the 15th to 77th bases of the human α-lactalbumin sequence A synthetic oligonucleotide of       (3) BglI site at the 77th base and XhoI in the 3'flank 6.2 from 1 clone 4a with a region between the sites a BglI / PstI fragment of human α-lactalbumin of kb;       (4) pSL1180 (Pharmacia) cleaved with PstI and SalI (Pharmacia)). Expression of human α-lactalbumin in transgenic mice   Two constructs, pHA-1 (human α-lactalbumin gene and fla Of the β-lactalbumin of sheep and pOBHA. Having a human α-lactoaluminin gene induced by a lomotor, It was injected into a mouse embryo to obtain a transformed animal.   The expression level of human α-lactalbumin in the milk of these mice was examined. It ranged from impermeable levels to about 3 mg / ml. Table 7 shows the transformed 1 shows a summary of the relative amounts of transgenic proteins. These animals Skimmed milk from SDS-PAGE stained with Coomassie blue, isoelectric focusing Electrophoresis, commercially available anti-human-α-lactalbumin antibody (manufactured by Sigma) Analyzed by visualized Western blotting and chromatofocusing did. From the results of these analyses, the transformed protein was identified as human α-lacto. The correct size when compared to a standard of tobumin (made by Sigma), It was shown to have pI and antigenicity.   Results for various mice are shown in FIGS. Figure 3 shows the untransformed SD of transgenic mouse skim milk run against Troll mouse milk 1 shows an S-PAGE analysis. Fig. 4 shows the standard of human α-lactalbumin. It shows the Western blotting of the run of the above milk to the quasi-substance. You. Example 2-Cloning and expression of bovine α-lactalbumin in mice Cloning of bovine α-lactalbumin   There are three known variants of bovine α-lactalbumin, of which B The type is the most common. Bos (Bos) Nomadicas F. Di. Index The 10th residue in the A mutant of nomadicus f.d. indicus is different from the B mutant That is, glutamic acid (Glu) at A is replaced by arginine (Arg) at B You. Sequence for the C variant of Bos (Bibos) javanicus Difference is not established [McKenzie & Whit e), Advances in Protein C hemistry), 41, pp. 173-315 (1991)].   The bovine α-lactalbumin gene (encoding the B form) is shown in FIG. Was cloned from the genomic DNA using the PCR primers. This primer Are given the following SEQ ID NOs:               Ba-2 SEQ ID NO: 1               Ba-7 SEQ ID NO: 2               Ba-8 SEQ ID NO: 3               Ba-9 SEQ ID NO: 4   The DNA source for all PCR reactions was Holstein-Friesian cow (Holstein-Friesian cow) blood.   Amplified using primer Ba-7 in combination with primer Ba-8 The length of the promoter region was 2.05 kb. This BamHI / EcoR The I fragment was cloned into Bluescript (pBA-P2 ).   All bovine α-lactoal containing a 300 bp 3'-flanking region Using the bumin gene, primer Ba-9 in combination with primer Ba-2 Amplified. These primers have a BamHI restriction enzyme recognition site. Which directly amplified the 3 kb fragment amplified in the BamHI site of pUC18. Subcloning yielded construct pBA-G3.   BamHI / EcoRV fragment BA-G3 EcoR of clone pBA-P2 Ligation to the V / BamHI fragment resulted in the construct pBA (Fig. 6).   Taq polymerase lacks proofreading activity and is therefore amplified. Bovine α-lactalbumin DNA is known to be isolated bovine α-lactalbumin DNA. It was essential that they match the gene. Sequence analysis for all exons and two Of the promoter fragment of. Bovine α-lactalbumin exon Three changes, compared to those published by Vilotte: It has been shown:       (1) in exon I, +759: C to A; 5'-noncoding region. This change is described by Hochi et al. In Mol. od. and Devel.), 33, p. 160-164 (1992). Was.       (2) at exon I, at +792: CTA to CTG; both are Roy Codes Shin.       (3) In exon II, +1231: GCG to ACG; alanine From threonine. This shows that it is the more common B form of the protein is there.   Mis-decoding of sequences during amplification by PCR can be ruled out, but the above-mentioned mispairing is bovine DN. It is believed that this is due to the difference in the source of A. Expression of bovine α-lactalbumin in transgenic mice   The construct pBA (FIG. 6) was injected into mouse embryos and 9 transformed animals were obtained. Real Milk was analyzed in the same manner as in Example 1. SDS-PAG stained with Coomassie blue Expression by analysis by E-gel and comparison with standard amount of α-lactalbumin Levels can range from undetectable to about 0.5-1 mg / ml. Was shown.Example 3 Site-directed mutagenesis Sequence number for PCR primer   The following SEQ ID NOs are used as PCR primers in this example and the following examples. Using: PKU-1 SEQ ID NO: 5 PKU-2 SEQ ID NO: 6 PKU-2L SEQ ID NO: 7 PKU-3 SEQ ID NO: 8 PKU-4 SEQ ID NO: 9 PKU-5 SEQ ID NO: 10 PKU-6 SEQ ID NO: 11 PKU-7 SEQ ID NO: 12 PKU-8 SEQ ID NO: 13 PKU-9 SEQ ID NO: 14 PKU-10 SEQ ID NO: 15 Substitution of phenylalanine residue   Four phenylalanine (Phe) residues in bovine α-lactalbumin Is within the first 3 exons of the gene.   7 oligos (PKU-PCR primers 1, 2, 2L, 3, 4, 7 and 8) The set is modeled for proteins, nutritional considerations, and natural α-lactoa. Any of the bubumin or lysozyme genes of various species It was designed for Phe substitution based on the amino acid variant present in Allocation of primer Sequence (complementary to either coding or non-coding strand), amino The expected changes in the no acids and the sites of mutagenesis for each are shown in FIG. These oligos act as PCR and mutagenic primers. PKU Lima-1 is used in combination with 7 in combination with 2 or 2L and 8. A 435 bp amplified fragment (A) was obtained in which the first two Phe were replaced. . With PKU primers 3 and 4 or 9 and 10, the second two Phe A displaced 601 bp PCR product (B) is produced (see Figure 8).   The PCR products were labeled with EcoRI / BamHI and BamHI / Xha, respectively. The I site was used for subcloning for sequence analysis. PKU Primer-2, 2L Encoding within this region by the PvuI site created in 3, 7, and 8. The 31st amino acid and the 53rd amino acid without changing the amino acid A single restriction enzyme site was created with the acid. Amino acid tail at the C-terminus   Addition of other amino acids at the C-terminus of the α-lactalbumin gene is endogenous Can be assisted in its separation from the α-lactalbumin protein. Other amino acids The purpose is to determine if it affects the expression of lancegene. Poly-Arg tail   Bovine α-lac was prepared using primers PKU-5 and 6 (see FIGS. 7 and 8). Amplification of the 3'end of the toalbumin gene gave a 0.44 kb PCR product C . Primer PKU-6 is the last native in bovine α-lactalbumin sequence. Immediately after the Leu amino acid has a coding region for 6 Arg residues Which results in a single SalI And SnaBI sites were created. This single SalI / SnaBI site is deleted And insert a linker to obtain the required length of poly-A An rg or poly-Arg / Lys tail can be made. Natural stop codon And about 30 bp of 3'sequence after the end of the restriction enzyme site of BspMI.Example 4-Assembly of bovine α-lactalbumin construct in vivo and Expression Constructs for expression in the mammary gland of transformed animals   All five constructs were made. These are the poly-Arg tail and two mutations. Poly-A having a bovine α-lactalbumin gene (pBARG-H) Native bovine α with and without rg tails (PKU-1 to PKU-4) -The lactalbumin gene. Mutations of Phe in these constructs Was based on protein modeling and nutritional considerations. Construction design The outline is shown in FIG.   pBARG-H was constructed from the following 5 DNA fragments:       (1) A 2.04 kb SstI to HpaI fragment derived from pBA;       (2) 1.25 kb cleavage from HBAI to HindIII derived from pBA Piece;       (3) 0.4 from HindIII to BspMI derived from PCR product C 4 kb fragment;       (4) pBA-derived BspMI to BgIII fragment of 0.51 kb ;as well as       (5) Vector pSL1180 digested with SstI and BgIII.   PKU-1 was constructed from the following 6 DNA fragments:       (1) A 2.04 kb SstI to HpaI fragment derived from pBA;       (2) HpaI from PCR product A (PKU-primer pair 1 and 2) To PvuI 0.46 kb fragment;       (3) PvuI to Bs from PCR product B (primer pair 3 and 4) a 0.60 kb fragment up to aBI;       (4) 0.22 kb from BsaBI to HindIII derived from pBA fragment;       (5) 0.95 kb from HindIII to BgIII derived from pBA Fragments; and       (6) Vector pSL1180 digested with SstI and BgIII.   PKU-2 was constructed in the same manner as pKU-1 except for fragment (5), and pBAR It was derived from GH and contained a poly-Arg tail.   PKU-3 was prepared in the same manner as pKU-1 except for fragment (2) (PCR product A). As constructed, PKU-Primer-1 was amplified in combination with 2L.   PKU-4 was constructed in the same manner as pKU-3 except for fragment (5), and pBAR It was derived from GH and contained a poly-Arg tail.   The substitution of the amino acid obtained by the above-mentioned mutagenesis was carried out using the plasmid PKU-1. Table 8 for PKU-4 et al. 3'-flanking region of human α-lactalbumin   The 9.3 kb BamHI fragment from λ clone 4a (FIG. 1) was isolated from human α-la. It has a 3'flanking region of the lactalbumin gene and is transferred to transformed embryos. All PKs as suitable targets for preimplantation screening Included in U construct. This 9.3 kb disconnection The piece was inserted into a single Bglll site present in all the above constructs (Figure 8). .   DNA for microinjection was digested with BamHI to It was removed from the vector sequence by digestion. DNA for microinjection The prepared and transformed mice are known [Hogan et al., “Manipulating   The Mouse Embryo: Laboratory Manual (Manipulating the Mouse Embryo: A Laboratory Manual) ", Cold Spring Harvara Noratorie Press (Cold Spring Harbor Laboratory Press) (1986)] Was obtained in the same manner as. Phenylalanine-free bovine .ALPHA.-lactoalbumi in transgenic mice Manifestation of   Transformed animals are milked 10 days after delivery and the milk is Western blotted. Analysis was performed (Fig. 9). All constructs, namely wild-type bovine α-lac Toalbumin pBARG-H and four mutagenized bovine α-lactos The albumin constructs (PKU1-4) are bovine α-lactalbumin tantalum in milk. The protein could be expressed and secreted. The strong upper band (see Figure 9) is glycosylated Due to the variant Is also present in pBARG-H (data Not shown). Table 9 contains no phenylalanine in the milk of the transformed mice. 1 shows the relative amount of bovine α-lactalbumin protein. Protein Run various dilutions of milk samples on a Wester blot to quantify quality And compared with a bovine α-lactalbumin standard. Figure 10 shows PKU-1 type Fig. 3 shows the above method when used for a sample of a quality-converted mouse.   Furthermore, this protein was analyzed by ELISA, pI, sequence analysis, circular dichroism, etc. Analyze and combine the established technologies of. By these methods, proteins Information about the size and charge of the But ideas will come to mind. RNA analysis   Transformed mammary gland total RNA was assayed for bovine α-lactalbumin-specific RNA. Analyzed by Northern blotting using a lobe. As expected, all Since animal samples had detectable levels of recombinant protein, bovine Expression of α-lactalbumin mRNA It was shown that. There is no distinction between mRNA and abundance of transformed protein. There is no white relationship.   Confirm that the expressed protein is derived from the mutant α-lactalbumin gene To confirm, of bovine α-lactalbumin present in the extracted RNA The cloning sequence of the mRNA was determined by the RT-PCR method. From the results, The mRNA was shown to be transcribed from the PKU-4 construct and thus translated The protein was found to contain no phenylalanine residues.Example 5-Under the control of the human alpha-lactalbumin promoter in vivo Of mutagenized bovine α-lactalbumin in Escherichia coli   The data from Examples 1, 2 and 4 demonstrate that the endogenous bovine and human genes Reflecting the difference in the expression level, the transgene of human α-lactalbumin Expression is significantly higher than that of the native bovine α-lactalbumin transgene. It was shown that It was thought that this was due to the difference in the control region of 5 '. Therefore, the 5'region of the transcription initiation site of bovine α-lactalbumin was replaced with human α-lactalbumin. It was replaced with a sequence derived from the toalbumin gene.   Two constructs, namely, those with the amino acid substitutions shown in Table 10 were made. PKU-5 and PKU-1H were prepared. Diagram of the design of these constructs 11 shows.   PKU-5: In the first cloning step, the following 3 fragments of pUC18 Subcloned into the EcoRI / BamHI site:       (1) PCR amplification using PKU-primer 7 in combination with 8 Width-induced EcoRI to PvuI fragment (fragment A, FIG. 11);       (2) PC using PKU-Primer 9 in combination with 10 A fragment from PvuI to BsaBI induced by amplification with R (fragment B, FIG. 11); and       (3) A fragment from BsaBI to HindIII derived from pBA. The final construct contained the following 6 DNA fragments:       (1) Human α-lactalbumin pro-derived from λ clone 4a (FIG. 1) A 3.7 kb fragment from SalI to KpnI containing the motor;       (2) Human α-lactalbumin sequence from KpnI site to AUG And a bovine α-lactalbumin sequence from the AUG to the HpaI site. bp synthetic oligonucleotides;       (3) HpaI to HindI obtained from the first subcloning step A 1.25 kb fragment up to II;       (4) 0.95 kb from HindIII to BgIII derived from pBA fragment;       (5) Human α-derived from λ clone 4a used as a BamHI fragment 3.7 from BamHI to XhoI of the 3'flank of the lactalbumin gene a kb fragment; and       (6) Bluescript (Bluescrip) cut with SalI and BamHI t) KS^-vector.   PKU-1H was constructed in the same manner as PKU-5 except for fragment (3), and PKU- 1 (Example 4). Expression in transgenic mice   Two constructs, PKU-1H and PKU-5, were injected into mouse embryos. here Induced transgenic animals for the PKU-5 construct. Rearing these animals The milk was analyzed for the first time.Example 6-In Vitro Mutagenized Bovine α-Lactalbumin Expression of Constructs for transient expression   In order to analyze a wide range of mutations in the α-lactalbumin gene, in vitro experiments were performed. The current assay was established. For the above purposes, natural and mutated cattle Coding region of α-lactalbumin gene [Examples 2, 3, 4 and 5; 7 Sequences 56 to 3030; Vilotte et al., Biochem ie), Vol. 69, pp. 609-620 (1987)] for expression of COS cells. It was cloned into pcDNA3 (manufactured by Invitrogen) to obtain pC- -10 was obtained from BOVA and pC-PKU-5.   A detailed overview of the construction is given below with reference to FIG. 12:       (1) Bovine α-lacto from the 756th position to the 831st HpaI site A synthetic oligonucleotide containing an albumin sequence was used as a vector-pcDNA3 Hin cloned into the dIII / BamHI site;       (2) HpaI was added to the coding region of bovine α-lactalbumin gene. To BamHI to obtain the construct pC-BOVA;       (3) 1.25k from HpaI to HindIII in pC-BOVA The fragment of b from HpaI to HindIII containing the Phe substitution (FIG. 11) 1. Exchange with a 25 kb fragment yielded constructs pC-PKU-5 to -10.   PpuMI between the 9th and 30th amino acids and the 53rd and 80th amino acids Due to the presence of AvrII restriction enzyme sites between amino acids (Fig. 11), the results are shown in Table 11. PKU with 1 or 2 out of 4 phenylalanines substituted as listed A construct was obtained. Expression in COS cells   Cells were infected with 10 μg of plasmid; after 72 hours the protein was³⁵S- Labeled with methionine and -cysteine. Collect the supernatant and Lactalbumin was immunoprecipitated and then analyzed on a polyacrylamide gel.   FIG. 13 shows bovine native and mutated by transient expression in COS cells. Of α-lactalbumin was shown to be secreted. 9th Phe to Ser Of the wild-type bovine α whose expression level was -Equivalent to lactalbumin. 30th Phe to Tyr substitution and 53rd Phe to Leu substitution (pC-PKU6), and 80th P The expression level was suppressed by the substitution of he to Tyr (pC-PKU8). pC- Expression of PKU5, 7 and 9 was below the level of detection in the above assay system. The system described above allows amino acids for maximum expression in transgenic animals. Identification of acid substitutions became possible.Example 7-Method of Using Milk Containing Modified Protein   For four children with phenylketonuria, phenylalanine A special dietary supplement that does not contain is required. Follow food Because it is important during the early ages, it is a therapeutic product that has a good taste and improves the followability of the meal. Formulations are preferred.   Age and growth requirements should be considered when planning a diet for children with phenylketonuria. Daily protein, calorie and amino acid requirements to support your needs ( Protein obtained by determining (including phenylalanine) and adding special curing Calculate the quality and calories, and even what you need without exceeding the required amount of phenylalanine. But it is necessary to decide if food is needed. In general, such pheny Preparation for lualanine is accomplished by feeding a given amount of milk and other regular meals. Done.   Modified as a specific daily menu for children with phenylketonuria α-lactalbumin and other nutrients as shown in Table 3 above Up to 3 times / day of special supplements including elementary substances. Age, growth and A phenylalanine-restricted diet that is regularly adjusted to meet your needs It is generally recommended to continue for a lifetime.Example 8-Preparation / isolation of modified protein from milk   For the preparation of most but not all special formulations, the modified α- Separation and purification of regular α-lactalbumin from lactalbumin protein It is desirable. For example, in children, adolescents and pregnant women, the phenylalanine content is A formulation that is zero or minimal allows maximum flexibility during curing, Perhaps it will adhere to the management of PKU's diet for life.   Therefore, to the C-terminus of the tail of poly-Arg or poly-Arg / Lys The attachment was designed to allow for a more efficient and cheaper effective separation. this Such a design has been modified so that it can be separated from normal α-lactalbumin. The charge to the protein must be changed. Such separation is This is possible with commercial-scale whey fractions and even with liquid chromatography techniques. Yes, the latter separation method is possible based on the difference in the charge of the molecules. Liquid chromatograph Has already been promised for the separation and purification of proteins and other components from whey. I was   Other techniques are also used, especially for experimental or bench top properties. Can be used. These include, but are not limited to, SDS-PAGE chromatography. Graphy and radioimmunoassay.Example 9-Modified Protein Formulation   According to the invention, several special diets intended for the treatment of hyperphenylalanineemia. It is possible to formulate the formulation. These formulations are total protein-modified. Α-lactalbumin-is the main protein (N) and phenylalanine Amino acid source that does not contain, but general formula In some cases, the N source is either L-amino acids or highly hydrolyzed casein protein. Only in morphology, it is unique compared to all common formulations.   The major total protein properties of these novel therapeutic formulations are non-taste and palatability. Improves food followability because it is constantly improved; L-amino acid and peptide refinement The mixture made is generally bitter and not tolerable. Therefore from an infant Beginning with pregnancy and continuing throughout adulthood, a series of formulations for a lifelong diet It is preferable to produce.   These formulations contained similar modified α-lactalbumin, but L-amino acids to change protein content and, if necessary, to increase nutritional value , Non-protein energy sources such as sugar, corn syrup and / or fat, And vitamins, minerals and flavors may be added. Obviously for infants Special Curative Formulations Consistent With Human Milk Profile or Its Physiological Effects The result is a high proportion of fat-induced calories. Pediatric and adult formulations require more total protein than infants and young children need Quality and less fat, and, if any, varying proportions of vitamins And minerals. Ingested in combination with these non-infant group formulations Natural diets are commonly distributed among dietary groups with similar phenylalanine levels Can be exchanged with each other to further enhance the variety of meals, therefore , Becomes easier to follow.

[Procedure of Amendment] Article 184-8 of the Patent Act [Submission date] June 28, 1995 [Correction contents]                                The scope of the claims Human wild type α-lac in which phenylalanine at position 80 was replaced with lysine Does not contain phenylalanine residues that do not naturally give rise to variants of toalbumin. Or a repair consisting of fewer phenylalanine residues than wild-type α-lactalbumin. Wild-type α-lactalbumin in decorated form. 2. Claim 1 having an amino acid tail at the C-terminus to facilitate purification. Modified α-lactalbumin according to item. 3. The C-terminal tail is poly-Arg or poly-Arg / Lys. Modified α-lactalbumin according to claim 2. 4. A modified bovine α-lactalbumin, claim 1, 2 or Modified α-lactalbumin according to item 3. The 5.9th Phe is replaced with Tyr, Ser or Leu. Modified bovine α-lactalbumin according to item 4. 6. The 31st Phe is replaced by Leu, Ile, Trp or Tyr. Modified bovine α-lactalbumin according to claim 4 or 5. The 7.53rd Phe is replaced by Tyr, Leu, Met or Trp, Modified bovine α-lactalbumin according to claim 4, 5, or 6. 8. Phe of No.80 is replaced by Tyr, Met, Trp or Leu Modified bovine α-lactoal according to any one of claims 4 to 7 Bumin. 9. A modified human α-lactalbumin, claim 1, 2 or Modified α-lactalbumin according to item 3. 20.3th Phe is replaced with Tyr, Leu, Arg, Met or Trp. Modified human α-lactalbumin according to claim 9. 11.31 Phe is replaced by Leu, Ile, Trp or Tyr, Modified human α-lactalbumin according to claim 9 or 10. 12.53 Phe is replaced by Tyr, Leu, Met or Trp, Modified human α-lactoalbu according to claim 9, 10 or 11. Min. 13. Phe of No. 80 is replaced by Tyr, Met, Trp or Leu, Modified human α-lacto according to any one of claims 9 to 12. albumin. 14． Contiguous amino acids are linked, and / or oligo- and / or Any of claims 1 to 13 which comprises linking a re-peptide. A method for preparing the modified α-lactalbumin according to 1. 15. Modified α-la defined in any one of claims 1 to 13. An isolated or recombinant nucleic acid encoding octalbumin. 16. The modified α-lacto according to any one of claims 1 to 13. An isolated or recombinant nucleic acid encoding albumin. 17． A vector comprising the nucleic acid according to claim 15 or 16. 18. Linking consecutive nucleotides and / or oligos and / or The method according to claim 15 or 16, which comprises linking poly-nucleotides. A method for preparing the described nucleic acid. 19. A recombinant nucleic acid according to claims 15 or 16 is included or claimed. A host comprising the vector according to box 17. 20. Has a DNA encoding a germline-modified α-lactalbumin A non-human mammal having a placenta -Alpha-lactolace modified in the mammary gland so that lactalbumin accumulates in milk The host according to claim 19, which is capable of expressing rubumin. . 21. A cow (or bull), sheep, goat, camel or pig 21. A host mammal according to claim 20. 22. Expression of the host wild-type α-lactalbumin gene is inhibited or suppressed 22. The host according to claim 19, 20 or 21. 23. At least one modification according to any of claims 2 to 13. It is used in patients with hyperphenylalaninemia, which consists of Groceries to be taken. 24. A curative supplement used for infants or children with hyperphenylalaninemia or 24. A food product according to claim 23 which is a formulation. 25. The milk of the female mammal according to claim 19, 20 or 21, 25. The food product according to claim 23 or 24. [Procedure of Amendment] Article 184-8 of the Patent Act [Submission date] July 11, 1995 [Correction contents] 26. The food product according to any one of claims 23 to 25 is made high phenyl. To patients with hyperphenylalaninemia consisting of administration to patients with alanineemia Supply method.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI C07K 14/76 9637-4B C12P 21/02 C C12N 5/10 9281-4B C12N 5/00 B C12P 21/02 9455-4C A61K 37/02 ADD // (C12P 21/02 C12R 1:91) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD) , AM, AU, BB, BG, BR, BY, CA, CN, CZ, FI, GE, HU, JP, KE, KG, KP, KR, KZ, LK, LT, LV, MD, MG MN, MW, NO, NZ, PL, RO, RU, SD, SI, SK, TJ, TT, UA, US, UZ, VN. Side Park 14 (72) Inventor Rigden, Daniel, Joan England Cheshire Shidabureau 2 7 P, Clew, Denver Avenue 38

Claims (1)

[Claims] Human α-lactoalbu in which phenylalanine at position 80 is substituted with lysine Non-mutated form of Min, containing no phenylalanine residue or wild-type α- Modified α-lacto containing fewer phenylalanine residues than lactalbumin albumin. 2. Claim 1 having an amino acid tail at the C-terminus to facilitate purification. Modified α-lactalbumin according to item. 3. The C-terminal tail is poly-Arg or poly-Arg / Lys. Modified α-lactalbumin according to claim 2. 4. A modified bovine α-lactalbumin, claim 1, 2 or Modified α-lactalbumin according to item 3. The 5.9th Phe is replaced with Tyr, Ser or Leu. Modified bovine α-lactalbumin according to item 4. 6. The 31st Phe is replaced by Leu, Ile, Trp or Tyr. Modified bovine α-lactalbumin according to claim 4 or 5. The 7.53rd Phe is replaced by Tyr, Leu, Met or Trp, Modified bovine α-lactalbumin according to claim 4, 5, or 6. 8. Phe of No.80 is replaced by Tyr, Met, Trp or Leu Modified bovine α-lactoal according to any one of claims 4 to 7 Bumin. 9. A modified human α-lactalbumin, claim 1, 2 or Modified α-lactalbumin according to item 3. 10.3 Phe is Tyr, Leu, Arg, Met or Trp 10. The modified human α-lactoalbummy according to claim 9 substituted with N. 11.31 Phe is replaced by Leu, Ile, Trp or Tyr, Modified human α-lactalbumin according to claim 9 or 10. 12.53 Phe is replaced by Tyr, Leu, Met or Trp, Modified human α-lactoalbu according to claim 9, 10 or 11. Min. 13. Phe of No. 80 is replaced by Tyr, Met, Trp or Leu, Modified human α-lacto according to any one of claims 9 to 12. albumin. 14． Connects consecutive amino acids and / or oligos and / or Any of claims 1 to 13 which comprises linking a re-peptide. A method for preparing the modified α-lactalbumin according to 1. 15. Α-lactalbumin containing no phenylalanine residue or wild-type Coated modified α-lactalbumin containing less phenylalanine residues An isolated or recombinant nucleic acid to be converted into a nucleic acid. 16. The modified α-lacto according to any one of claims 1 to 13. An isolated or recombinant nucleic acid encoding albumin. 17． Recombinant D according to claim 15 or 16 having the form of a vector. NA. 18. Linking consecutive nucleotides and / or oligo- and / or The method according to claim 15 or 16, which comprises linking poly-nucleotides. A method for preparing the described nucleic acid. 19. A recombinant DNA according to claim 15, 16 or 17 Host. 20. Has a DNA encoding a germline-modified α-lactalbumin A non-human mammal having a placenta -Alpha-lactoal modified in the mammary gland so that lactalbumin accumulates in milk The host according to claim 19, which is capable of expressing bumin. 21. A cow (or bull), sheep, goat, camel or pig 21. A host mammal according to claim 20. 22. Expression of the host wild-type α-lactalbumin gene is inhibited or suppressed 22. The host according to claim 19, 20 or 21. 23. Α-lactalbumin containing no phenylalanine residue or wild-type At least one modified α-lacto containing less phenylalanine residues A food product consisting of albumin used in patients with hyperphenylalanineemia. 24. Modified α-lactalbumin or modified α-lactoalbumy At least one of the claims is defined in any of claims 2 to 13. 24. A food product according to claim 23, which is derived. 25. A curative supplement used for infants or children with hyperphenylalaninemia or 25. A food product according to claim 23 or 24 which is a formulation. 26. The milk of the female mammal according to claim 19, 20 or 21, 25. The food product according to claim 23 or 24. 27. Α-lactalbumin containing no phenylalanine residue or wild-type Contains modified α-lactalbumin containing fewer phenylalanine residues Administering one or more food products to a patient with hyperphenylalanineemia A method for supplying a patient with hyperphenylalanineemia, which comprises: 28. The food product according to any one of claims 23 to 26 28. The method of claim 27, wherein