WO1995018224A1 - Recombinant production of modified proteins lacking certain amino acids - Google Patents
Recombinant production of modified proteins lacking certain amino acids Download PDFInfo
- Publication number
- WO1995018224A1 WO1995018224A1 PCT/EP1994/004343 EP9404343W WO9518224A1 WO 1995018224 A1 WO1995018224 A1 WO 1995018224A1 EP 9404343 W EP9404343 W EP 9404343W WO 9518224 A1 WO9518224 A1 WO 9518224A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- phe
- polypeptide
- expression cassette
- recombinant expression
- sequence
- Prior art date
Links
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title abstract description 10
- 102000035118 modified proteins Human genes 0.000 title description 8
- 108091005573 modified proteins Proteins 0.000 title description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 116
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 77
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 64
- 235000018102 proteins Nutrition 0.000 claims abstract description 62
- 238000000034 method Methods 0.000 claims abstract description 55
- 230000009261 transgenic effect Effects 0.000 claims abstract description 44
- 239000000203 mixture Substances 0.000 claims abstract description 21
- 108090000942 Lactalbumin Proteins 0.000 claims abstract description 19
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims abstract description 19
- 102000004407 Lactalbumin Human genes 0.000 claims abstract description 18
- 235000021241 α-lactalbumin Nutrition 0.000 claims abstract description 17
- 201000011252 Phenylketonuria Diseases 0.000 claims abstract description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 8
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 83
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 83
- 229920001184 polypeptide Polymers 0.000 claims description 82
- 210000004027 cell Anatomy 0.000 claims description 55
- 108091033319 polynucleotide Proteins 0.000 claims description 38
- 102000040430 polynucleotide Human genes 0.000 claims description 38
- 239000002157 polynucleotide Substances 0.000 claims description 38
- 241000283690 Bos taurus Species 0.000 claims description 28
- 230000001105 regulatory effect Effects 0.000 claims description 27
- 238000003259 recombinant expression Methods 0.000 claims description 26
- 230000003248 secreting effect Effects 0.000 claims description 22
- 241000124008 Mammalia Species 0.000 claims description 19
- 235000013336 milk Nutrition 0.000 claims description 17
- 239000008267 milk Substances 0.000 claims description 17
- 210000004080 milk Anatomy 0.000 claims description 17
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 16
- 108020004705 Codon Proteins 0.000 claims description 15
- 108010011756 Milk Proteins Proteins 0.000 claims description 15
- 102000014171 Milk Proteins Human genes 0.000 claims description 14
- 235000021239 milk protein Nutrition 0.000 claims description 14
- 239000005018 casein Substances 0.000 claims description 11
- 238000009472 formulation Methods 0.000 claims description 11
- 235000021244 human milk protein Nutrition 0.000 claims description 9
- 235000013305 food Nutrition 0.000 claims description 8
- 210000002955 secretory cell Anatomy 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 235000021240 caseins Nutrition 0.000 claims description 7
- 210000004962 mammalian cell Anatomy 0.000 claims description 6
- 235000005911 diet Nutrition 0.000 claims description 5
- 101000946384 Homo sapiens Alpha-lactalbumin Proteins 0.000 claims description 4
- 108050001786 Alpha-s2 casein Proteins 0.000 claims description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 3
- 230000037213 diet Effects 0.000 claims description 3
- 239000004474 valine Substances 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims description 3
- 102000008192 Lactoglobulins Human genes 0.000 claims description 2
- 108010060630 Lactoglobulins Proteins 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 23
- 235000005550 amino acid supplement Nutrition 0.000 abstract description 5
- 108700019146 Transgenes Proteins 0.000 description 34
- 235000001014 amino acid Nutrition 0.000 description 27
- 229940024606 amino acid Drugs 0.000 description 27
- 230000033228 biological regulation Effects 0.000 description 19
- 235000008729 phenylalanine Nutrition 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 description 16
- 239000013598 vector Substances 0.000 description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 12
- 241000894007 species Species 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 108091026890 Coding region Proteins 0.000 description 11
- 108020004511 Recombinant DNA Proteins 0.000 description 11
- 238000010561 standard procedure Methods 0.000 description 11
- 230000028327 secretion Effects 0.000 description 10
- 210000002459 blastocyst Anatomy 0.000 description 9
- 239000003623 enhancer Substances 0.000 description 9
- 238000011144 upstream manufacturing Methods 0.000 description 9
- 102000011632 Caseins Human genes 0.000 description 8
- 108010076119 Caseins Proteins 0.000 description 8
- 108091092195 Intron Proteins 0.000 description 8
- 241001529936 Murinae Species 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 210000002257 embryonic structure Anatomy 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 210000001161 mammalian embryo Anatomy 0.000 description 7
- 150000007523 nucleic acids Chemical group 0.000 description 7
- 210000000287 oocyte Anatomy 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000233866 Fungi Species 0.000 description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 210000004602 germ cell Anatomy 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 208000029751 Amino acid metabolism disease Diseases 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- -1 aromatic amino acids Chemical class 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 235000013365 dairy product Nutrition 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 230000000050 nutritive effect Effects 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 241000206602 Eukaryota Species 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 108010069013 Phenylalanine Hydroxylase Proteins 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 235000015872 dietary supplement Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003754 fetus Anatomy 0.000 description 3
- 238000002509 fluorescent in situ hybridization Methods 0.000 description 3
- 235000020256 human milk Nutrition 0.000 description 3
- 210000004251 human milk Anatomy 0.000 description 3
- 238000002513 implantation Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 210000001938 protoplast Anatomy 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 102000015781 Dietary Proteins Human genes 0.000 description 2
- 108010010256 Dietary Proteins Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- 102000010445 Lactoferrin Human genes 0.000 description 2
- 108010063045 Lactoferrin Proteins 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 102000003792 Metallothionein Human genes 0.000 description 2
- 108090000157 Metallothionein Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 239000005862 Whey Substances 0.000 description 2
- 102000007544 Whey Proteins Human genes 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000000378 dietary effect Effects 0.000 description 2
- 235000021245 dietary protein Nutrition 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 239000003701 inert diluent Substances 0.000 description 2
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 2
- 235000021242 lactoferrin Nutrition 0.000 description 2
- 229940078795 lactoferrin Drugs 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 210000000472 morula Anatomy 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 150000002994 phenylalanines Chemical class 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000007909 solid dosage form Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N Aspartic acid Chemical compound OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 101100228206 Caenorhabditis elegans gly-6 gene Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 101000766307 Gallus gallus Ovotransferrin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 101000801481 Homo sapiens Tissue-type plasminogen activator Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102100033468 Lysozyme C Human genes 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- 235000006679 Mentha X verticillata Nutrition 0.000 description 1
- 235000002899 Mentha suaveolens Nutrition 0.000 description 1
- 235000001636 Mentha x rotundifolia Nutrition 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 101100426589 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) trp-3 gene Proteins 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 238000002669 amniocentesis Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004952 blastocoel Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940021722 caseins Drugs 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000009025 developmental regulation Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 235000020243 first infant milk formula Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005428 food component Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021472 generally recognized as safe Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010087005 glusulase Proteins 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 102000050459 human LTF Human genes 0.000 description 1
- 102000047823 human PLAT Human genes 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 208000008245 hypermethioninemia Diseases 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000015978 inherited metabolic disease Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 108010056929 lyticase Proteins 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000001216 nucleic acid method Methods 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 201000011296 tyrosinemia Diseases 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
- 108010082737 zymolyase Proteins 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/19—Dairy proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/101—Bovine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous proteins
Definitions
- the present invention relates to the recombinant pro d uction of proteins with desired properties. In particular, it relates to the production of proteins which lack specific amino acids.
- a number of amino acid metabolism disorders are known. Examples of such diseases include phenylketonuria (PK U) , tyrosinaemias, disorders of branched chain amino acid catabolism, disorders affecting sulfur-containing amino acids, an d t h e like.
- PK U phenylketonuria
- tyrosinaemias disorders of branched chain amino acid catabolism
- disorders affecting sulfur-containing amino acids an d t h e like.
- the most common amino acid metabolism disorder is PKU.
- the disease is usually caused by a deficiency of the enzyme phenylalanine hydroxylase and results in mental subnormality.
- Phenylalanine hydroxylase converts phenylalanine into tyrosine and is responsible for the first step in the catabolic pathway of phenylalanine.
- Patients with partial deficiencies have also been described.
- the disease resulting from partial reduction of phenylalanine hydroxylase is calle d hyperphenylalaninemia. Mental retardation in this group is often less severe.
- phenylalanine levels are near normal at the time of birth, but rise rapidly when the infant receives feeding.
- phenylalanine is entirely of dietary origin. Treatment of patients is, therefore, possible by strictly regulating phenylalanine intake. As a consequence, the amount of protein these patients may consume is limited and they require amino acid-supplements for normal growth. Dietary treatment is usually required to the age of 10-12 years.
- phenylketonuric women require a low phenylalanine diet throughout pregnancy to protect the fetus. It is currently a matter of debate whether continued treatment in older patients is advisable.
- the amino acid supplements typically contain certain amounts (depending on the age of the patient) of vitamins and trace elements and provide up to 80% of the total protein requirement of patients.
- These dietary supplements are often derived from hydrolysates of natural protein from which aromatic amino acids are removed by charcoal treatment. In order to maintain the full complement of amino acids, tyrosine and tryptophan must be added back to the hydrolysates. These products, however, suffer from a very unpleasant taste. More recent products are composed of mixed purified amino acids. While their patient acceptance is higher than for the hydrolysate products, the peculiar taste of these products is still a significant problem.
- a protein (or mixture of proteins) lacking a specific amino acid such as phenylalanine would be the best form of giving amino acid supplements for the treatment of amino acid metabolism disorders.
- phenylalanine there are no known proteins with a high nutritional value that do not contain phenylalanine. The present invention addresses these and other needs.
- the present invention provides recombinant polypeptides substantially lacking certain amino acids which can be used for the treatment of various amino acid metabolism disorders.
- Exemplary polypeptides are those lacking phenylalanine (Phe ⁇ polypeptides) .
- the invention provides recombinant expression cassettes comprising a regulatory sequences operably linked to a polynucleotide sequence encoding a Phe " polypeptide.
- the Phe " polypeptide is usually a human milk protein which substantially lacks phenylalanine, such as Phe " ⁇ -lactalbumin.
- ⁇ -lactalbumin the phenylalanine codons in the polynucleotide are typically replaced by valine codons, to improve the nutritional value of the polypeptide.
- the expression cassette will typically comprise a regulatory sequences which regulate expression of the Phe" polypeptide i " h a mammalian cell although sequences functional in prokaryotes, yeast and fungi may also be used.
- the expression cassette may further comprise a polynucleotide sequence encoding a secretory signal sequence which is functional in mammary secretory cells. Regulatory sequences from human or bovine milk proteins are preferred. Sequences from human ⁇ -lactalbumin is particularly preferred.
- transgenic nonhuman mammals comprising a recombinant expression cassette comprising a regulatory sequences operably linked to a polynucleotide sequence encoding a Phe " polypeptide, such as Phe " ⁇ - lactalbumin.
- the mammal is preferably a bovine which secretes the polypeptide in milk.
- the invention further provides food formulations comprising the recombinant Phe " polypeptides of the invention. Also provided are methods of supplementing the diet of a phenylketonuria patient. The methods comprise administering to the patient the food formulations of the invention.
- modified polypeptides are recombinantly produced proteins which substantially lack a preselected amino acid and are encoded by polynucleotide sequences in which substantially all of the codons encoding the amino acid have been mutated to encode a different amino acid.
- “Tyr " polypeptides” substantially lack tyrosine and are useful for the treatment of tyrosinae ias
- “Mefpolypeptides” substantially lack methionine for the treatment of hypermethioninaemia
- “Phe ⁇ polypeptides” substantially lack phenylalanine and are useful for the treatment of " PKU.
- substantially lacks refers to proteins encoded by polynucleotide sequences in which all or substantially all target codons are mutated.
- the term also specifically encompasses proteins in which a small number of the target codons are retained, so long as the desired amino acid accounts for less than 1 mol %, preferably less than about 0.5 mol%, and more preferably less than about 0.25 mol % of the protein. Typically, the desired amino acid will not be present in the modified polypeptide.
- human milk proteins are preferably used. Such proteins have a nutritionally optimal amino acid composition and ensure optimal growth and development.
- the polynucleotide sequences which encode the polypeptides of the invention are derived from genes which occur naturally in living organisms. The proteins are thus mutated forms of naturally-occurring functional proteins isolated from living organisms.
- the recombinant proteins of the invention may or may not retain all the functions of the naturally occurring protein.
- the proteins of the invention may also comprise other modifications such as substitutions, deletions and insertions as described below.
- the proteins of the invention also include recombinant proteins that lack a particular amino acid (e.g., phenylalanine) and are substantially identical (as defined below) to those specifically disclosed here.
- a "polynucleotide sequence encoding" a modified polypeptide of the invention is a subsequence or full length polynucleotide sequence which, when present in a cell, expresses a modified protein of the invention such as a Phe" polypeptide.
- a modified protein of the invention such as a Phe
- the inserted polynucleotide sequence need not be identical and may be “substantially identical" to a sequence of the gene from which it was derived. As explained below, these variants are specifically covered by this term.
- proteins and polypeptides of the present invention can consist of a full length protein (e.g., ⁇ - lactalbumin) , or a fragment thereof.
- nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
- complementary to is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence. Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a "comparison window" to identify and compare local regions of sequence similarity.
- a “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Optimal alignment of sequences for comparison may be conducted by the local ho ology algorithm of Smith and Waterman Adv. Appl . Math . 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol . Biol . 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl . Acad. Sci . (U.S.A.
- Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) " as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
- substantially identity of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using the programs described above (e.g., GENEWORKS) using standard parameters.
- amino acid sequences for these purposes normally means sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
- stringent conditions are sequence dependent and will be different in different circumstances.
- stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
- Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60°C.
- proteins of the invention include proteins immunologically reactive with antibodies raised against polypeptides.
- operably linked refers to a linkage of polynucleotide elements in a functional relationship.
- a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
- a promoter or enhancer is operably linked to a coding sequence if it affects or regulates the transcription of the coding sequence.
- Operably linked DNA sequences are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
- a structural gene (e.g., a gene encoding a protein which lacks phenylalanine) which is operably linked to regulatory sequences from a gene encoding a milk protein is generally expressed in substantially the same temporal and cell type-specific pattern as is the naturally-occurring milk protein gene.
- a “recombinant expression cassette” is a polynucleotide sequence containing a coding sequence which is capable of affecting expression of the coding sequence in hosts compatible with the sequence. Such cassettes include the coding sequence and regulatory sequences such as promoters transcription termination signals as well as other sequences (e.g. enhancers) necessary or helpful in affecting expression.
- a "secretory signal sequence” is defined as any polynucleotide sequence which when operably linked to a coding sequence encodes a signal peptide which is capable of causing the secretion of the recombinant polypeptide encoded by the coding sequence.
- An example of a secretory signal sequence is a sequence which allows secretion by mammary gland cells.
- “Operably linked” in the context of linking a secretory signal sequence to a coding sequence means that the secretory sequence is linked to the coding sequence so that the resultant secretory-recombinant DNA sequence encodes 5' to 3' for the secretory signal sequence and recombinant Phe " polypeptide. Accordingly, the reading frame for the secretory sequence and the recombinant DNA sequence must be covalently combined such that an open reading frame exists from the 5* end of the mRNA sequence formed after transcription and processing of the primary RNA transcript.
- naturally-occurring as used herein as applied to a compound refers to the fact that the compound can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) and that can be isolated from a source in nature is naturally-occurring.
- This invention provides compositions and methods for producing recombinant proteins that lack certain amino acids such as phenylalanine (Phe ⁇ proteins) .
- the methods involve construction of recombinant expression vectors in which the desired codons are replaced with codons for other amino acids in a gene encoding the desired protein.
- the mutated gene is subsequently expressed in a host cell or transgenic animal and the protein is purified.
- Phe proteins they can serve as amino acid supplements with largely improved taste characteristics for PKU-patients.
- Polynucleotide sequences encoding any number of proteins of interest can be used in the present invention.
- the particular protein used to produce a mutant of the invention is not a critical aspect of the invention, although human milk proteins are generally preferred because of their nutritional value.
- the nucleic acids used to make the constructs of the invention may be isolated from natural sources or may be synthesized in vitro.
- oligonucleotides used in the methods of the invention can be chemically synthesized using for instance an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.
- the proteins of the invention are used as dietary supplements, proteins with high nutritional value are typically preferred.
- the recombinant protein is expressed in the milk of transgenic mammals, such as cows, sheep, goats, mice and the like.
- a protein normally expressed in milk is particularly preferred.
- milk proteins that can be engineered in this way include human milk proteins such as lactoferrin, lysozyme, secreted immunoglobulins, lactalbumin, caseins and the like.
- milk proteins that can be engineered in this way include human milk proteins such as lactoferrin, lysozyme, secreted immunoglobulins, lactalbumin, caseins and the like.
- other proteins that may be used in the present invention include serum albumin, transferrin and ovalbumin.
- ⁇ -lactalbumin ( ⁇ -Lac) .
- This protein is the most important nutritive protein of human milk.
- Table I shows the amino acid composition of human ⁇ -Lac as compared with the amino acid composition of mature human milk.
- Table I Comparison of amino acid composition of human ⁇ - lactalbumin and mature human milk. * A spartic acid and asparagine combined, ** Glutamic acid and glutamine combined.
- nucleotide sequences used for production of recombinant Phe " polypeptides or other mutants can be modified according to standard techniques to yield desired polypeptides, with a variety of desired properties, in addition to lacking particular amino acid residues.
- the modified polypeptides of the invention can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art.
- the polypeptides can vary from the naturally-occurring sequence at the primary structure level by amino acid insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
- the amino acid sequence variants can be prepared with various objectives in mind, including increasing nutritive value or facilitating purification and preparation of the recombinant polypeptides.
- the amino acid sequence variants are usually predetermined variants not found in nature but exhibit the many of the same properties (e.g., immunogenic activity, nutritive value, and the like) as the naturally occurring polypeptides. For instance, polypeptide fragments comprising only a portion (usually at least about 60-80%, typically 90-95%) of the primary structure may be produced.
- modifications of the sequences encoding the polypeptides of the invention may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Kunkel, Proc . Natl . Acad. Sci . USA 82:488- 492 (1985); Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al . , Nature 328:731-734 (1987)).
- site-directed mutagenesis see, Kunkel, Proc . Natl . Acad. Sci . USA 82:488- 492 (1985); Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al . , Nature 328:731-734 (1987)
- Kunkel Proc . Natl . Acad. Sci . USA 82:488- 492 (1985)
- Gillman and Smith Gene 8:81-97 (1979)
- Roberts S. et al . , Nature
- the proteins of the invention can be produced in prokaryotic or eukaryotic host cells by expression of the appropriate polynucleotides.
- the cloned DNA sequences are expressed in hosts after the sequences have been operably linked to an expression control sequence in an expression vector.
- Expression vectors are typically replicable in the host organisms either as episo es or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see, e . g. , U.S. Patent 4,704,362).
- nucleic acids encoding e.g., Phe " proteins
- Phe proteins
- a promoter which is either constitutive or inducible
- the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
- Typical expression cassettes will also contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the polynucleotide sequence encoding Phe " polypeptides.
- transcription and translation terminators useful for regulation of the expression of the polynucleotide sequence encoding Phe " polypeptides.
- promoters useful for regulation of the expression of the polynucleotide sequence encoding Phe " polypeptides.
- microbial strains such as E. coli , bacilli, for example Bacillus subtilis , various species of Pseudomonas, or other bacterial strains.
- Preferred strains are generally recognized as safe strains (GRAS strains) such as Lactobacilli used in cheese production.
- Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA.
- the recombinant polypeptides produced by prokaryote cells may not necessarily fold properly.
- the expressed polypeptides may first be denatured and then renatured. This can be accomplished by solubilizing the bacterially produced proteins in a chaotropic agent such as guanidine HC1 and reducing all the cysteine residues with a reducing agent such as beta-mercaptoethanol.
- the polypeptides are then renatured, either by slow dialysis or by gel filtration.
- Detection of the expressed protein is achieved by methods known in the art as radioimmunoassays. Western blotting techniques or immunoprecipitation. Purification from E. coli can be achieved following procedures described in U.S. Patent No. 4,511,503.
- yeast strain that are currently used to produce food components (e.g., Kluyveromyces species) are preferred.
- yeast expression plasmids can be used as vectors.
- a gene of interest can be fused to any of the promoters in various yeast vectors.
- suitable vectors are described in the literature (Botstein, et al . , 1979, Gene, 8:17-24; Broach, et al., 1979, Gene, 8:121-133) . Two procedures are used in transforming yeast cells.
- yeast cells are first converted into protoplasts using zymolyase, lyticase or glusulase, followed by addition of DNA and polyethylene glycol (PEG) .
- PEG polyethylene glycol
- the PEG-treated protoplasts are then regenerated in a 3% agar medium under selective conditions. Details of this procedure are given in the papers by J.D. Beggs, 1978, Nature (London), 275:104-109; and Hinnen, A., et al . , 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933.
- the second procedure does not involve removal of the cell wall. Instead the cells are treated with lithium chloride or acetate and PEG and put on selective plates (Ito, H., et al . , 1983, J. Bact. , 153:163-168).
- polypeptides of the invention can be isolated from yeast and other fungi by lysing the cells or hyphae and applying standard protein isolation techniques to the lysates.
- the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassays of other standard immunoassay techniques.
- the polynucleotides of the invention can be ligated to various expression vectors for use in transforming host mammalian or insect cell cultures.
- the vectors preferably contain a marker to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or metallothionein.
- the vector e.g., a plasmid, which is used to transform the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the antigen gene sequence. These sequences are referred to as expression control sequences.
- expression control sequences When the host cell is of insect or mammalian origin illustrative expression control sequences are obtained from the SV-40 promoter (Science , 222:524-527, 1983), the CMV I.E. Promoter (Proc . Natl . Acad . Sci .
- the cloning vector containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable and ligated with polynucleotides coding for the Phe " polypeptides by means well known in the art.
- polyadenlyation or transcription terminator sequences from known mammalian genes need to be incorporated into the vector.
- An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
- An example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et al . , 1983, J. Virol. 45: 773-781).
- gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M. , 1985, "Bovine Papilloma virus DNA a
- the host cells are competent or rendered competent for transformation by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation and micro-injection of the DNA directly into the cells.
- the transformed cells are cultured by means well known in the art. Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., (1977) .
- the expressed polypeptides are isolated from cells grown as suspensions or as onolayers. The latter are recovered by well known mechanical, chemical or enzymatic means.
- polypeptides of the invention produced by recombinant DNA technology from cell culture may be purified by standard techniques well known to those of skill in the art.
- Recombinantly produced polypeptides can be directly expressed or expressed as a fusion protein.
- the protein is then purified by a combination of cell lysis (e.g., sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme release the desired polypeptide.
- polypeptides of this invention may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification : Principles and Practice , Springer-Verlag: New York (1982).
- the invention also encompasses methods and polynucleotide constructs which are employed for generating nonhuman transgenic animals which produce Phe " proteins.
- the constructs are used to produce transgenic nonhuman mammals, such as cows, sheep, goats, pigs, rabbits or mice.
- the coding sequence of interest is operably linked to expression regulatory sequences.
- the expression regulatory sequence is at least the minimal sequences required for efficient cell-type specific expression, which generally are at least a promoter and at least about 1 kilobase (kb) upstream of the promoter, preferably at least about 2 to 3 kb upstream of the promoter, more preferably at least about 5 kb upstream of the promoter. and frequently at least about 8 or more kb upstream of the promoter.
- sequences downstream of the promoter are included in the transgene constructs (Brinster et al. (1988) Proc . Natl . Acad . Sci . USA 85: 836.
- sequences upstream of the promoter are used contiguously, although various deletions and rearrangements can be employed.
- Some desired regulatory elements e.g., enhancers, silencers
- an enhancer may be located at a different distance from a promoter, in a different orientation, and/or in a different linear order.
- an enhancer that is located 3' to a promoter in germline configuration might be located 5' to the promoter in a transgene.
- the expression regulatory sequence(s) selected are relatively inefficient in transcribing the gene encoding the Phe " protein, it may be desirable to incorporate multiple copies of a transgene or targeting construct to compensate with an enhanced gene dosage of the transgene.
- expression regulation sequences are chosen to produce tissue-specific or cell type-specific expression of the recombinant or secretory-recombinant DNA.
- tissue or cell type is chosen for expression.
- expression regulation sequences are chosen.
- such expression regulation sequences are derived from genes that are expressed primarily in the tissue or cell type chosen.
- the genes from which these expression regulation sequences are obtained are expressed substantially only in the tissue or cell type chosen, although secondary expression in other tissue and/or cell types is acceptable if expression of the recombinant DNA in the transgene in such tissue or cell type is not detrimental to the transgenic animal.
- Particularly preferred expression regulation sequences are those endogenous to the species of animal to be manipulated.
- expression regulation sequences from other species such as those from human genes may also be used.
- the expression regulation sequences and the recombinant DNA sequences are from the same species, e.g., each from bovine species or from a human source.
- the expression regulation sequences and recombinant DNA sequences are obtained from different species, e.g., expression regulation sequence from bovine species and a recombinant DNA sequence from a human source) . In such cases, the expression regulation and recombinant DNA sequence are heterologous to each other.
- the upstream expression regulation sequence includes the transcribed portion of the endogenous gene upstream from the translation initiation sequence and those flanking upstream sequences which comprise a functional promoter.
- Such sequences typically include a TATA sequence or box located generally about 25 to 30 nucleotides from the transcription initiation site.
- the constructs may also comprise one or more enhancer and/or other sequences which facilitate expression of the coding sequence and as a consequence facilitate expression.
- the constructs will usually also comprise downstream expression regulation sequences to supplement tissue or cell- type specific expression.
- the downstream expression regulation sequences include polyadenylation sequences (either from the endogenous gene or from other sources such as SV40) and sequences that may affect RNA stability as well as enhancer and/or other sequences which enhance expression.
- sequences comprise about 2 kb, more preferably 8 kb and most preferably about 15 kb of 3• flanking sequence.
- the constructs of the invention preferably also comprise intervening sequences.
- intervening sequences can be derived, for example, from bovine ⁇ Sl casein and from human ⁇ Lac.
- Recombinant intervening sequences may comprise hybrid intervening sequences which comprise a 5• RNA splice signal and 3• RNA splice signal from intervening sequences from different sources.
- Hybrid intervening sequences are not limited to transgenes utilizing cDNA sequences but are also useful when the recombinant polypeptide is encoded by a genomic sequence.
- genomic DNA sequences express at higher levels than sequences derived from cDNA, it is expected that such hybrid intervening sequences used in conjunction with genomic recombinant DNA will further enhance expression levels above that which would otherwise be obtained with genomic sequence alone.
- a secretory signal sequence encoding a functional secretion signal peptide is also operably linked to the sequences encoding the Phe" polypeptide to direct secretion of the recombinant polypeptide from one or more cell types within the transgenic animal.
- the signal peptide generally is removed in vivo during secretion to produce an extracellular form of the recombinant Phe" polypeptide.
- Secretory DNA sequences in general are derived from genes encoding secreted proteins of the same species of the transgenic animal. Such secretory sequences are preferably derived from genes encoding polypeptides secreted from the cell type targeted for tissue-specific expression, e.g. secreted milk proteins for expression in and secretion from mammary secretory cells. Secretory DNA sequences, however, are not limited to such sequences and may be derived from proteins secreted from other cell types within the species of transgenic animal, e.g., the native signal sequence of a gene encoding a protein secreted other than in the mammary glands.
- Heterologous secretory sequences which encode signal secretion peptides from species other than the transgenic animals may also be used e.g., human t-PA, human serum albumin human lactoferrin and human ⁇ Lac and secretion signals from microbial genes encoding secreted polypeptides such as from yeast, filamentous fungi, and bacteria.
- a secretory DNA sequence encoding a secretory signal sequence functional in the mammary secretory cells of bovine species is used to cause secretion of recombinant polypeptide from bovine mammary secretory cells.
- the secretory signal sequence is operably linked to the recombinant DNA sequence.
- secretory sequences examples include DNA sequences encoding signal secretion sequences for bovine ⁇ Sl casein, murine lactoferrin and human transferrin.
- the preferred secretory signal sequence is that encoding the secretory sequence of human ⁇ Lac.
- a Phe" polypeptide or other modified protein of the invention is expressed predominantly in the mammary secretory cells of transgenic bovine species.
- tissue-specific expression is obtained by operably linking mammary specific expression regulation sequences to recombinant sequences comprising a secretory signal sequence and a polynucleotide encoding a Phe" protein.
- mammary specific regulation sequences can be derived from various bovine genes preferentially expressed in the mammary secretory cells of the species.
- mammary specific genes include ⁇ Sl casein; ⁇ S2-casein; ⁇ -casein; K-casein; ⁇ -lactalbumin; and /3-lactoglobulin.
- gene targeting mediated by homologous recombination between a targeting polynucleotide construct and a homologous chromosomal sequence, to replace an endogenous gene with the gene encoding, e .g. , a Phe " mutant of the gene.
- Methods and materials for preparing such constructs are known by those of skill in the art and are described in various references. See, e.g., Thomas et al. , Cell 51:503 (1987) and Capecchi, Science 244:1288 (1989).
- Homologous targeting constructs have at least one region having a sequence that substantially corresponds to, or is substantially complementary to, a predetermined endogenous target gene sequence (e.g., an exon sequence, an enhancer, a promoter, an intronic sequence, or a flanking sequence of the target gene) .
- a homology region serves as a template for homologous pairing and recombination with substantially identical endogenous gene sequence(s) .
- such homology regions typically flank the replacement region, which is a region of the targeting transgene that is to undergo replacement with the targeted endogenous gene sequence.
- a segment of the targeting transgene flanked by homology regions can replace a segment of the endogenous gene sequence by double crossover homologous recombination.
- embryonic stem (ES) cells or fertilized oocytes can be used as the transgene recipients.
- this technology involves the insertion of the desired transgene construct into a pluripotent cell line (e.g., bovine, porcine, ovine, or murine cell) that is capable of differentiating into germ cell tissue.
- a pluripotent cell line e.g., bovine, porcine, ovine, or murine cell
- Methods of introducing transgenes into embryonal target cells include microinjection of the transgene into the pronuclei of fertilized oocytes or nuclei of ES cells of the non-human animal. Such methods are well known to those skilled in the art.
- the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc . Natl . Acad . Sci . USA 73:1260-1264) .
- the preferred method is microinjection of the fertilized bovine oocyte.
- the fertilized oocytes are first microinjected by standard techniques. They are thereafter cultured in vitro until a "pre-implantation embryo" is obtained.
- pre-implantation embryos preferably contain approximately 16 to 150 cells.
- the 16 to 32 cell stage of an embryo is commonly referred to as a morula.
- blastpcysts Those pre-implantation embryos containing more than 32 cells. They are generally characterized as demonstrating the development of a blastocoel cavity typically at the 64 cell stage.
- Methods for culturing fertilized oocytes to the pre-implantation stage include those described by Gordon, et al.
- one aspect of the invention methods are provided for identifying embryos wherein transgenesis has occurred and which permit implantation of transgenic embryos to form transgenic animals.
- one or more cells are removed from the pre-implantation embryo.
- the embryo is preferably not cultivated past the morula stage (32 cells) . Division of the pre-implantation embryo (reviewed by Williams et al.
- Theriogenology 22:521-531) results in two "hemi-embryos" (hemi-morula or hemi-blastocyst) one of which is capable of subsequent development after implantation into the appropriate female to develop in utero to term.
- hemi-embryos hemi-morula or hemi-blastocyst
- equal division of the pre- implantation embryo is preferred, it is to be understood that such an embryo may be unequally divided either intentionally or unintentionally into two hemi-embryos which are not necessarily of equal cell number. Essentially, all that is required is that one of the embryos which is not analyzed as hereinafter described be of sufficient cell number to develop to full term in utero .
- the hemi- embryo which is not analyzed as described herein, if shown to be transgenic is used to generate a clonal population of transgenic non-human animals.
- a preferred method for detecting transgenesis at this early stage in the embryo's using an endonuclease that recognizes a restriction site only when one of the bases in the site is methylated This method if described in detail in WO/08216, supra .
- biopsies are taken from 16-32 cell embryos and analyzed using standard techniques for detecting the presence or absence of a target sequence.
- Fluorescent In Situ Hybridization FISH
- FISH Fluorescent In Situ Hybridization
- Several guides to FISH techniques are available, e.g.. Gall et al . Meth . Enzymol . , 21:470-480 (1981) and Angerer et al . in Genetic Engineering: Principles and Methods Setlow and Hollaender, Eds. Vol 7, pgs 43-65 (plenum Press, New York 1985).
- sequences can also be detected by PCR using primers and probes specific for the transgene.
- Standard PCR methods useful in the present invention are described in PCR Protocols : Guide to Methods and Applications (Innis et al., eds.. Academic Press, San Diego).
- transgenes of the invention are practically limited to linearized DNA having a length not greater than about 50 kb.
- the transgenes of the invention may be readily generated by introducing two or more overlapping fragments of the desired transgene into an embryonal target cell. When so introduced, the overlapping fragments undergo homologous recombination which results in integration of the fully reconstituted transgene in the genome of the target cell. In general, it is preferred that such overlapping transgene fragments have 100% homology in those regions which overlap. However, lower sequence homology may be tolerated provided efficient homologous recombination occurs.
- non-homology does exist between the homologous sequence portions, it is preferred that the non-homology not be spread throughout the homologous sequence portion but rather be located in discrete areas. Although as few as 14 base pairs at 100% homology are sufficient for homologous recombination in mammalian cells (Rubnitz, J. and Subramani, S. (1984) Mol . Cell . Biol . 4:2253-2258), longer homologous sequence portions are preferred, e.g. 500bp, more preferably lOOObp, next most preferably 2000bp and most preferably greater than 2000bp for each homologous sequence portion.
- C. Isolation of Modified Proteins Methods for isolating the recombinant polypeptides are well known.
- the polypeptides are purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice , supra .
- the transgenic milk so obtained may be either used as is or further treated to purify the recombinant polypeptide. This depends, in part, on the recombinant polypeptide contained in the transgenic milk and the ultimate use for that protein. For example, a protocol suitable for the purification of ⁇ Lac is described below.
- the recombinant polypeptides contained in the transgenic milk may also be used in food formulations which will typically be the major source of dietary protein for the patient.
- a number of formulations can be used as described for instance in Milupa Special Products : For the treatment of inherited disorders of amino acid metabolism (Milupa AG, March 1988) .
- a typical food formulation will comprise a Phe " polypeptide of the invention, vitamins, minerals, trace elements, sucrose and water.
- the formulation may be in the form of syrup, emulsion or suspension, to which a sweetening agent such as sucrose, fructose, glucose, mannitol, sorbitol, and/or flavoring agents such as cherry, fruit, orange, mint, may be added.
- Solid dosage forms for oral administration may also be used, such as capsules, tablets, pills, powders, and granules.
- the polypeptides may be admixed with at least one inert diluent such as sucrose lactose or starch.
- Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
- the dosage forms may also comprise buffering agents.
- the total daily amount of the food formulations will depend upon the body weight and age as well as on the individual tolerance to the particular amino acid of interest. Generally, the formulations are designed to provide total dietary protein and will thus be consumed at least once a day.
- transgene construction and generation of transgenic mice Generation of transgenic mice expressing human ⁇ Lac generally follows the methods described by Vilotte et al. 1989, Eur. J. Biochem. 186:43 and Soulier et al. (FEBS Lett. 1992, 297: 13). Using these mice, the tissue-specificity, developmental regulation and levels of transgene expression is analyzed in offspring from genetic founders. The effects of transgene expression on murine milk are also evaluated.
- transgenes are constructed in which the ⁇ Lac gene is fused to regulatory sequences of the bovine ⁇ Sl-casein gene. These sequences are also capable of driving expression of the introduced gene at a high level.
- Milk is analyzed for levels of Phe " ⁇ Lac by a specific immunoassay using commercially available antibodies for human ⁇ Lac (e.g., polyclonal rabbit antihuman ⁇ Lac available from Sigma) .
- the Phe " ⁇ Lac is purified from the other milk proteins.
- the first step of this protocol is to separate the whey from the casein fraction.
- the Phe " ⁇ Lac will appear in the whey fraction (based on its similarity to natural ⁇ Lac) .
- the purification protocol is optimized empirically using standard techniques. For instance, chromatographic matrices that selectively interact with aromatic amino acid residues can be used, since the amount of the aromatic amino acids in the Phe " protein is 40% less than in bovine ⁇ Lac.
- Another chromatographic separation step e.g., size exclusion
- to remove other abundant whey proteins such as betalactoglobulin and albumin may also be used.
- Immature oocytes are retrieved in large numbers from slaughterhouse ovaries or from live animals using echoscopic procedures. Oocytes obtained in either way are matured and fertilized with sperm from selected elite dairy bulls. Resulting zygotes are microinjected and subsequently cultured to the late orula/early blastocyst stage. At that stage selected embryos are transferred to recipient cattle for the balance of gestation, as described above. If necessary, early detection of transgenesis can be achieved using the methods described above. Recipients carrying putative transgenic fetuses are kept on a farm equipped to generate and tend transgenic dairy cattle. During pregnancy, amniocentesis samples may be taken to determine which animals carry transgenic fetuses. After birth the presence of an integrated transgene is confirmed by analysis of DNA purified from placenta, blood and skin tissue.
- cows do not lactate until they have calved. Calving does not typically occur until 20 months, at the earliest. Using a hormonal treatment, however, female calves can be induced to lactate as early as 6 months after birth. The milk produced by these animals fully resembles mature milk (McFadden et al., 1989, J " . Dairy Sci . , 72, 1754- 1763) and is used for initial characterization of the transgenic protein.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Nutrition Science (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
This invention provides compositions and methods for producing recombinant proteins that lack certain amino acids such as phenylalanine (Phe- proteins). The proteins are expressed in a host cell or transgenic animal and serve as an amino acid supplement with largely improved taste characteristics for, e.g., PKU-patients. The production of Phe- αlactalbumin is exemplified
Description
RECOMBINANT PRODUCTION OF MODIFIED PROTEINS LACKING CERTAIN AMINO ACIDS
BACKGROUND OF THE INVENTION The present invention relates to the recombinant production of proteins with desired properties. In particular, it relates to the production of proteins which lack specific amino acids.
A number of amino acid metabolism disorders are known. Examples of such diseases include phenylketonuria (PKU) , tyrosinaemias, disorders of branched chain amino acid catabolism, disorders affecting sulfur-containing amino acids, and the like. For a review of such diseases, see, Pollitt "Amino Acid Disorders" in The Inherited Metabolic Diseases Holton, J.B. , ed. (Churchill-Livingstone, 1987) .
The most common amino acid metabolism disorder is PKU. The disease is usually caused by a deficiency of the enzyme phenylalanine hydroxylase and results in mental subnormality. Phenylalanine hydroxylase converts phenylalanine into tyrosine and is responsible for the first step in the catabolic pathway of phenylalanine. Patients with partial deficiencies have also been described. The disease resulting from partial reduction of phenylalanine hydroxylase is called hyperphenylalaninemia. Mental retardation in this group is often less severe.
In affected individuals, phenylalanine levels are near normal at the time of birth, but rise rapidly when the infant receives feeding. In humans, phenylalanine is entirely of dietary origin. Treatment of patients is, therefore, possible by strictly regulating phenylalanine intake. As a consequence, the amount of protein these patients may consume is limited and they require amino acid-supplements for normal growth. Dietary treatment is usually required to the age of 10-12 years. In addition, phenylketonuric women require a low phenylalanine diet throughout pregnancy to protect the fetus.
It is currently a matter of debate whether continued treatment in older patients is advisable.
The amino acid supplements typically contain certain amounts (depending on the age of the patient) of vitamins and trace elements and provide up to 80% of the total protein requirement of patients. These dietary supplements are often derived from hydrolysates of natural protein from which aromatic amino acids are removed by charcoal treatment. In order to maintain the full complement of amino acids, tyrosine and tryptophan must be added back to the hydrolysates. These products, however, suffer from a very unpleasant taste. More recent products are composed of mixed purified amino acids. While their patient acceptance is higher than for the hydrolysate products, the peculiar taste of these products is still a significant problem.
Ideally, a protein (or mixture of proteins) lacking a specific amino acid such as phenylalanine would be the best form of giving amino acid supplements for the treatment of amino acid metabolism disorders. In the case of PKU, there are no known proteins with a high nutritional value that do not contain phenylalanine. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION The present invention provides recombinant polypeptides substantially lacking certain amino acids which can be used for the treatment of various amino acid metabolism disorders. Exemplary polypeptides are those lacking phenylalanine (Phe~ polypeptides) .
The invention provides recombinant expression cassettes comprising a regulatory sequences operably linked to a polynucleotide sequence encoding a Phe" polypeptide. The Phe" polypeptide is usually a human milk protein which substantially lacks phenylalanine, such as Phe" α-lactalbumin. In the case of α-lactalbumin, the phenylalanine codons in the polynucleotide are typically replaced by valine codons, to improve the nutritional value of the polypeptide.
The expression cassette will typically comprise a regulatory sequences which regulate expression of the Phe"
polypeptide i"h a mammalian cell although sequences functional in prokaryotes, yeast and fungi may also be used. The expression cassette may further comprise a polynucleotide sequence encoding a secretory signal sequence which is functional in mammary secretory cells. Regulatory sequences from human or bovine milk proteins are preferred. Sequences from human α-lactalbumin is particularly preferred.
Also provided are transgenic nonhuman mammals comprising a recombinant expression cassette comprising a regulatory sequences operably linked to a polynucleotide sequence encoding a Phe" polypeptide, such as Phe" α- lactalbumin. The mammal is preferably a bovine which secretes the polypeptide in milk.
The invention further provides food formulations comprising the recombinant Phe" polypeptides of the invention. Also provided are methods of supplementing the diet of a phenylketonuria patient. The methods comprise administering to the patient the food formulations of the invention.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below. The "modified polypeptides" of the invention are recombinantly produced proteins which substantially lack a preselected amino acid and are encoded by polynucleotide sequences in which substantially all of the codons encoding the amino acid have been mutated to encode a different amino acid. For example, "Tyr"polypeptides" substantially lack tyrosine and are useful for the treatment of tyrosinae ias, "Mefpolypeptides" substantially lack methionine for the treatment of hypermethioninaemia and "Phe~polypeptides" substantially lack phenylalanine and are useful for the
treatment of "PKU. The term "substantially lacks" refers to proteins encoded by polynucleotide sequences in which all or substantially all target codons are mutated. The term also specifically encompasses proteins in which a small number of the target codons are retained, so long as the desired amino acid accounts for less than 1 mol %, preferably less than about 0.5 mol%, and more preferably less than about 0.25 mol % of the protein. Typically, the desired amino acid will not be present in the modified polypeptide. Where the proteins of the invention are used as dietary supplements for humans, human milk proteins are preferably used. Such proteins have a nutritionally optimal amino acid composition and ensure optimal growth and development. The polynucleotide sequences which encode the polypeptides of the invention are derived from genes which occur naturally in living organisms. The proteins are thus mutated forms of naturally-occurring functional proteins isolated from living organisms. The recombinant proteins of the invention may or may not retain all the functions of the naturally occurring protein. The proteins of the invention may also comprise other modifications such as substitutions, deletions and insertions as described below. The proteins of the invention also include recombinant proteins that lack a particular amino acid (e.g., phenylalanine) and are substantially identical (as defined below) to those specifically disclosed here.
A "polynucleotide sequence encoding" a modified polypeptide of the invention is a subsequence or full length polynucleotide sequence which, when present in a cell, expresses a modified protein of the invention such as a Phe" polypeptide. In the expression of transgenes one of skill will recognize that the inserted polynucleotide sequence need not be identical and may be "substantially identical" to a sequence of the gene from which it was derived. As explained below, these variants are specifically covered by this term.
In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional
polypeptide, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically covered by the above term. The proteins and polypeptides of the present invention can consist of a full length protein (e.g., α- lactalbumin) , or a fragment thereof.
Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term "complementary to" is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence. Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted by the local ho ology algorithm of Smith and Waterman Adv. Appl . Math . 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol . Biol . 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl . Acad. Sci . (U.S.A. ) 85: 2444 (1988), by computerized implementations of these algorithms. Preferred programs include the FASTA program issued by the University of Wisconsin and GENEWORKS issued by Intelligenetics Inc. using standard parameters. "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions
(i.e., gaps) "as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using the programs described above (e.g., GENEWORKS) using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60°C.
Another indication that protein sequences are substantially identical is if one protein is immunologically reactive with antibodies raised against the other protein.
Thus, the proteins of the invention include proteins immunologically reactive with antibodies raised against polypeptides.
As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects or regulates the transcription of the coding sequence. Operably linked DNA sequences are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous. A structural gene (e.g., a gene encoding a protein which lacks phenylalanine) which is operably linked to regulatory sequences from a gene encoding a milk protein is generally expressed in substantially the same temporal and cell type-specific pattern as is the naturally-occurring milk protein gene.
A "recombinant expression cassette" is a polynucleotide sequence containing a coding sequence which is capable of affecting expression of the coding sequence in hosts compatible with the sequence. Such cassettes include the coding sequence and regulatory sequences such as promoters transcription termination signals as well as other sequences (e.g. enhancers) necessary or helpful in affecting expression. As used herein a "secretory signal sequence" is defined as any polynucleotide sequence which when operably linked to a coding sequence encodes a signal peptide which is capable of causing the secretion of the recombinant polypeptide encoded by the coding sequence. An example of a secretory signal sequence is a sequence which allows secretion by mammary gland cells. "Operably linked" in the context of linking a secretory signal sequence to a coding sequence means that the secretory sequence is linked to the coding sequence
so that the resultant secretory-recombinant DNA sequence encodes 5' to 3' for the secretory signal sequence and recombinant Phe" polypeptide. Accordingly, the reading frame for the secretory sequence and the recombinant DNA sequence must be covalently combined such that an open reading frame exists from the 5* end of the mRNA sequence formed after transcription and processing of the primary RNA transcript. The term "naturally-occurring" as used herein as applied to a compound refers to the fact that the compound can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) and that can be isolated from a source in nature is naturally-occurring.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention provides compositions and methods for producing recombinant proteins that lack certain amino acids such as phenylalanine (Phe~ proteins) . Generally the methods involve construction of recombinant expression vectors in which the desired codons are replaced with codons for other amino acids in a gene encoding the desired protein. The mutated gene is subsequently expressed in a host cell or transgenic animal and the protein is purified. In the case of Phe"proteins, they can serve as amino acid supplements with largely improved taste characteristics for PKU-patients.
Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry described below are those well known and commonly employed in the art. Standard techniques are uised for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation (e.g., electroporation, microinjection, lipofection) . Generally enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this document. The procedures therein are believed
to be well known in the art and are provided for the convenience of the reader. Much of the nomenclature and general laboratory procedures described below can be found in Sambrook, et al . , Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. The manual is hereinafter referred to as "Sambrook, et al . "
Polynucleotide sequences encoding any number of proteins of interest can be used in the present invention. The particular protein used to produce a mutant of the invention is not a critical aspect of the invention, although human milk proteins are generally preferred because of their nutritional value. The nucleic acids used to make the constructs of the invention, whether RNA, cDNA, genomic DNA, or a hybrid of the various combinations, may be isolated from natural sources or may be synthesized in vitro. In general, oligonucleotides used in the methods of the invention can be chemically synthesized using for instance an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.
Since the proteins of the invention are used as dietary supplements, proteins with high nutritional value are typically preferred. In certain embodiments, the recombinant protein is expressed in the milk of transgenic mammals, such as cows, sheep, goats, mice and the like. In these embodiments, a protein normally expressed in milk is particularly preferred. Examples of milk proteins that can be engineered in this way include human milk proteins such as lactoferrin, lysozyme, secreted immunoglobulins, lactalbumin, caseins and the like. Examples of other proteins that may be used in the present invention include serum albumin, transferrin and ovalbumin.
The engineering of one exemplary milk protein is described below, α-lactalbumin (α-Lac) . This protein is the most important nutritive protein of human milk. Table I shows the amino acid composition of human α-Lac as compared with the amino acid composition of mature human milk.
Table I. Comparison of amino acid composition of human α- lactalbumin and mature human milk.
*Aspartic acid and asparagine combined, ** Glutamic acid and glutamine combined.
amino acid symbol freq. mol% mol% in in in αLac αLac milk
Essential amino acids
Histidine H His 2 1.6 1.4
Isoleucine I lie 12 9.8 5.7
Leucine L Leu 14 11.4 9.6
Lysine K Lys 12 9.8 5.6
Methionine M Met 2 1.6 1.8
Threonine T Thr 7 5.7 5.1
Tryptophan W Trp 3 2.4 1.1
Valine V Val 2 1.6 7.0
Phenylalanine F Phe 4 3.3 3.2
Nonessential amino acids
Alanine A Ala 5 4.1 5.7
Arginine R Arg 1 0.8 2.1
Aspartic acid D Asp 12 9.8 9.3*
Asparagine N Asn 4 3.3 ——*
Cysteine C Cys 8 6.5 2.1
Glutamic Ac. E Glu 8 6.5 18.0**
Glutamine Q Gin 7 5.7 —**
Glycine G Gly 6 4.9 3.9
Proline P Pro 2 1.6 8.7
Serine S Ser 8 6.5 6.1
Tyrosine Y Tyr 4 3.3 3.5
I. Modification of polynucleotide sequences
The nucleotide sequences used for production of recombinant Phe" polypeptides or other mutants can be modified according to standard techniques to yield desired polypeptides, with a variety of desired properties, in addition to lacking particular amino acid residues. The modified polypeptides of the invention can be readily designed
and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art. For example, the polypeptides can vary from the naturally-occurring sequence at the primary structure level by amino acid insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
The amino acid sequence variants can be prepared with various objectives in mind, including increasing nutritive value or facilitating purification and preparation of the recombinant polypeptides. The amino acid sequence variants are usually predetermined variants not found in nature but exhibit the many of the same properties (e.g., immunogenic activity, nutritive value, and the like) as the naturally occurring polypeptides. For instance, polypeptide fragments comprising only a portion (usually at least about 60-80%, typically 90-95%) of the primary structure may be produced.
In general, modifications of the sequences encoding the polypeptides of the invention may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Kunkel, Proc . Natl . Acad. Sci . USA 82:488- 492 (1985); Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al . , Nature 328:731-734 (1987)). One of ordinary skill will appreciate that the effect of many mutations is difficult to predict. Thus, most modifications are evaluated by routine screening in a suitable assay for the desired characteristic (e.g., immunogenic activity, nutritive value, and the like) . II. In vitro expression of modified proteins.
The proteins of the invention can be produced in prokaryotic or eukaryotic host cells by expression of the appropriate polynucleotides. The cloned DNA sequences are expressed in hosts after the sequences have been operably linked to an expression control sequence in an expression vector. Expression vectors are typically replicable in the host organisms either as episo es or as an integral part of the host chromosomal DNA. Commonly, expression vectors will
contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see, e . g. , U.S. Patent 4,704,362). It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of the DNA encoding recombinant proteins in cell cultures. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes is made here.
In brief summary, the expression of natural or synthetic nucleic acids encoding, e.g., Phe" proteins will typically be achieved by operably linking the DNA or cDNA to regulatory sequences, such as a promoter (which is either constitutive or inducible) to form an expression cassette.
The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes will also contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the polynucleotide sequence encoding Phe" polypeptides. To obtain high level expression of a cloned gene, such as those polynucleotide sequences encoding Phe" proteins, it is desirable to construct expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a riboso e binding site for translational initiation, and a transcription/translation terminator.
A. Expression in Prokaryotes
Various microbial strains may be used, such as E. coli , bacilli, for example Bacillus subtilis , various species of Pseudomonas, or other bacterial strains. Preferred strains are generally recognized as safe strains (GRAS strains) such as Lactobacilli used in cheese production.
The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked
phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA.
The recombinant polypeptides produced by prokaryote cells may not necessarily fold properly. During purification, the expressed polypeptides may first be denatured and then renatured. This can be accomplished by solubilizing the bacterially produced proteins in a chaotropic agent such as guanidine HC1 and reducing all the cysteine residues with a reducing agent such as beta-mercaptoethanol. The polypeptides are then renatured, either by slow dialysis or by gel filtration. U.S. Patent No. 4,511,503.
Detection of the expressed protein is achieved by methods known in the art as radioimmunoassays. Western blotting techniques or immunoprecipitation. Purification from E. coli can be achieved following procedures described in U.S. Patent No. 4,511,503.
B. Expression of Modified Proteins in Eukaryotes A variety of eukaryotic expression systems such as yeast, insect cell lines and mammalian cells, are known to those of skill in the art.
1. Expression in Yeast and Funαi Synthesis of heterologous proteins in yeast is well known and described. Methods in Yeast Genetics , Sherman, F. , et al . , Cold Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods available to produce recombinant polypeptides in yeast. Typically, yeast strain that are currently used to produce food components (e.g., Kluyveromyces species) are preferred.
A number of well known yeast expression plasmids can be used as vectors. A gene of interest can be fused to any of the promoters in various yeast vectors. For instance, suitable vectors are described in the literature (Botstein, et al . , 1979, Gene, 8:17-24; Broach, et al., 1979, Gene, 8:121-133) . Two procedures are used in transforming yeast cells.
In one case, yeast cells are first converted into protoplasts using zymolyase, lyticase or glusulase, followed by addition of DNA and polyethylene glycol (PEG) . The PEG-treated
protoplasts are then regenerated in a 3% agar medium under selective conditions. Details of this procedure are given in the papers by J.D. Beggs, 1978, Nature (London), 275:104-109; and Hinnen, A., et al . , 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933. The second procedure does not involve removal of the cell wall. Instead the cells are treated with lithium chloride or acetate and PEG and put on selective plates (Ito, H., et al . , 1983, J. Bact. , 153:163-168).
Techniques for transforming other fungi are well known in the literature, and have been described, for instance, by Beggs (Nature 275:104-108 (1978)), Hinnen et al. (Proc. Natl . Acad. Sci . USA 75: 1929-1933, 1978), Yelton et al. (Proc . Natl . Acad . Sci . USA 81: 1740-1747, 1984), Russell (Nature 301: 167-169, 1983) and U.S. Patent 4,935,349. Expression in fungi can be accomplished using for example, Aspergillus , and the like.
The polypeptides of the invention can be isolated from yeast and other fungi by lysing the cells or hyphae and applying standard protein isolation techniques to the lysates. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassays of other standard immunoassay techniques.
2. Expression in Mammalian and Insect Cell Cultures The polynucleotides of the invention can be ligated to various expression vectors for use in transforming host mammalian or insect cell cultures. The vectors preferably contain a marker to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or metallothionein.
Mammalian cell systems often will be in the form of monolayers of cells although cell suspensions may also be used. As indicated above, the vector, e.g., a plasmid, which is used to transform the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the antigen gene sequence. These sequences are referred to as expression control sequences. When the host cell is of insect or mammalian origin illustrative
expression control sequences are obtained from the SV-40 promoter (Science , 222:524-527, 1983), the CMV I.E. Promoter (Proc . Natl . Acad . Sci . 81:659-663, 1984) or the metallothionein promoter (Nature 296:39-42, 1982). The cloning vector containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable and ligated with polynucleotides coding for the Phe" polypeptides by means well known in the art. As with yeast, when higher animal host cells are employed, polyadenlyation or transcription terminator sequences from known mammalian genes need to be incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et al . , 1983, J. Virol. 45: 773-781).
Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M. , 1985, "Bovine Papilloma virus DNA a
Eukaryotic Cloning Vector" in DNA Cloning Vol. II a Practical Approach Ed. D.M. Glover, IRL Press, Arlington, Virginia pp. 213-238.
The host cells are competent or rendered competent for transformation by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation and micro-injection of the DNA directly into the cells.
The transformed cells are cultured by means well known in the art. Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., (1977) . The expressed polypeptides are isolated from cells grown as suspensions or as onolayers. The latter are recovered by well known mechanical, chemical or enzymatic means.
C. Purification of Modified Polypeptides
The polypeptides of the invention produced by recombinant DNA technology from cell culture may be purified by standard techniques well known to those of skill in the art. Recombinantly produced polypeptides can be directly expressed or expressed as a fusion protein. The protein is then purified by a combination of cell lysis (e.g., sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme release the desired polypeptide.
The polypeptides of this invention may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification : Principles and Practice , Springer-Verlag: New York (1982).
III. Expression of Phe" Proteins in Transgenic Nonhuman Mammals
The invention also encompasses methods and polynucleotide constructs which are employed for generating nonhuman transgenic animals which produce Phe" proteins. The constructs are used to produce transgenic nonhuman mammals, such as cows, sheep, goats, pigs, rabbits or mice. A. Vector construction
Appropriate constructs and methods for production of transgenic animals are described in International Application Nb. WO/08216 and Krimpenfort, et al . , Biotechnology 9:844-847 (1991) . Typically, the coding sequence of interest is operably linked to expression regulatory sequences. In such transgenes, the expression regulatory sequence is at least the minimal sequences required for efficient cell-type specific expression, which generally are at least a promoter and at least about 1 kilobase (kb) upstream of the promoter, preferably at least about 2 to 3 kb upstream of the promoter, more preferably at least about 5 kb upstream of the promoter.
and frequently at least about 8 or more kb upstream of the promoter. Frequently, sequences downstream of the promoter, especially intronic sequences, are included in the transgene constructs (Brinster et al. (1988) Proc . Natl . Acad . Sci . USA 85: 836. Usually the sequences upstream of the promoter are used contiguously, although various deletions and rearrangements can be employed. Some desired regulatory elements (e.g., enhancers, silencers) may be relatively position-insensitive, so that the regulatory element will function correctly even if positioned differently in a transgene than in the corresponding germline gene. For example, an enhancer may be located at a different distance from a promoter, in a different orientation, and/or in a different linear order. For example, an enhancer that is located 3' to a promoter in germline configuration might be located 5' to the promoter in a transgene.
If the expression regulatory sequence(s) selected are relatively inefficient in transcribing the gene encoding the Phe" protein, it may be desirable to incorporate multiple copies of a transgene or targeting construct to compensate with an enhanced gene dosage of the transgene.
Typically, expression regulation sequences are chosen to produce tissue-specific or cell type-specific expression of the recombinant or secretory-recombinant DNA. Once a tissue or cell type is chosen for expression, expression regulation sequences are chosen. Generally, such expression regulation sequences are derived from genes that are expressed primarily in the tissue or cell type chosen. Preferably, the genes from which these expression regulation sequences are obtained are expressed substantially only in the tissue or cell type chosen, although secondary expression in other tissue and/or cell types is acceptable if expression of the recombinant DNA in the transgene in such tissue or cell type is not detrimental to the transgenic animal. Particularly preferred expression regulation sequences are those endogenous to the species of animal to be manipulated. However, expression regulation sequences from other species such as those from human genes may also be used.
In some instances, the expression regulation sequences and the recombinant DNA sequences (either genomic or cDNA) are from the same species, e.g., each from bovine species or from a human source. Alternatively, the expression regulation sequences and recombinant DNA sequences (either cDNA or genomic) are obtained from different species, e.g., expression regulation sequence from bovine species and a recombinant DNA sequence from a human source) . In such cases, the expression regulation and recombinant DNA sequence are heterologous to each other.
In general, the upstream expression regulation sequence includes the transcribed portion of the endogenous gene upstream from the translation initiation sequence and those flanking upstream sequences which comprise a functional promoter. Such sequences typically include a TATA sequence or box located generally about 25 to 30 nucleotides from the transcription initiation site. The constructs may also comprise one or more enhancer and/or other sequences which facilitate expression of the coding sequence and as a consequence facilitate expression.
The constructs will usually also comprise downstream expression regulation sequences to supplement tissue or cell- type specific expression. The downstream expression regulation sequences include polyadenylation sequences (either from the endogenous gene or from other sources such as SV40) and sequences that may affect RNA stability as well as enhancer and/or other sequences which enhance expression. Preferably, such sequences comprise about 2 kb, more preferably 8 kb and most preferably about 15 kb of 3• flanking sequence.
Examples of expression regulation sequences suitable in various species are provided in Table 2.
TABLE 2
Expression Regulation Tissue Animal Sequence Specificity Species
16 kb of bovine αSl Mammary bovine casein 5• to structural secretory gene and 8 kb 3 ' to cells structural gene
«15 kb 5' to murine Liver murine albumin gene
«15 kb 5' to murine Muscle murine α-actin gene
«15 kb upstream of Spermatids murine murine protamine gene
In addition to the above expression regulation sequences the constructs of the invention preferably also comprise intervening sequences. Such intervening sequences can be derived, for example, from bovine αSl casein and from human αLac. Recombinant intervening sequences may comprise hybrid intervening sequences which comprise a 5• RNA splice signal and 3• RNA splice signal from intervening sequences from different sources. Hybrid intervening sequences are not limited to transgenes utilizing cDNA sequences but are also useful when the recombinant polypeptide is encoded by a genomic sequence. Based on the results obtained with the cDNA recombinant DNA and the general expectation that genomic DNA sequences express at higher levels than sequences derived from cDNA, it is expected that such hybrid intervening sequences used in conjunction with genomic recombinant DNA will further enhance expression levels above that which would otherwise be obtained with genomic sequence alone.
If the Phe" polypeptide is to be secreted in, for example, the milk of the transgenic animal, a secretory signal sequence encoding a functional secretion signal peptide is also operably linked to the sequences encoding the Phe" polypeptide to direct secretion of the recombinant polypeptide from one or more cell types within the transgenic animal. The signal peptide generally is removed in vivo during secretion
to produce an extracellular form of the recombinant Phe" polypeptide.
Secretory DNA sequences in general are derived from genes encoding secreted proteins of the same species of the transgenic animal. Such secretory sequences are preferably derived from genes encoding polypeptides secreted from the cell type targeted for tissue-specific expression, e.g. secreted milk proteins for expression in and secretion from mammary secretory cells. Secretory DNA sequences, however, are not limited to such sequences and may be derived from proteins secreted from other cell types within the species of transgenic animal, e.g., the native signal sequence of a gene encoding a protein secreted other than in the mammary glands. Heterologous secretory sequences which encode signal secretion peptides from species other than the transgenic animals may also be used e.g., human t-PA, human serum albumin human lactoferrin and human αLac and secretion signals from microbial genes encoding secreted polypeptides such as from yeast, filamentous fungi, and bacteria. In one of the preferred embodiments, a secretory DNA sequence encoding a secretory signal sequence functional in the mammary secretory cells of bovine species is used to cause secretion of recombinant polypeptide from bovine mammary secretory cells. The secretory signal sequence is operably linked to the recombinant DNA sequence. Examples of such secretory sequences include DNA sequences encoding signal secretion sequences for bovine αSl casein, murine lactoferrin and human transferrin. The preferred secretory signal sequence is that encoding the secretory sequence of human αLac.
In the preferred embodiments of the invention, a Phe" polypeptide or other modified protein of the invention is expressed predominantly in the mammary secretory cells of transgenic bovine species. Such tissue-specific expression is obtained by operably linking mammary specific expression regulation sequences to recombinant sequences comprising a secretory signal sequence and a polynucleotide encoding a Phe" protein. In the case of human milk proteins such as αLac,
human regulatory sequences are preferably used. Alternatively, such mammary specific regulation sequences can be derived from various bovine genes preferentially expressed in the mammary secretory cells of the species. Such mammary specific genes include αSl casein; αS2-casein; β-casein; K-casein; α-lactalbumin; and /3-lactoglobulin.
In certain embodiments, it is desirable to use gene targeting, mediated by homologous recombination between a targeting polynucleotide construct and a homologous chromosomal sequence, to replace an endogenous gene with the gene encoding, e .g. , a Phe" mutant of the gene. Methods and materials for preparing such constructs are known by those of skill in the art and are described in various references. See, e.g., Thomas et al. , Cell 51:503 (1987) and Capecchi, Science 244:1288 (1989). Homologous targeting constructs have at least one region having a sequence that substantially corresponds to, or is substantially complementary to, a predetermined endogenous target gene sequence (e.g., an exon sequence, an enhancer, a promoter, an intronic sequence, or a flanking sequence of the target gene) . Such a homology region serves as a template for homologous pairing and recombination with substantially identical endogenous gene sequence(s) . In the targeting of transgenes, such homology regions typically flank the replacement region, which is a region of the targeting transgene that is to undergo replacement with the targeted endogenous gene sequence. Thus, a segment of the targeting transgene flanked by homology regions can replace a segment of the endogenous gene sequence by double crossover homologous recombination. B. Generation of transgenic animals
The constructs described above are introduced into embryonal target cells using standard techniques. For example, embryonic stem (ES) cells or fertilized oocytes can be used as the transgene recipients. Briefly, this technology involves the insertion of the desired transgene construct into a pluripotent cell line (e.g., bovine, porcine, ovine, or murine cell) that is capable of differentiating into germ cell tissue. Methods of introducing transgenes into embryonal
target cells "include microinjection of the transgene into the pronuclei of fertilized oocytes or nuclei of ES cells of the non-human animal. Such methods are well known to those skilled in the art. Alternatively, the transgene may be introduced into an animal by infection of zygotes with a retrovirus containing the transgene (Jaenisch, R. (1976) Proc . Natl . Acad . Sci . USA 73:1260-1264) .
The preferred method is microinjection of the fertilized bovine oocyte. In this preferred embodiment, the fertilized oocytes are first microinjected by standard techniques. They are thereafter cultured in vitro until a "pre-implantation embryo" is obtained. Such pre-implantation embryos preferably contain approximately 16 to 150 cells. The 16 to 32 cell stage of an embryo is commonly referred to as a morula. Those pre-implantation embryos containing more than 32 cells are commonly referred to as blastpcysts. They are generally characterized as demonstrating the development of a blastocoel cavity typically at the 64 cell stage. Methods for culturing fertilized oocytes to the pre-implantation stage include those described by Gordon, et al. (1984) Methods in Enzymology 101:414; Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (for the mouse embryo) ; and Hammer, et al. (1985) Nature 315:680 (for rabbit and porcine embryos) Gandolfi, et al. (1987) J. Reprod. Fert . 81:23-28; Rexroad, et al. (1988) J. Anim . Sci . 66:947-953 (for ovine embryos) and Eyestone, W.H. et al. (1989) J. Reprod. Fert . 85:715-720; Camous., et al. (1984) J. Reprod . Fert . 72:779-785; and Heyman, Y. , et al. (1987) Theriogenology 27:5968 (for bovine embryos). Such pre- implantation embryos are thereafter transferred to an appropriate female by standard methods to permit the birth of a transgenic or mosaic animal depending upon the stage of development when the transgene is introduced. As is well known, mosaic animals can be bred to form true germline transgenic animals.
Since the frequency of transgene incorporation is often low, the detection of transgene integration or transgenesis in the pre-implantation embryo is highly
desirable. ϊh one aspect of the invention methods are provided for identifying embryos wherein transgenesis has occurred and which permit implantation of transgenic embryos to form transgenic animals. In this method, one or more cells are removed from the pre-implantation embryo. When equal division is used, the embryo is preferably not cultivated past the morula stage (32 cells) . Division of the pre-implantation embryo (reviewed by Williams et al. (1986) Theriogenology 22:521-531) results in two "hemi-embryos" (hemi-morula or hemi-blastocyst) one of which is capable of subsequent development after implantation into the appropriate female to develop in utero to term. Although equal division of the pre- implantation embryo is preferred, it is to be understood that such an embryo may be unequally divided either intentionally or unintentionally into two hemi-embryos which are not necessarily of equal cell number. Essentially, all that is required is that one of the embryos which is not analyzed as hereinafter described be of sufficient cell number to develop to full term in utero . In a specific embodiment, the hemi- embryo which is not analyzed as described herein, if shown to be transgenic, is used to generate a clonal population of transgenic non-human animals. A preferred method for detecting transgenesis at this early stage in the embryo's using an endonuclease that recognizes a restriction site only when one of the bases in the site is methylated. This method if described in detail in WO/08216, supra .
Alternatively, biopsies are taken from 16-32 cell embryos and analyzed using standard techniques for detecting the presence or absence of a target sequence. For instance, Fluorescent In Situ Hybridization (FISH) can be used to detect the transgene. Several guides to FISH techniques are available, e.g.. Gall et al . Meth . Enzymol . , 21:470-480 (1981) and Angerer et al . in Genetic Engineering: Principles and Methods Setlow and Hollaender, Eds. Vol 7, pgs 43-65 (plenum Press, New York 1985).
The sequences can also be detected by PCR using primers and probes specific for the transgene. Standard PCR methods useful in the present invention are described in PCR
Protocols : Guide to Methods and Applications (Innis et al., eds.. Academic Press, San Diego).
Based on the present technology for cloning and manipulating DNA, the construction and microinjection of transgenes is practically limited to linearized DNA having a length not greater than about 50 kb. However, the transgenes of the invention, especially those having a length greater than about 50 kb, may be readily generated by introducing two or more overlapping fragments of the desired transgene into an embryonal target cell. When so introduced, the overlapping fragments undergo homologous recombination which results in integration of the fully reconstituted transgene in the genome of the target cell. In general, it is preferred that such overlapping transgene fragments have 100% homology in those regions which overlap. However, lower sequence homology may be tolerated provided efficient homologous recombination occurs. If non-homology does exist between the homologous sequence portions, it is preferred that the non-homology not be spread throughout the homologous sequence portion but rather be located in discrete areas. Although as few as 14 base pairs at 100% homology are sufficient for homologous recombination in mammalian cells (Rubnitz, J. and Subramani, S. (1984) Mol . Cell . Biol . 4:2253-2258), longer homologous sequence portions are preferred, e.g. 500bp, more preferably lOOObp, next most preferably 2000bp and most preferably greater than 2000bp for each homologous sequence portion. C. Isolation of Modified Proteins Methods for isolating the recombinant polypeptides are well known. Generally, the polypeptides are purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, Protein Purification: Principles and Practice , supra . In those embodiments where the recombinant Phe" polypeptide is expressed and secreted into the milk of transgenic animal, the transgenic milk so obtained may be either used as is or further treated to purify the recombinant polypeptide. This
depends, in part, on the recombinant polypeptide contained in the transgenic milk and the ultimate use for that protein. For example, a protocol suitable for the purification of αLac is described below. The recombinant polypeptides contained in the transgenic milk may also be used in food formulations which will typically be the major source of dietary protein for the patient. In the case of PKU patients, a number of formulations can be used as described for instance in Milupa Special Products : For the treatment of inherited disorders of amino acid metabolism (Milupa AG, March 1988) . A typical food formulation will comprise a Phe" polypeptide of the invention, vitamins, minerals, trace elements, sucrose and water. The formulation may be in the form of syrup, emulsion or suspension, to which a sweetening agent such as sucrose, fructose, glucose, mannitol, sorbitol, and/or flavoring agents such as cherry, fruit, orange, mint, may be added.
Solid dosage forms for oral administration may also be used, such as capsules, tablets, pills, powders, and granules. In such solid dosage forms, the polypeptides may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
The total daily amount of the food formulations will depend upon the body weight and age as well as on the individual tolerance to the particular amino acid of interest. Generally, the formulations are designed to provide total dietary protein and will thus be consumed at least once a day.
Examples The following example describes the production of transgenic mice and cows which secrete a Phe" mutant of human αLac.
I. Cloning of the human alpha lactalbumin gene. Alpha lactalbumin genes from several species (including human) have been cloned and sequenced. The sequence of the human
αLac gene is "disclosed in Hall et al . , Biochem . J. 242:735-742 (1987) . The genomic gene encoding human αLac is composed of four exons and three intervening sequences. The total length of the gene is approximately 2.5 kb. Since the gene is relatively small and due to the fact that the entire DNA sequence is known, it can readily be cloned from a phage library (e.g., human DNA cloned in phage EMBL3; commercially available from Clontech Inc.).
II. Site directed mutagenesis. The human αLac protein contains four phenylalanines (see Table 1) . In order to produce a protein free of phenylalanines all Phe-codons are replaced with Val-codons. This requires the introduction of the following four point mutations: Codon 3 [UUC] will be changed into [GUC] Codon 31 [UUU] will be changed into [GUU] Codon 53 [UUC) will be changed into [GUC] Codon 80 [UUC] will be changed into [GUC]
These mutations are introduced into the cloned gene via an established method described by e.g. Kunkel (1985, Proc. Natl. Acad. Sci. USA 82, 488-492). After introducing the mutations the DNA sequence of the entire gene will be determined to ensure the integrity of the introduced changes and to confirm that no additional mutations have occurred.
III. Transgene construction and generation of transgenic mice. Generation of transgenic mice expressing human αLac generally follows the methods described by Vilotte et al. 1989, Eur. J. Biochem. 186:43 and Soulier et al. (FEBS Lett. 1992, 297: 13). Using these mice, the tissue-specificity, developmental regulation and levels of transgene expression is analyzed in offspring from genetic founders. The effects of transgene expression on murine milk are also evaluated.
The entire gene surrounded by its own flanking sequences is first introduced into the germline of mice. This results in several mouse lines expressing in the mg/ml range. As an alternative, transgenes are constructed in which the αLac gene is fused to regulatory sequences of the bovine
αSl-casein gene. These sequences are also capable of driving expression of the introduced gene at a high level.
Milk is analyzed for levels of Phe" αLac by a specific immunoassay using commercially available antibodies for human αLac (e.g., polyclonal rabbit antihuman αLac available from Sigma) .
Next, the Phe" αLac is purified from the other milk proteins. The first step of this protocol is to separate the whey from the casein fraction. The Phe" αLac will appear in the whey fraction (based on its similarity to natural αLac) . As a next step the purification protocol is optimized empirically using standard techniques. For instance, chromatographic matrices that selectively interact with aromatic amino acid residues can be used, since the amount of the aromatic amino acids in the Phe" protein is 40% less than in bovine αLac. Another chromatographic separation step (e.g., size exclusion) to remove other abundant whey proteins such as betalactoglobulin and albumin may also be used. IV. Generation of transgenic dairy calves. Immature oocytes are retrieved in large numbers from slaughterhouse ovaries or from live animals using echoscopic procedures. Oocytes obtained in either way are matured and fertilized with sperm from selected elite dairy bulls. Resulting zygotes are microinjected and subsequently cultured to the late orula/early blastocyst stage. At that stage selected embryos are transferred to recipient cattle for the balance of gestation, as described above. If necessary, early detection of transgenesis can be achieved using the methods described above. Recipients carrying putative transgenic fetuses are kept on a farm equipped to generate and tend transgenic dairy cattle. During pregnancy, amniocentesis samples may be taken to determine which animals carry transgenic fetuses. After birth the presence of an integrated transgene is confirmed by analysis of DNA purified from placenta, blood and skin tissue.
Normally, cows do not lactate until they have calved. Calving does not typically occur until 20 months, at the earliest. Using a hormonal treatment, however, female
calves can be induced to lactate as early as 6 months after birth. The milk produced by these animals fully resembles mature milk (McFadden et al., 1989, J". Dairy Sci . , 72, 1754- 1763) and is used for initial characterization of the transgenic protein.
V. Analysis and testing of Phe" αLac from bovine milk. The first milk obtained from transgenic calves (induced for lactation) is used for standard biochemical characterization. For instance, the protein is sequenced and characterized for all parameters that are needed to commence clinical trials (such as concentration of residual phenylalanine) . Material from this milk may also be used to perform feeding studies in rats to assess its safety and toxicity. The founder cows are then used to generate production herds according to standard techniques.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.
Claims
1. A recombinant expression cassette comprising a regulatory sequence operably linked to a polynucleotide sequence encoding a Phe" polypeptide.
2. The recombinant expression cassette of claim 1, wherein the Phe" polypeptide is a Phe" human milk protein.
3. The recombinant expression cassette of claim 2, wherein the human milk protein is Phe" human α-lactalbumin.
4. The recombinant expression cassette of claim 1, wherein at least one phenylalanine codon is replaced by a valine codon.
5. The recombinant expression cassette of claim 1, wherein the regulatory sequence regulates expression of the Phe" polypeptide in a mammalian cell.
6. The recombinant expression cassette of claim 5, further comprising a polynucleotide sequence encoding a secretory signal sequence which is functional in mammary secretory cells.
7. The recombinant expression cassette of claim 5, wherein the regulatory sequence is from a human gene encoding a milk protein.
8. The recombinant expression cassette of claim 7, wherein the milk protein is α-lactalbumin.
9. The recombinant expression cassette of claim 5, wherein the regulatory sequence is from a bovine gene encoding a milk protein.
10. The recombinant expression cassette of claim 9, wherein the milk protein is selected from the group consisting of αSl-casein, αS2-casein, jS-casein, /c-casein, α-lactalbumin, and 3-lactoglobulin.
11. The recombinant expression cassette of claim 1, wherein the regulatory sequence regulates expression of the
Phe" polypeptide in a prokaryote.
12. The recombinant expression cassette of claim 11, wherein the Phe" polypeptide is Phe" α-lactalbumin.
13. The recombinant expression cassette of claim 1, wherein the regulatory sequence regulates expression of the Phe" polypeptide in a yeast cell.
14. The recombinant expression cassette of claim
13, wherein the Phe" polypeptide is Phe" α-lactalbumin.
15. The recombinant expression cassette of claim 1, wherein the regulatory sequence regulates expression of the Phe" polypeptide in a fungal cell.
16. The recombinant expression cassette of claim 15, wherein the Phe" polypeptide is Phe" α-lactalbumin.
17. A transgenic nonhuman mammal comprising a recombinant expression cassette comprising a regulatory sequence operably linked to a polynucleotide sequence encoding a Phe" polypeptide.
18. The transgenic mammal of claim 17, wherein the
Phe" polypeptide is a Phe" human milk protein.
19. The transgenic mammal of claim 18, wherein the human milk protein is Phe" α-lactalbumin.
20. The transgenic mammal of claim 17, wherein the mammal is bovine.
21. The transgenic mammal of claim 17, wherein the recombinant expression cassette further comprises a polynucleotide sequence encoding a secretory signal sequence which is functional in mammary secretory cells.
22. The transgenic mammal of claim 17, wherein the mammal produces the Phe" polypeptide in milk.
23. The transgenic mammal of claim 17, wherein the regulatory sequence is from a human gene encoding a milk protein.
24. The transgenic mammal of claim 23, wherein the milk protein is α-lactalbumin.
25. The transgenic mammal of claim 17, wherein the regulatory sequence is from a bovine gene encoding a milk protein.
26. The transgenic mammal of claim 25, wherein the milk protein is selected from the group consisting of αSl- casein, αS2-casein, jScasein, #c-casein, α-lactalbumin, and β- lactoglobulin.
27. A composition comprising a recombinant Phe" polypeptide.
28. The composition of claim 27, wherein the recombinant protein is Phe" α-lactalbumin.
29. The composition of claim 27, wherein the Phe" polypeptide is in milk from a transgenic mammal.
30. A food formulation comprising a recombinant Phe" protein.
31. The food formulation of claim 30, wherein the recombinant protein is Phe" α-lactalbumin.
32. A method of supplementing the diet of a phenylketonuria patient, the method comprising administering to the patient the food formulation of claim 30.
33. A recombinant expression cassette comprising a regulatory, sequence operably linked to a polynucleotide sequence encoding a modified polypeptide which substantially lacks a preselected amino acid.
34. The recombinant expression cassette of claim
33, wherein the modified polypeptide is a Phe" polypeptide.
35. The recombinant expression cassette of claim 33, wherein the regulatory sequence regulates expression of the Phe" polypeptide in a mammalian cell.
36. A transgenic nonhuman mammal comprising the recombinant expression cassette of claim 33.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU14544/95A AU1454495A (en) | 1993-12-29 | 1994-12-29 | Recombinant production of modified proteins lacking certain amino acids |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17510893A | 1993-12-29 | 1993-12-29 | |
US08/175,108 | 1993-12-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995018224A1 true WO1995018224A1 (en) | 1995-07-06 |
Family
ID=22638937
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1994/004343 WO1995018224A1 (en) | 1993-12-29 | 1994-12-29 | Recombinant production of modified proteins lacking certain amino acids |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU1454495A (en) |
WO (1) | WO1995018224A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2117243B2 (en) * | 1971-04-08 | 1981-03-19 | Heinr. Auer, Mühlenwerke KGaA, 5000 Köln | Process for the production of tasteless or tasty dry products from protein hydrolyzate solutions |
WO1993004165A1 (en) * | 1991-08-13 | 1993-03-04 | Wisconsin Milk Marketing Board | DNA SEQUENCE ENCODING BOVINE α-LACTALBUMIN AND METHODS OF USE |
WO1993017581A1 (en) * | 1992-03-13 | 1993-09-16 | Valio Oy | A method for removing phenylalanine from proteinaceous compositions, a product so obtained and use thereof |
WO1993025567A1 (en) * | 1992-06-15 | 1993-12-23 | Gene Pharming Europe B.V. | Production of recombinant polypeptides by bovine species and transgenic methods |
WO1995002692A1 (en) * | 1993-07-16 | 1995-01-26 | Ppl Therapeutics (Scotland) Limited | Modified alpha-lactalbumin |
-
1994
- 1994-12-29 WO PCT/EP1994/004343 patent/WO1995018224A1/en active Application Filing
- 1994-12-29 AU AU14544/95A patent/AU1454495A/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2117243B2 (en) * | 1971-04-08 | 1981-03-19 | Heinr. Auer, Mühlenwerke KGaA, 5000 Köln | Process for the production of tasteless or tasty dry products from protein hydrolyzate solutions |
WO1993004165A1 (en) * | 1991-08-13 | 1993-03-04 | Wisconsin Milk Marketing Board | DNA SEQUENCE ENCODING BOVINE α-LACTALBUMIN AND METHODS OF USE |
WO1993017581A1 (en) * | 1992-03-13 | 1993-09-16 | Valio Oy | A method for removing phenylalanine from proteinaceous compositions, a product so obtained and use thereof |
WO1993025567A1 (en) * | 1992-06-15 | 1993-12-23 | Gene Pharming Europe B.V. | Production of recombinant polypeptides by bovine species and transgenic methods |
WO1995002692A1 (en) * | 1993-07-16 | 1995-01-26 | Ppl Therapeutics (Scotland) Limited | Modified alpha-lactalbumin |
Non-Patent Citations (1)
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 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996002640A1 (en) * | 1994-07-13 | 1996-02-01 | Ppl Therapeutics (Scotland) Ltd. | Alpha-lactalbumin gene constructs |
AU700224B2 (en) * | 1994-07-13 | 1998-12-24 | Ppl Therapeutics (Scotland) Ltd | Alpha-lactalbumin gene constructs |
US5852224A (en) * | 1994-12-15 | 1998-12-22 | Ppl Therapeutics (Scotland) Limited | α-lactalbumin gene constructs |
Also Published As
Publication number | Publication date |
---|---|
AU1454495A (en) | 1995-07-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6066725A (en) | Production of recombinant polypeptides by bovine species and transgenic methods | |
JP3467272B2 (en) | DNA encoding κ-casein, method for obtaining the protein, and use | |
RU2095414C1 (en) | Transgene for preparing the recombinant polypeptide in transgenic cow milk, a method of obtaining the transgenic cow (variants), milk from transgenic cow, food composition | |
US5739407A (en) | Human β-casein, process for producing it and use thereof | |
JP3670003B2 (en) | Production of recombinant polypeptides by bovine species and transgenic methods | |
US20110023158A1 (en) | Bovine genome editing with zinc finger nucleases | |
Park et al. | Recombinant human erythropoietin produced in milk of transgenic pigs | |
EP1252184A2 (en) | C1 inhibitor produced in the milk of transgenic mammals | |
WO1993025567A9 (en) | Production of recombinant polypeptides by bovine species and transgenic methods | |
US20130131317A1 (en) | Expression of secreted human alpha-fetoprotein in transgenic animals | |
US6545198B1 (en) | Transgenically produced prolactin | |
WO1995018224A1 (en) | Recombinant production of modified proteins lacking certain amino acids | |
JPH10502816A (en) | α-lactalbumin gene construct | |
US20040231010A1 (en) | Lysozyme transgenic ungulates | |
AU6935694A (en) | Transgenic production of ec-sod | |
US20050043530A1 (en) | Seminal vesicle tissue-specific promoters and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA US UZ VN |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): KE MW SD SZ AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |