EP0964918A2 - Reversibly inactive synthetic beta-galactosidase - Google Patents
Reversibly inactive synthetic beta-galactosidaseInfo
- Publication number
- EP0964918A2 EP0964918A2 EP97953997A EP97953997A EP0964918A2 EP 0964918 A2 EP0964918 A2 EP 0964918A2 EP 97953997 A EP97953997 A EP 97953997A EP 97953997 A EP97953997 A EP 97953997A EP 0964918 A2 EP0964918 A2 EP 0964918A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- enzyme
- galactosidase
- milk
- synthetic
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108010005774 beta-Galactosidase Proteins 0.000 title abstract description 77
- 102000005936 beta-Galactosidase Human genes 0.000 title abstract 3
- 102000004190 Enzymes Human genes 0.000 claims abstract description 56
- 108090000790 Enzymes Proteins 0.000 claims abstract description 56
- 235000013336 milk Nutrition 0.000 claims abstract description 29
- 239000008267 milk Substances 0.000 claims abstract description 29
- 210000004080 milk Anatomy 0.000 claims abstract description 29
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 7
- 238000012545 processing Methods 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 5
- 235000013351 cheese Nutrition 0.000 claims abstract description 4
- 229940088598 enzyme Drugs 0.000 claims description 55
- 108020004414 DNA Proteins 0.000 claims description 30
- 238000003776 cleavage reaction Methods 0.000 claims description 23
- 230000007017 scission Effects 0.000 claims description 23
- 241000228245 Aspergillus niger Species 0.000 claims description 22
- 230000028327 secretion Effects 0.000 claims description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 230000009261 transgenic effect Effects 0.000 claims description 8
- 108090000746 Chymosin Proteins 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 210000005075 mammary gland Anatomy 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 5
- 229940080701 chymosin Drugs 0.000 claims description 5
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 claims description 5
- 241000282412 Homo Species 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical group 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 238000013518 transcription Methods 0.000 claims description 2
- 230000035897 transcription Effects 0.000 claims description 2
- 102100026189 Beta-galactosidase Human genes 0.000 description 55
- 239000013598 vector Substances 0.000 description 31
- 108090000284 Pepsin A Proteins 0.000 description 30
- 102000057297 Pepsin A Human genes 0.000 description 30
- 229940111202 pepsin Drugs 0.000 description 30
- 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 22
- 239000002299 complementary DNA Substances 0.000 description 22
- 239000008101 lactose Substances 0.000 description 22
- 108090000623 proteins and genes Proteins 0.000 description 22
- 238000010276 construction Methods 0.000 description 17
- 239000000047 product Substances 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 230000002496 gastric effect Effects 0.000 description 13
- 230000001105 regulatory effect Effects 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 12
- 241000283690 Bos taurus Species 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 102000035195 Peptidases Human genes 0.000 description 10
- 108091005804 Peptidases Proteins 0.000 description 10
- 238000006460 hydrolysis reaction Methods 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- 108010076119 Caseins Proteins 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 230000007062 hydrolysis Effects 0.000 description 9
- 101000798100 Bos taurus Lactotransferrin Proteins 0.000 description 8
- 229940072440 bovine lactoferrin Drugs 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 7
- 108010059881 Lactase Proteins 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 210000000936 intestine Anatomy 0.000 description 7
- 229940116108 lactase Drugs 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 210000002784 stomach Anatomy 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 102000011632 Caseins Human genes 0.000 description 5
- 102100022624 Glucoamylase Human genes 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 240000006439 Aspergillus oryzae Species 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 201000010538 Lactose Intolerance Diseases 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- 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 4
- 235000021240 caseins Nutrition 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000002538 fungal effect Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000003301 hydrolyzing effect Effects 0.000 description 4
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 239000005862 Whey Substances 0.000 description 3
- 102000007544 Whey Proteins Human genes 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 235000012054 meals Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 102000004407 Lactalbumin Human genes 0.000 description 2
- 108090000942 Lactalbumin Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108010019653 Pwo polymerase Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000004913 chyme Anatomy 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 210000004051 gastric juice Anatomy 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108010064037 prorennin Proteins 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 101000757144 Aspergillus niger Glucoamylase Proteins 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101000946377 Bos taurus Alpha-lactalbumin Proteins 0.000 description 1
- 101000914103 Bos taurus Chymosin Proteins 0.000 description 1
- 102100025590 Chromosome transmission fidelity protein 8 homolog Human genes 0.000 description 1
- 108090000205 Chymotrypsin C Proteins 0.000 description 1
- 102100039511 Chymotrypsin-C Human genes 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000015781 Dietary Proteins Human genes 0.000 description 1
- 108010010256 Dietary Proteins Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 241000074748 Gagrella grandis Species 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases 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
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 229920002306 Glycocalyx Polymers 0.000 description 1
- 102100029100 Hematopoietic prostaglandin D synthase Human genes 0.000 description 1
- 101000856249 Homo sapiens Chromosome transmission fidelity protein 8 homolog Proteins 0.000 description 1
- 101001012451 Homo sapiens Enteropeptidase Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010023648 Lactase deficiency Diseases 0.000 description 1
- 108010059801 Lactase-Phlorizin Hydrolase Proteins 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 108010085169 Lysine carboxypeptidase Proteins 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 101800003414 Pro-elastase Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108090000783 Renin Proteins 0.000 description 1
- 102100028255 Renin Human genes 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach 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
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229940021722 caseins Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 1
- 235000021001 fermented dairy product Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
- 210000005095 gastrointestinal system Anatomy 0.000 description 1
- 235000021474 generally recognized As safe (food) 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
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000004517 glycocalyx Anatomy 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 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 1
- 230000006651 lactation Effects 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000013777 protein digestion Effects 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 235000021246 κ-casein Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- 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)
Definitions
- Lactose is one of the main milk components and it is found exclusively in dairy
- Lactose cannot be absorbed directly by the intestine. Rather, it must be cleaved into its
- lactase-phlorizin hydrolase lactase-phlorizin hydrolase
- lactose intolerance is characterized by a number of gastrointestinal disorders when the
- lactose malabsorption and milk intolerance affects the majority of the world's population.
- the present invention features the engineering of a ⁇ -galactosidase protein
- pancreatic secretions ⁇ -galactosidase genes are available that encode enzymes which
- ⁇ -galactosidase is 3-4, that of Aspergillus oryzae is about 5.0, while that o ⁇ K. lactis is
- the time parameter in the stomach is governed by the rate of flow of the chyme
- the volume of the meal is the predominant regulator of
- the invention provides a synthetic ⁇ -galactosidase enzyme which differs from
- cleavage site preferably an enzyme-cleavable peptide bond
- regulatory domain is to reversibly inactivate/suppress the hydrolyzing activity of
- the cleaving enzyme is one which is present preferably in the human
- the cleavage site should be one
- the invention also features DNA molecules encoding the synthetic enzymes of the
- the invention also features transgenic mammals whose mammary glands express the
- DNA molecule encoding the synthetic enzyme includes a
- mammary-specific promoter controlling transcription of the DNA encoding the enzyme
- Fig. 1 is a diagrammatic representation of the construction of a plasmid used as the
- Fig. 2 is a diagrammatic representation of the construction of a vector containing A. niger ⁇ -galactosidase-encoding DNA under the control of the ⁇ -Gla ( ⁇ -glucoamylase)
- Fig. 3 is a diagrammatic representation of the construction of a vector containing
- DNA encoding A. niger ⁇ -galactosidase fused to a bovine lactoferrin tail.
- Fig. 4 is a diagrammatic representation of the construction of a vector containing
- DNA encoding A. niger ⁇ -galactosidase fused to a polyamino acid tail-encoding sequence.
- Fig. 5 is a diagrammatic representation of the construction of a vector containing a
- FIG. 6 is a diagrammatic representation of an expression cassette in which the A. niger
- ⁇ -galactosidase gene is fused to A. niger ⁇ -galactosidase cDNA (tail) through a pepsin
- Fig. 7 is a diagrammatic representation of a vector in which the 3' end of the
- ⁇ -galactosidase gene is modified to encode a protein with a cleavage site recognizable by
- Fig. 8 is a diagrammatic representation of the fusion of the modified ⁇ -galactosidase
- Fig. 9 is a diagrammatic representation of the strategy of the construction of a vector
- genomic A. niger ⁇ -galactosidase-encoding DNA fused through a pepsin site to
- A. niger ⁇ -galactosidase cDNA (tail).
- Fig. 10 is a diagrammatic representation of the overall strategy for constructing a
- the enzyme to be used must be acid resistant and resist denaturation within the
- stomach from pH 1.0 (fasting state) to 5.0.
- the enzyme must retain activity in the presence of
- proteolytic enzymes pepsin in the stomach and the pancreatic proteases in the intestine
- the ⁇ -galactosidase of A. niger has a pH optimum in the range of 3-5, which makes it
- This enzyme has relatively high
- the ⁇ -galactosidase of A. niger is a suitable ⁇ -galactosidase of A. niger.
- the pro-enzyme is completely inactive. Following secretion from the cell, the pro-enzyme is
- Gastrointestinal protease structure and function are well understood.
- the target is Gastrointestinal protease structure and function.
- the target is Gastrointestinal protease structure and function.
- the target is Gastrointestinal protease structure and function.
- the target is Gastrointestinal protease structure and function.
- Pepsin is found in significant quantities within the gastrointestinal system. Its presence is a
- construct involved a bovine casein promoter driving the expression of bovine chymosin
- Chymosin was synthesized as a prochymosin which is
- Fusion-tail systems have been developed using
- polyamino acid tails have been generated in order to facilitate their purification (Niederauer
- ⁇ -galactosidase for example, through an enzyme recognition/cleavage recognition site
- the cleavage sequence is engineered for recognition by a proteolytic enzyme.
- constructs include a promoter region
- antibiotic resistant genes for example amp
- sequences depends on the host organism to be used as the expression system. These components are addressed below:
- the signal peptide can be one from the endogenous ⁇ -galactosidase gene if it is a
- prolactase is to be produced (i.e. from the A. niger glucoamylase gene). If the prolactase is to be produced
- mammary epithelial cells into milk Such systems are described, e.g., in Gordon et al., 1987,
- the ⁇ -galactosidase e.g., from A. niger or A. oryzae, has desirable functional
- the fused gene can be engineered to include a cleavage site
- the engineered tail will render the ⁇ -galactosidase molecule reversibly inactive due to
- the enzyme may also serve as an affinity handle during purification of the enzyme from culture broth.
- the tail domain may be added at the 3'-, 5'- or both ends of the gene whose activity is
- Proteins of known length and function which are normal constituents of milk such as caseins, lactoferrin or lactalbumin can be used as tails.
- tails Proteins of known length and function which are normal constituents of milk such as caseins, lactoferrin or lactalbumin can be used as tails.
- caseins lactoferrin or lactalbumin
- lactoferrin or lactalbumin can be used as tails.
- polyaminoacid tails can be used that add either a positive or negative charge to the
- Truncation can be either at the amino or carboxyl
- prolactase molecules (with the tail part having a series of deletions) are provided.
- the antibodies used can recognize either the ⁇ -galactosidase or the
- a preferred fungal host is for example the one
- the cleavage sequence is engineered to act as a recognition site for the specific
- Table 1 provides a partial list of such proteolytic enzymes and their target
- cleavage sites can be added for a specific application. For example, activation of the prolactase in the gastric environment would require a site that is recognized
- regulatory domain can generate a fusion molecule that could be added in milk during the
- the tail is removed, and the ⁇ -galactosidase is released, thereby hydrolyzing the lactose
- Prokaryotic fermentation typically utilizes Escherichia coli
- Saccharomyces cerevisiae (Goff et al, 1984) is routinely used. Fungal fermentation also
- the expression vector was derived from the pGla expression vector kindly provided
- step 1) was first digested with Sal restriction enzyme (Promega, Madison, WI, USA) and
- kb band containing the pUC18 backbone was purified using the QiaxII kit. The two purified
- ⁇ -galactosidase cDNA or structural gene was restricted with Xhol which excises the A. niger
- bovine lactoferrin (BLF) cDNA was cloned from mRNA isolated from bovine
- the PCR was performed in a final volume of 100 ⁇ l reaction using
- the underlined sequences represent overlapping sequences so that the amplified 3' sequence
- ⁇ -gal genomic DNA was fused with the BLF sequence using PCR without primers.
- the fusion product was then subcloned into a pGEMT vector that allows direct subcloning of PCR products. The resulting
- vector pGEMT/bgblfPCR was restricted with Nael and SpHI and ligated into the Nael, SpHI
- GTF7 genomic ⁇ -galactosidase gene
- Tails are to be fused to the 3'-end of the ⁇ -galactosidase
- prolactase containing bovine -lactalbumin (used as the regulatory domain fused at the 3'-end of the ⁇ -galactosidase gene with or without a pepsin recognition site
- Bovine ⁇ -lactalbumin was cloned by RT-PCR. Total RNA was first extracted from
- PCR fragment was digested with Agel and Notl and ligated to the Agel, Notl sites of the
- the resulting plasmid contains the a-lac gene fused at the
- the objective was to construct an expression cassette, pGgbpb (Fig. 6), based on the
- the glucoamylase promoter (Element 1), full length genomic ⁇ -galactosidase gene
- the starting vector is pGTF7 (Fig. 9). This vector contains pUC18 backbone, Element
- pSPanb Fig. 8 which are pSP72 vectors containing genomic and cDNA ⁇ -galactosidase
- the construction replaced Element 2 of the plasmid pGTF7 (Fig. 9) with a DNA
- the DNA template is the plasmid pSPganb, which contains the A. niger ⁇ -galactosidase
- the 5' primer was 5'-CAAGAACGGCATCTGGTCAG-3', and the 3' primer
- the pepsin recognition site (Element 3) and suitable cloning sites are (Agel and BssHII)
- Suitable cloning sites are designed to fuse the ⁇ -galactosidase cDNA sequences at the
- the DNA template for PCR was plasmid pSPanb
- the 5' primer is 5'-ATTAGCGCGCGAACTGTTGCAGAAATACGTC-3*, and
- deoxyribonucleotide(dNTP) 0.25 uM of each primer and the thermo DNA polymerase buffer.
- the reaction was carried out in MiniCycler (MJ Research Inc.) using the following
- plasmid pSPgbpb (Fig.8) which contains Element 2, Element 3 and Element 4.
- cDNA served as a template for the cloning of the ⁇ -casein gene.
- the second 3' end primer contains a unique Not I site to facilitate
- the PCR product (.56kb) was restricted with bpnl/Notl (step 2) and subcloned into
- pepsin will be cleaving the pepsin recognition site (between the b-gal gene and
- Lactose hydrolysis is measured by a
- the model system developed is based on conditions prevailing in the stomach. Gastric
- juice is a simple fluid containing 150 meq/ml of hydrogen (pH of 1.5) and 0.5 to 1 mg/ml of
- the pure juice at high rates of secretion can have a pH below 1 , but upon
- pH has been estimated to range between 1.5 and 2.5.
- a range of pepsin concentration from 0.1 to 10 mg/ml is used in order to take into account the great variability of pepsin found in
- the activity of the prolactase is tested using 400 ul of milk by the addition 3 different
- hydrochloric acid e.g., hydrochloric acid, pepsin and prolactase or lactase (used as the positive control).
- Varying the quantity of hydrochloric acid added allow us to cover a range of pH from about
- Lactose concentrations are determined by HPLC analysis.
- pepsin stock solution (100 mg/ml).
- prochymosin derivatives containing extension of various length of the pro-part.
- subtilis FEMS Microbiol. Lett. 61:243-249.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Dairy Products (AREA)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US775842 | 1985-09-13 | ||
US77584296A | 1996-12-31 | 1996-12-31 | |
PCT/IB1997/001658 WO1998029536A2 (en) | 1996-12-31 | 1997-12-29 | Reversibly inactive synthetic beta-galactosidase |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0964918A2 true EP0964918A2 (en) | 1999-12-22 |
Family
ID=25105684
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97953997A Withdrawn EP0964918A2 (en) | 1996-12-31 | 1997-12-29 | Reversibly inactive synthetic beta-galactosidase |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0964918A2 (ja) |
JP (1) | JP2001506136A (ja) |
AU (1) | AU5775798A (ja) |
WO (1) | WO1998029536A2 (ja) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120230973A1 (en) * | 2009-09-22 | 2012-09-13 | Amano Enzyme Inc. | Lactase preparation |
CN107857801B (zh) * | 2017-10-23 | 2020-09-22 | 华南理工大学 | 一种可用于提高分泌效率的信号肽及其应用 |
CN107698666A (zh) * | 2017-10-23 | 2018-02-16 | 华南理工大学 | 一种可有效提高分泌的信号肽及其应用 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US551459A (en) * | 1895-12-17 | Counter-fixture | ||
EP0832981A1 (en) * | 1987-02-17 | 1998-04-01 | Pharming B.V. | DNA sequences to target proteins to the mammary gland for efficient secretion |
GB8905674D0 (en) * | 1989-03-13 | 1989-04-26 | Imperial College | Dna construct and modified yeast |
JP3662948B2 (ja) * | 1992-12-09 | 2005-06-22 | ニユー・イングランド・バイオレイブス・インコーポレイテツド | 改質タンパク質及びその製造法 |
US5780009A (en) * | 1995-01-20 | 1998-07-14 | Nexia Biotechnologies, Inc. | Direct gene transfer into the ruminant mammary gland |
US5821350A (en) * | 1995-11-01 | 1998-10-13 | Nexia Biotechnologies, Inc. | Aspergillus niger beta-galactosidase gene |
-
1997
- 1997-12-29 WO PCT/IB1997/001658 patent/WO1998029536A2/en not_active Application Discontinuation
- 1997-12-29 EP EP97953997A patent/EP0964918A2/en not_active Withdrawn
- 1997-12-29 AU AU57757/98A patent/AU5775798A/en not_active Abandoned
- 1997-12-29 JP JP52977598A patent/JP2001506136A/ja active Pending
Non-Patent Citations (1)
Title |
---|
See references of WO9829536A3 * |
Also Published As
Publication number | Publication date |
---|---|
WO1998029536A3 (en) | 1998-09-11 |
AU5775798A (en) | 1998-07-31 |
JP2001506136A (ja) | 2001-05-15 |
WO1998029536A2 (en) | 1998-07-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Mohanty et al. | Bovine chymosin: Production by rDNA technology and application in cheese manufacture | |
US5959171A (en) | Method for the production of biologically active polypeptides in a mammal's | |
US20180371440A1 (en) | Constructs for expressing lysosomal polypeptides | |
US7718851B2 (en) | Expression of human milk proteins in transgenic plants | |
Garg et al. | Rennet: current trends and future research | |
WO1996014413A1 (en) | Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in aspergillus | |
US6727084B1 (en) | Cold-active beta-galactosidase, the process for its preparation and the use thereof | |
US5821104A (en) | Tripeptidyl aminopeptidase | |
Roche et al. | Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae | |
Zevaco et al. | Properties and specificity of a cell-wall proteinase from Lactobacillus helveticus | |
JP2004517643A (ja) | 酵母におけるTritirachiumalbum由来の組換えプロテイナーゼKの発現 | |
JP4120972B2 (ja) | 融合蛋白質を開裂させる方法 | |
Houen et al. | The primary structure and enzymic properties of porcine prochymosin and chymosin | |
JPH10511845A (ja) | 細菌において細胞外タンパク質を製造するための方法 | |
EP0964918A2 (en) | Reversibly inactive synthetic beta-galactosidase | |
JP3550409B2 (ja) | ジクチオステリウムのジペプチジルアミノペプチダーゼ | |
JP7008767B2 (ja) | 低温酸性プロテアーゼPsAPA並びにその調製方法及び応用 | |
JP4346368B2 (ja) | 組換え枯草菌 | |
EP2976356B1 (en) | Aspartic proteases | |
RU2157847C2 (ru) | Молекула днк для экспрессии, стимулируемой солями желчи липазы (bssl) | |
Roller et al. | Food enzymes | |
Shibui et al. | Periplasmic production of human pancreatic prokallikrein in Escherichia coli | |
Balabova et al. | Can recombinant tree shrew (Tupaia belangeri chinensis) chymosin coagulate cow (Bos taurus) milk? | |
Verheij et al. | [19] Cloning, expression, and purification of porcine pancreatic phospholipase A2 and mutants | |
Foltmann et al. | Comparative investigations on pig gastric proteases and their zymogens |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19990728 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): DE GB |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20010703 |