JP2560969B2 - Human insulin-like growth factor I gene - Google Patents
Human insulin-like growth factor I geneInfo
- Publication number
- JP2560969B2 JP2560969B2 JP5111559A JP11155993A JP2560969B2 JP 2560969 B2 JP2560969 B2 JP 2560969B2 JP 5111559 A JP5111559 A JP 5111559A JP 11155993 A JP11155993 A JP 11155993A JP 2560969 B2 JP2560969 B2 JP 2560969B2
- Authority
- JP
- Japan
- Prior art keywords
- igf
- gene
- fused
- peptide
- coli
- 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.)
- Expired - Lifetime
Links
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 title description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 42
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 claims description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 32
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- 239000013613 expression plasmid Substances 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 3
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims 4
- 102000004877 Insulin Human genes 0.000 claims 2
- 108090001061 Insulin Proteins 0.000 claims 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims 2
- 239000003102 growth factor Substances 0.000 claims 2
- 229940125396 insulin Drugs 0.000 claims 2
- 239000012634 fragment Substances 0.000 description 21
- 239000013612 plasmid Substances 0.000 description 15
- 230000004927 fusion Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 241000588724 Escherichia coli Species 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 229960000723 ampicillin Drugs 0.000 description 7
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 238000003379 elimination reaction Methods 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 241000671016 Escherichia coli F11 Species 0.000 description 4
- 241001131785 Escherichia coli HB101 Species 0.000 description 4
- 101150017040 I gene Proteins 0.000 description 4
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- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 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 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- 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 3
- 235000021240 caseins Nutrition 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
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- 239000002953 phosphate buffered saline Substances 0.000 description 3
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- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 239000012506 Sephacryl® Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
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- 239000001963 growth medium Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- XNCSCQSQSGDGES-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]propyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)C(C)CN(CC(O)=O)CC(O)=O XNCSCQSQSGDGES-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-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
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 244000309718 Escherichia coli F6 Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 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
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- 102100024319 Intestinal-type alkaline phosphatase Human genes 0.000 description 1
- 101710184243 Intestinal-type alkaline phosphatase Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 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 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
- 229930195725 Mannitol Natural products 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000015278 beef Nutrition 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
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- -1 diethylaminoethyl group Chemical group 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- RYCLIXPGLDDLTM-UHFFFAOYSA-J tetrapotassium;phosphonato phosphate Chemical compound [K+].[K+].[K+].[K+].[O-]P([O-])(=O)OP([O-])([O-])=O RYCLIXPGLDDLTM-UHFFFAOYSA-J 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】Detailed Description of the Invention
【0001】[0001]
【産業上の利用分野】この発明は、ヒトインスリン様成
長因子I(以下IGF−Iという)の改良された製法に
関するものである。より詳しくは保護ペプチドが最終の
アミノ酸としてメチオニン残基を有するぺプチドであっ
て、その保護ペプチドの該メチオニン残基を介してIG
FIと融合している保護ペプチド融合IGF−I(以
下、融合IGF−Iという)をコードする2個以上のシ
ストロンを有する多シストロン性遺伝子を有する発現プ
ラスミドにより形質転換された微生物を培地に培養し、
得られる培養液から融合IGF−Iを回収し、次いで得
られる融合IGF−Iを保護ペプチドの臭化シアンを用
いた脱離反応に付し、IGF−Iを得ることからなるI
GF−Iの製造に用いられる遺伝子、プラスミドおよび
形質転換体に関するものである。IGF−Iが次の配列
の70個のアミノ酸から成ることは既知である:FIELD OF THE INVENTION The present invention relates to an improved process for producing human insulin-like growth factor I (hereinafter referred to as IGF-I). More specifically, the protected peptide is a peptide having a methionine residue as the final amino acid, and the IG peptide is introduced via the methionine residue of the protected peptide.
A microorganism transformed with an expression plasmid having a polycistronic gene having two or more cistrons encoding a protective peptide fused IGF-I fused with FI (hereinafter referred to as fused IGF-I) is cultured in a medium. ,
The fusion IGF-I is recovered from the resulting culture medium, and the obtained fusion IGF-I is then subjected to an elimination reaction using the protected peptide cyanogen bromide to obtain IGF-I.
The present invention relates to genes, plasmids and transformants used for producing GF-I. It is known that IGF-I consists of 70 amino acids of the following sequence:
【0002】[0002]
【化1】 この発明の発明者らは、次の各基本工程を用いることに
より、IGF−Iを好収率で製造することに成功した:工程1 融合IGF−Iをコードする遺伝子(以下融合IGF−
I遺伝子という)を製造する工程。工程2 多シストロン性融合IGF−I遺伝子を有する発現プラ
スミドを製造する工程。工程3 該発現プラスミドにより宿主生物を形質転換することか
らなる形質転換体製造工程。Embedded image The inventors of the present invention succeeded in producing IGF-I in good yield by using the following basic steps: Step 1 Gene encoding fusion IGF-I (hereinafter referred to as fusion IGF-
I gene). Step 2 A step of producing an expression plasmid having a polycistronic fusion IGF-I gene. Step 3 A step of producing a transformant, which comprises transforming a host organism with the expression plasmid.
【0003】工程4 該形質転換体を適当な培地中で培養することからなる融
合IGF−I製造工程。工程5 宿主生物細胞から融合IGF−Iを単離する工程。工程6 該融合IGF−Iを保護ペプチドの脱離反応に付し、I
GF−Iを得るIGF−Iの製造工程。保護ペプチド
は、宿主生物の細胞中のプロテアーゼによる分解からI
GF−Iを保護するために使用され、前記融合IGF−
Iの保護ペプチド脱離反応によって除去される。すなわ
ち、該融合IGF−Iは該脱離反応によってIGF−I
を製造するための中間体であり、従って、該保護ペプチ
ドには、天然または合成のペプチドまたはそれらの断片
が包含される。好適な「融合IGF−I」には、保護ペ
プチドのメチオニンを介して保護ペプチドと融合したI
GF−Iが包含される。保護ペプチドの好適な例の一つ
は「ペプチドLH」であり、そのアミノ酸配列は次の通
りである。 Step 4 A step of producing fused IGF-I which comprises culturing the transformant in an appropriate medium. Step 5 Isolating the fused IGF-I from the host organism cell. Step 6 subject the fused IGF-I to elimination reaction of the protected peptide,
A process for producing IGF-I to obtain GF-I. Protective peptides are derived from degradation by proteases in cells of the host organism.
Used to protect GF-I, said fusion IGF-
It is removed by the protected peptide elimination reaction of I. That is, the fusion IGF-I is converted to IGF-I by the elimination reaction.
Thus, the protected peptide includes natural or synthetic peptides or fragments thereof. A suitable "fused IGF-I" is I fused to a protected peptide via the protected peptide methionine.
GF-I is included. One suitable example of a protected peptide is "Peptide LH", the amino acid sequence of which is as follows.
【0004】[0004]
【化2】 ペプチドLH融合IGF−Iをコードする好適遺伝子
(422bp)は次の通りである。Embedded image A preferred gene (422 bp) encoding the peptide LH fusion IGF-I is as follows.
【0005】[0005]
【化3】 Embedded image
【0006】[0006]
【化4】 Embedded image
【0007】[0007]
【化5】 この発明で用いられるプロモーター遺伝子は、通常用い
られる種々のプロモーター系から適宜選択されるもので
あってよい。その好適な例としては、たとえば大腸菌の
トリプトファンプロモーターのヌクレオチド配列に基づ
く、この発明の発明者により合成された合成プロモータ
ー遺伝子(例えば合成trpプロモーターI遺伝子等)
が包含される。該合成trpプロモーターI遺伝子のD
NA配列は次の通りである。Embedded image The promoter gene used in the present invention may be appropriately selected from various commonly used promoter systems. A preferred example thereof is a synthetic promoter gene (for example, a synthetic trp promoter I gene, etc.) synthesized by the inventor of the present invention based on the nucleotide sequence of the E. coli tryptophan promoter.
Is included. D of the synthetic trp promoter I gene
The NA sequences are as follows.
【0008】[0008]
【化6】 合成trpプロモーターI遺伝子中のプロモーター領域
およびシャインダルガノ領域(SD領域)を上記式中に
示した。[Chemical 6] The promoter region and Shine-Dalgarno region (SD region) in the synthetic trp promoter I gene are shown in the above formula.
【0009】この発明の製造法では、2個以上の融合I
GF−I遺伝子をプラスミドに順次挿入して、多シスト
ロン性融合IGF−I遺伝子を有する発現プラスミドを
得る。プロモーター遺伝子および融合IGF−I遺伝子
を適切なプラスミドと、所望により適切なDNA断片
(たとえばリンカー、他の制限部位等)を用いて、常法
(たとえば制限酵素による消化、T4ポリヌクレオチド
キナーゼを用いてのホスホリル化、T4DNAリガーゼ
を用いてのライゲーション)により、順次、環状に連結
することにより、多シストロン性遺伝子を有する発現プ
ラスミドを得ることができる。適当なプラスミドにはp
BR322等がある。このようにして得た多シストロン
性遺伝子を有する発現プラスミドを常法(たとえば形質
転換、顕微注射等)により微生物(宿主細胞)に挿入す
ることにより、形質転換体を得ることができる。適当な
宿主生物には、大腸菌、すなわちエシエリキア(Esc
herichia;以下E.という)コリ(coli)
(たとえばE.coli HB101等)などが包含さ
れる。この発明の方法によって融合IGF−Iを製造す
るには、このようにして得た発現プラスミド含有形質転
換体を栄養培地中で培養する。In the manufacturing method of the present invention, two or more fused I
The GF-I gene is sequentially inserted into the plasmid to obtain an expression plasmid having the polycistronic fusion IGF-I gene. Using the promoter gene and the fused IGF-I gene with an appropriate plasmid and, if desired, an appropriate DNA fragment (eg, linker, other restriction sites, etc.), a conventional method (eg, digestion with a restriction enzyme, T4 polynucleotide kinase) Phosphorylation and ligation using T4 DNA ligase) to successively ligate in a circular form to obtain an expression plasmid having a polycistronic gene. P is the appropriate plasmid
There are BR322 and the like. A transformant can be obtained by inserting the expression plasmid having a polycistronic gene thus obtained into a microorganism (host cell) by a conventional method (eg, transformation, microinjection, etc.). Suitable host organisms include E. coli, Escherichia ( Esc
herichia ; hereinafter E. That) E. coli (coli)
(For example, E. coli HB101 etc.) and the like are included. In order to produce the fused IGF-I by the method of the present invention, the expression plasmid-containing transformant thus obtained is cultured in a nutrient medium.
【0010】栄養培地は炭素源(たとえばグルコース、
グリセリン、マンニトール、フルクトース、ラクトース
等)および無機または有機の窒素源(硫酸アンモニウ
ム、塩化アンモニウム、カゼイン加水分解物、酵母エキ
ス、ポリペプトン、バクトトリプトン、牛肉エキス等)
を含有する。所望により、他の栄養源[たとえば無機塩
(たとえば重リン酸ナトリウムまたはカリウム、リン酸
水素二カリウム、塩化マグネシウム、硫酸マグネシウ
ム、塩化カルシウム)、ビタミン類(たとえばビタミン
B1 )、抗生物質(たとえばアンピシリン)等]を培地
に加えてもよい。形質転換体の培養は一般に、pH5.
5〜8.5(好ましくはpH7〜7.5)、18〜40
℃(好ましくは25〜38℃)で、5〜50時間にわた
って行えばよい。こうして生産された融合IGF−Iは
培養された形質転換体の細胞中に存在するのが普通であ
るので、細胞を濾過または遠心分離によって集め、それ
らの細胞壁および/または細胞膜を常法(たとえば超音
波処理および/またはリソチーム処理等)により破壊し
て、デブリスを得る。このデブリスから、天然または合
成の蛋白を単離、精製するのに一般に採用される常法
[たとえば適当な溶媒(たとえば8M尿素水溶液、6M
塩酸グアニジン等)を用いての蛋白の溶解、透析、ゲル
濾過、カラムクロマトグラフィー、高速液体クロマトグ
ラフィー等]により、融合IGF−Iを単離、精製でき
る。かくして得られる融合IGF−Iの好適な例の一つ
として、「ペプチドLHと融合したIGF−I」が挙げ
られ、そのアミノ酸配列は次の通りである。The nutrient medium contains a carbon source (eg glucose,
Glycerin, mannitol, fructose, lactose, etc.) and inorganic or organic nitrogen sources (ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactotryptone, beef extract, etc.)
Contains. If desired, other nutritional sources [eg inorganic salts (eg sodium or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg vitamin B 1 ), antibiotics (eg ampicillin). ) Etc.] may be added to the medium. Cultivation of transformants is generally at pH 5.
5 to 8.5 (preferably pH 7 to 7.5), 18 to 40
It may be performed at 5 ° C (preferably 25 to 38 ° C) for 5 to 50 hours. Since the fusion IGF-I thus produced is usually present in the cells of the transformed transformants, the cells are collected by filtration or centrifugation and their cell walls and / or cell membranes are routinely (eg Sonication and / or lysozyme treatment, etc.) to obtain debris. Conventional methods commonly used to isolate and purify natural or synthetic proteins from this debris [eg suitable solvent (eg 8M urea solution, 6M
Fusion IGF-I can be isolated and purified by protein dissolution using guanidine hydrochloride, etc.), dialysis, gel filtration, column chromatography, high performance liquid chromatography, etc.]. One preferable example of the fused IGF-I thus obtained is "IGF-I fused with peptide LH", and its amino acid sequence is as follows.
【0011】[0011]
【化7】 次いで、上記で得られた融合IGF−Iを保護ペプチド
の脱離反応に付いてIGF−Iを得る。本脱離反応は、
反応に悪影響を及ぼさない慣用の溶媒中で、緩和な条件
下に実施することができる。[Chemical 7] Then, the fused IGF-I obtained above is subjected to elimination reaction of the protective peptide to obtain IGF-I. This elimination reaction is
It can be carried out under mild conditions in a conventional solvent that does not adversely influence the reaction.
【0012】この明細書中の配列において、A、G、C
およびTはそれぞれ式In the sequences herein, A, G, C
And T are expressions
【化8】 を意味し、5’−末端A、G、CおよびTはそれぞれ式Embedded image And 5′-terminals A, G, C and T are each of the formula
【化9】 を意味し、3’−末端A、G、CおよびTはそれぞれ式[Chemical 9] And 3′-terminals A, G, C and T are each of the formula
【化10】 を意味する。[Chemical 10] Means
【0013】[0013]
【実施例】以下の実施例は本発明をより詳細に説明する
ために記載するものである。実施例1 プラスミドpUC−SS1の構築 特開昭61−1396号公報に記載された方法によって
得られたペプチドLH融合IGF−I発現プラスミド
(pLHSdMmtrp)(100μg)をHpaI
(180単位)によりHpaI消化用緩衝液中37℃で
1時間消化した。0.8%アガロースゲル上で完全消化
を検知したのち、1MNaClとPstI(180単
位)を混合物に加え、混合物を37℃で1時間インキュ
ベートして、2種のDNA断片(3.7kbpおよび
0.8kbp)を得た。大きい方のDNA断片(3.7
kbp)を0.8%アガロースゲル上で分離し、DEA
Eトヨパール650Mカラム(ジエチルアミノエチル基
を有する陰イオン交換樹脂、東洋曹達工業株式会社製)
クロマトグラフィーにより精製し、続いてエタノールで
沈殿させ、HpaI−PstI消化DNA断片(3.7
kbp)(40μg)を得た。このDNA断片(35μ
g)をHincII緩衝液中HincII(63単位)
により37℃で18分間部分消化して、6種のDNA断
片(3690、3417、2958、732、459お
よび273bp)を得た。732bpのDNA断片(2
μg)を分取用の0.8%アガロースゲル電気泳動およ
びDEAEトヨパール650Mカラムクロマトグラフィ
ーにより精製した。他方、プラスミドpUC9(ファル
マシアから購入)(10μg)をHincII(120
単位)を用いて37℃で1時間消化し、pUC9のHi
ncIIDNA断片(2μg)を得た。このDNA断片
(1.9μg)を仔ウシ腸アルカリホスファターゼ(以
下CIPという)(ベーリンガーマンハイムから購入)
(20単位)と共に30分間37℃でインキュベート
し、つぎにCIP(20単位)を追加して30分間、3
7℃でインキュベートした。65℃で15分間加熱して
酵素を不活化したのち、フェノール−クロロホルム抽出
およびそれに続くセファデックスG−50スーパーファ
イン(ファルマシア製)スパンカラムクロマトグラフィ
ーおよびエタノール沈殿によって精製して、pUC9の
脱ホスホリルHincII消化DNA断片(1.5μ
g)を得た。このDNA断片(1.2μg)を、上で調
製したペプチドLH融合IGF−I遺伝子含有HpaI
−HincII消化DNA断片(732bp)と、ライ
ゲーション緩衝液中、T4DNAリガーゼ(8単位)存
在下に、16℃で20時間にわたってライゲートさせ
た。ライゲーション混合物をE.coli MM294
に導入して形質転換し、多数のアンピシリン耐性コロニ
ーを得た。22コロニー中の1つが所望のプラスミドp
UC−SS1であった。これはPstI(3.4kb
p)およびBamHI(2.9kbpおよび0.45k
bp)を用いての制限エンドヌクレアーゼ分析によって
確認した。このプロセスを図1に示す。The following examples are provided to illustrate the present invention in more detail. Example 1 Construction of plasmid pUC-SS1 The peptide LH-fused IGF-I expression plasmid (pLHSdMmtrp) (100 μg) obtained by the method described in JP-A-61-1396 was HpaI.
(180 units) digested in HpaI digestion buffer at 37 ° C. for 1 hour. After detecting complete digestion on a 0.8% agarose gel, 1M NaCl and PstI (180 units) were added to the mixture and the mixture was incubated at 37 ° C. for 1 hour and the two DNA fragments (3.7 kbp and 0. 8 kbp) was obtained. Larger DNA fragment (3.7
kbp) was separated on a 0.8% agarose gel and DEA
E Toyopearl 650M column (anion exchange resin having diethylaminoethyl group, manufactured by Toyo Soda Kogyo Co., Ltd.)
Purification by chromatography followed by ethanol precipitation and HpaI-PstI digested DNA fragment (3.7
kbp) (40 μg) was obtained. This DNA fragment (35μ
g) HincII (63 units) in HincII buffer
Was partially digested at 37 ° C. for 18 minutes to obtain 6 DNA fragments (3690, 3417, 2958, 732, 459 and 273 bp). 732 bp DNA fragment (2
μg) was purified by preparative 0.8% agarose gel electrophoresis and DEAE Toyopearl 650M column chromatography. On the other hand, plasmid pUC9 (purchased from Pharmacia) (10 μg) was added to HincII (120
Unit) and digested for 1 hour at 37 ° C. to obtain pUC9 Hi
An ncII DNA fragment (2 μg) was obtained. Calf intestinal alkaline phosphatase (hereinafter referred to as CIP) (purchased from Boehringer Mannheim) was prepared from this DNA fragment (1.9 μg).
Incubate with (20 units) for 30 minutes at 37 ° C, then add CIP (20 units) for 30 minutes, 3
Incubated at 7 ° C. After heating at 65 ° C. for 15 minutes to inactivate the enzyme, it was purified by phenol-chloroform extraction followed by Sephadex G-50 Superfine (Pharmacia) span column chromatography and ethanol precipitation to dephosphorylate HincII of pUC9. Digested DNA fragment (1.5μ
g) was obtained. This DNA fragment (1.2 μg) was added to the above-prepared peptide LH-fused IGF-I gene-containing HpaI.
-HincII digested DNA fragment (732 bp) was ligated in the presence of T4 DNA ligase (8 units) in ligation buffer at 16 ° C for 20 hours. The ligation mixture was transformed into E. coli MM294
Then, a large number of ampicillin resistant colonies were obtained. One of the 22 colonies contains the desired plasmid p
It was UC-SS1. This is PstI (3.4 kb
p) and BamHI (2.9 kbp and 0.45 k
bp) and confirmed by restriction endonuclease analysis. This process is shown in FIG.
【0014】発現プラスミドpLS−T2およびpLS
−T3の構築 プラスミドpLHSdMmtrp(50μg)をBam
HI(120単位)によりBamHI消化用緩衝液中、
37℃で1時間消化し、続いて0.8%アガロースゲル
を用いて電気泳動を行って、2種のDNA断片を得た。
大きい方のDNA断片(4.5kbp)(16μg)を
CIPで処理して、pLHSdMmtrpの脱ホスホリ
ル化BamHI消化断片(4μg)を得た。他方、pU
C−SS1(10μg)をBamHI(60単位)で消
化して、2種のDNA断片(2.6kbpおよび464
bp)を得た。小さい方のDNA断片(464bp)を
0.8%アガロースゲルを用いる電気泳動によって精製
した。得られたBamHI消化DNA断片(464b
p)(36ng)および上記pLHSdMmtrpの脱
ホスホリル化BamHI消化DNA断片(200ng)
とをT4DNAリガーゼ(4単位)の存在下に16℃で
20時間インキュベートした。このライゲーション混合
物によりE.coli HB101を形質転換し、アン
ピシリン耐性コロニーを得た。12コロニーのうちの7
つは、2シストロン性のペプチドLH融合IGF−I遺
伝子を有するプラスミド(pLS−T2と名付ける)で
あり、1つは3シストロン性のペプチドLH融合IGF
−I遺伝子を有するプラスミド(pLS−T3と名付け
る)であった。これらは制限エンドヌクレアーゼ分析に
より確認された[pLS−T2:PstI−PvuII
(1.5、3.4kbp)、EcoRI(198、26
6bp)、PstI−SalI(3.0、2.0kb
p)、HpaI(4.98kbp);pLS−T3:P
stI−PvuII(1.5、3.9kbp)、Eco
RI(198、266bp)、PstI−SalI
(3.0、2.5kbp)、HpaI(5.45kb
p)]。このプロセスを図2に示す。プラスミドpLS
−T2を含有するE.coli HB101をE.co
liF−10、プラスミドpLS−T3を含有するE.
coli HB101をE.coli F−11と名付
けた。 Expression plasmids pLS-T2 and pLS
Construction of -T3 Plasmid pLHSdMmtrp (50 μg) was Bam
HI (120 units) in BamHI digestion buffer,
It was digested at 37 ° C. for 1 hour, and then electrophoresed using 0.8% agarose gel to obtain two kinds of DNA fragments.
The larger DNA fragment (4.5 kbp) (16 μg) was treated with CIP to obtain the dephosphorylated BamHI digested fragment of pLHSdMmtrp (4 μg). On the other hand, pU
C-SS1 (10 μg) was digested with BamHI (60 units) to give two DNA fragments (2.6 kbp and 464).
bp) was obtained. The smaller DNA fragment (464 bp) was purified by electrophoresis on a 0.8% agarose gel. The resulting BamHI-digested DNA fragment (464b
p) (36 ng) and the dephosphorylated BamHI digested DNA fragment (200 ng) of the above pLHSdMmtrp.
Were incubated in the presence of T4 DNA ligase (4 units) for 20 hours at 16 ° C. This ligation mixture allowed E. E. coli HB101 was transformed to obtain ampicillin resistant colonies. 7 out of 12 colonies
One is a plasmid having a bicistronic peptide LH-fused IGF-I gene (named pLS-T2), and one is a tricistronic peptide LH-fused IGF.
It was a plasmid having the -I gene (named pLS-T3). These were confirmed by restriction endonuclease analysis [pLS-T2: PstI-PvuII.
(1.5, 3.4 kbp), EcoRI (198, 26)
6 bp), PstI-SalI (3.0, 2.0 kb
p), HpaI (4.98 kbp); pLS-T3: P
stI-PvuII (1.5, 3.9 kbp), Eco
RI (198,266 bp), PstI-SalI
(3.0, 2.5 kbp), HpaI (5.45 kb)
p)]. This process is shown in FIG. Plasmid pLS
E. containing T2- E. coli HB101 to E. co
li F-10, E containing the plasmid pLS-T3.
E. coli HB101 to E. It was named E. coli F-11.
【0015】実施例2 ペプチドLH融合IGF−IのE.coli F−10
での生産 50μg/mlのアンピシリンを含有するLブロス中で
のE.coli F−10の一夜培養物を、グルコース
0.2%、カザミノ酸(酸加水分解カゼイン)0.5
%、ビタミンB1 50μg/mlおよびアンピシリン2
5μg/mlを含有するM9培地で1:20に稀釈し
た。A600 が約0.5となったとき、β−インドールア
クリル酸を最終濃度10μg/mlになるよう添加し
た。つぎに、細胞をインキュベートし、アリコート(培
養液20ml)を遠心分離(7krpm、40℃、5分
間)で集めた。 Example 2 Peptide LH-fused IGF-I E. coli F-10
Production in E. coli in L broth containing 50 μg / ml ampicillin. E. coli F-10 overnight culture, glucose 0.2%, casamino acid (acid hydrolyzed casein) 0.5
%, Vitamin B 1 50 μg / ml and ampicillin 2
It was diluted 1:20 with M9 medium containing 5 μg / ml. When the A 600 was about 0.5, β-indole acrylic acid was added to a final concentration of 10 μg / ml. Next, the cells were incubated, and an aliquot (20 ml of culture solution) was collected by centrifugation (7 krpm, 40 ° C., 5 minutes).
【0016】実施例3 ペプチドLH融合IGF−IのE.coli F−11
での生産 50μg/mlのアンピシリンを含有するLブロス中で
のE.coli F−11の一夜培養物をグルコース
0.2%、カザミノ酸(酸加水分解カゼイン)0.5
%、ビタミンB1 50μg/mlおよびアンピシリン2
5μg/mlを含有するM9培地で1:20に稀釈し
た。A600 が約0.5となったとき、β−インドールア
クリル酸を最終濃度10μg/mlとなるよう添加し
た。つぎに、細胞をインキュベートし、アリコート(培
養液20ml)を遠心分離(7krpm、4℃、5分
間)により集めた。 Example 3 Peptide LH-fused IGF-I E. coli F-11
Production in E. coli in L broth containing 50 μg / ml ampicillin. E. coli F-11 overnight culture with glucose 0.2%, casamino acid (acid hydrolyzed casein) 0.5
%, Vitamin B 1 50 μg / ml and ampicillin 2
It was diluted 1:20 with M9 medium containing 5 μg / ml. When the A 600 reached about 0.5, β-indole acrylic acid was added to a final concentration of 10 μg / ml. The cells were then incubated and aliquots (20 ml of culture) were collected by centrifugation (7 krpm, 4 ° C, 5 minutes).
【0017】実施例4 ペプチドLH融合IGF−Iの単離と精製E.coli
F−11を誘導後4時間培養し、遠心分離(14kr
pm、4℃)で集めた。湿潤細胞ペースト(120g:
培養液20リットルから)を10mMリン酸塩緩衝食塩
水(以下PBSという)−10mM EDTA(pH
8.0)300mlに懸濁し、−80℃で凍結した。こ
の混合物を融解し、0.5M EDTA 50mlおよ
び10mg/mlリソチーム溶液50mlに加えた。0
℃で1時間かき混ぜたのち、混合物をホモジナイズし
た。細胞デブリスを25mM PBS−10mM ED
TA−0.5%サルコシルナトリウム(pH8.0)2
リットル中に懸濁し、つぎにこの混合物をホモジナイズ
した。0℃で1時間かき混ぜたのち、混合物を7,00
0rpm、4℃で35分間遠心分離にかけた。ペレット
を10mM PBS−10mM EDTA(pH8.
0)に懸濁した。混合物をホモジナイズし、上と同じ方
法で遠心分離した。ペレットを6M塩酸グアニジン−1
00mMトリス−塩酸−10mM EDTA−100m
M DTT(pH8.0)200mlに溶解し、40k
rpm、20℃で1時間遠心分離した。上澄みを集め、
0.1Mトリス−塩酸(pH8.0)/8M尿素および
10mM2−メルカプトエタノールで平衡化したセファ
クリルS300スーパーファインカラム(5.0×8
6.6cm;樹脂1700ml)にかけた。溶出は、平
衡化緩衝液を用い、流速0.6ml/分で、4℃で行っ
た。セファクリルS300(ファルマシア製)クロマト
グラフィーを行って、35mlの画分を集めた。全ての
クロマトグラフィー工程について、分画の直後にアッセ
イを行った。活性画分を集め、プールした画分500m
lを、1M酢酸水溶液16リットルに対して室温で3時
間透析し、つぎに新鮮な1M酢酸水溶液16リットルに
対して一夜透析した。透析ずみ画分を凍結乾燥して、所
望の成分を含有するペプチドLH融合IGF−I(1.
26g)を得た。このペプチドLH融合IGF−Iは、
15%SDS PAGEにおいて分子量15,500の
位置にバンドを示す。 Example 4 Isolation and purification of peptide LH-fused IGF-I E. coli
F-11 was cultured for 4 hours after induction and centrifuged (14 kr
pm, 4 ° C.). Wet cell paste (120 g:
From 20 liters of the culture solution, 10 mM phosphate buffered saline (hereinafter referred to as PBS) -10 mM EDTA (pH
8.0) Suspended in 300 ml and frozen at -80 ° C. This mixture was thawed and added to 50 ml 0.5 M EDTA and 50 ml 10 mg / ml lysozyme solution. 0
After stirring for 1 hour at ° C, the mixture was homogenized. Cell debris was added to 25 mM PBS-10 mM ED.
TA-0.5% sarcosyl sodium (pH 8.0) 2
Suspended in liter and then homogenized this mixture. After stirring at 0 ° C. for 1 hour, the mixture was mixed with 7,000
It was centrifuged at 0 rpm, 4 ° C. for 35 minutes. The pellet was mixed with 10 mM PBS-10 mM EDTA (pH 8.
0). The mixture was homogenized and centrifuged in the same manner as above. Pellets with 6M guanidine hydrochloride-1
00 mM Tris-hydrochloric acid-10 mM EDTA-100 m
Dissolve in 200 ml of M DTT (pH 8.0), 40k
Centrifugation was performed for 1 hour at 20 rpm and rpm. Collect the supernatant,
Sephacryl S300 Superfine column (5.0 × 8) equilibrated with 0.1 M Tris-hydrochloric acid (pH 8.0) / 8 M urea and 10 mM 2-mercaptoethanol.
6.6 cm; resin 1700 ml). Elution was performed at 4 ° C. with equilibration buffer at a flow rate of 0.6 ml / min. Sephacryl S300 (Pharmacia) chromatography was performed to collect 35 ml fractions. Assays were performed immediately after fractionation for all chromatographic steps. Active fractions collected, pooled fraction 500m
1 was dialyzed against 16 liters of 1M aqueous acetic acid for 3 hours at room temperature and then against 16 liters of fresh 1M aqueous acetic acid overnight. The dialyzed fraction was lyophilized to give the peptide LH-fused IGF-I (1.
26 g) was obtained. This peptide LH fusion IGF-I is
A band is shown at a position of a molecular weight of 15,500 in 15% SDS PAGE.
【0018】実施例5 培養液中のペプチドLH融合IGF−Iの含量を、放射
免疫検定法(以下RIAという)を用いて測定し、IG
F−I含量として計算した。IGF−Iの放射免疫検定
は、矢内原の方法[矢内原ら:ペプチド・ホルモンズ・
イン・パンクレアス,3,28(1983)]に従って
実施した。 方法:上記の試料または標準試料[IGF−I断片(2
6−46)]0.1mlと試料緩衝液[0.01M P
BSおよび0.025MEDTA中の0.5%牛血清ア
ルブミン(以下、BSAという)(0.4ml)]、I
GF−I(26−46)のウサギ抗血清(0.1ml)
および 125I−IGF−I(26−46)(0.1m
l)を混合した。混合物を4℃で48時間放置し、つぎ
に、ウサギ血清(0.1ml)、ウサギγ−グロブリン
抗血清(0.1ml)および5%PEG6000 0.
9ml)を添加した。さらに2時間4℃に放置したの
ち、遠心分離(3krpm、4℃、30分間)によって
ペレットを集め、γ−カウンターにより放射能を測定し
た。この放射能からIGF−I含量を計算した。 Example 5 The content of the peptide LH-fused IGF-I in the culture medium was measured using a radioimmunoassay method (hereinafter referred to as RIA), and IG was determined.
Calculated as FI content. The radioimmunoassay for IGF-I is carried out by the method of Yanaihara [Yanaihara et al .: Peptide Hormones.
In Pancleas, 3 , 28 (1983)]. Method: The above sample or standard sample [IGF-I fragment (2
6-46)] 0.1 ml and sample buffer [0.01 MP
0.5% bovine serum albumin (hereinafter referred to as BSA) in BS and 0.025 MEDTA (0.4 ml)], I
GF-I (26-46) rabbit antiserum (0.1 ml)
And 125 I-IGF-I (26-46) (0.1 m
l) were mixed. The mixture was allowed to stand at 4 ° C. for 48 hours, then rabbit serum (0.1 ml), rabbit γ-globulin antiserum (0.1 ml) and 5% PEG6000.
9 ml) was added. After standing at 4 ° C for 2 hours, the pellets were collected by centrifugation (3krpm, 4 ° C, 30 minutes) and the radioactivity was measured by a γ-counter. The IGF-I content was calculated from this radioactivity.
【0019】結果:それぞれ実施例2および実施例3に
記載の方法によって調製した培養液(20ml)(M9
ブロス中でE.coli F−10およびE.coli
F−11を培養した液)を7,000rpmで遠心分
離した。得られた細胞を8M尿素、10mM EDTA
(pH8.0)(2ml)に懸濁し、つぎに超音波によ
り破壊した。懸濁液を遠心分離し(18,000rp
m、30分間、4℃)、上澄みを0.5%BSA、10
mM PBS、25ml EDTAで稀釈し、RIAお
よびHPLC用の試料として使用した。IGF−I含量
を、発現プラスミドpLHSdMmtrpを含有する
E.coli F−6を用いて同様にして調製した培養
液のそれと比較した。Results: Culture solution (20 ml) (M9) prepared by the method described in Example 2 and Example 3, respectively.
E. in Broth E. coli F-10 and E. coli
(F-11 culture solution) was centrifuged at 7,000 rpm. The obtained cells were treated with 8 M urea, 10 mM EDTA.
The cells were suspended in (pH 8.0) (2 ml) and then disrupted by ultrasonic waves. Centrifuge the suspension (18,000 rp
m, 30 minutes, 4 ° C.), and the supernatant was added with 0.5% BSA for 10 minutes.
Diluted with mM PBS, 25 ml EDTA and used as a sample for RIA and HPLC. IGF-I content contains the expression plasmid pLHSdMmtrp
E. E. coli F-6 was used for comparison with that of a culture solution prepared in the same manner.
【0020】[0020]
【表1】 [Table 1]
【0021】[0021]
【図1】 プラスミドpUC−SS1の構築のプロセス
を示す図である。FIG. 1 shows the process of construction of plasmid pUC-SS1.
【図2】 プラスミドpLS−T2およびプラスミドp
LS−T3の構築を示す図である。FIG. 2: Plasmid pLS-T2 and plasmid p
It is a figure which shows the construction of LS-T3.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 北口 忠司 尼崎市久々知西町1−9−9 (72)発明者 小野 裕樹 大阪府三島郡島本町青葉3−12 シャル マンコーポ水無瀬3−402 審査官 新見 浩一 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tadashi Kitaguchi 1-9-9 Kukuchinishimachi, Amagasaki City (72) Inventor Hiroki Ono 3-12 Aoba, Shimamoto-cho, Mishima-gun, Osaka Sharman Corp. 3-402 Minamise Examiner Niimi Koichi
Claims (2)
チオニン残基を有するペプチドであって、その保護ペプ
チドの該メチオニン残基を介してインスリン様成長因子
Iと融合している保護ペプチド融合インスリン成長因子
Iをコードする2個以上のシストロンを有する多シスト
ロン性遺伝子。1. A protected peptide-fused insulin growth factor I, wherein the protected peptide has a methionine residue as the final amino acid, and is fused with insulin-like growth factor I via the methionine residue of the protected peptide. A polycistronic gene having two or more cistrons that encodes.
チオニン残基を有するペプチドであって、その保護ペプ
チドの該メチオニン残基を介してインスリン様成長因子
Iと融合している保護ペプチド融合インスリン成長因子
Iをコードする2個以上のシストロンを有する多シスト
ロン性遺伝子を含有する発現プラスミドにより形質転換
された微生物。2. A protected peptide-fused insulin growth factor I, wherein the protected peptide has a methionine residue as the final amino acid and is fused with insulin-like growth factor I via the methionine residue of the protected peptide. A microorganism transformed with an expression plasmid containing a polycistronic gene having two or more cistrons encoding the gene.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8522977 | 1985-09-17 | ||
GB858522977A GB8522977D0 (en) | 1985-09-17 | 1985-09-17 | Production of insulin-like growth factor 1 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61214736A Division JPH0630614B2 (en) | 1985-09-17 | 1986-09-11 | Method for producing human insulin-like growth factor (I) |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH06319556A JPH06319556A (en) | 1994-11-22 |
JP2560969B2 true JP2560969B2 (en) | 1996-12-04 |
Family
ID=10585301
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61214736A Expired - Lifetime JPH0630614B2 (en) | 1985-09-17 | 1986-09-11 | Method for producing human insulin-like growth factor (I) |
JP5111559A Expired - Lifetime JP2560969B2 (en) | 1985-09-17 | 1993-05-13 | Human insulin-like growth factor I gene |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61214736A Expired - Lifetime JPH0630614B2 (en) | 1985-09-17 | 1986-09-11 | Method for producing human insulin-like growth factor (I) |
Country Status (2)
Country | Link |
---|---|
JP (2) | JPH0630614B2 (en) |
GB (1) | GB8522977D0 (en) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AT373281B (en) * | 1977-11-08 | 1984-01-10 | Genentech Inc | METHOD FOR PRODUCING A STRUCTURAL GENE |
US4713339A (en) * | 1983-01-19 | 1987-12-15 | Genentech, Inc. | Polycistronic expression vector construction |
JPS59203495A (en) * | 1983-04-28 | 1984-11-17 | Kyowa Hakko Kogyo Co Ltd | Novel method for developing exogenote |
SE8303626D0 (en) * | 1983-06-23 | 1983-06-23 | Kabigen Ab | A RECOMBINANT PLASMID AND A TRANSFORMANT MICROORGANISM, A POLYDOXYREBONUCLEOTIDE SEGMENT, A PROCESS FOR PRODUCING A BIOLOGICALLY ACTIVE PROTEIN, AND THE PROTEIN THUS PRODUCED |
-
1985
- 1985-09-17 GB GB858522977A patent/GB8522977D0/en active Pending
-
1986
- 1986-09-11 JP JP61214736A patent/JPH0630614B2/en not_active Expired - Lifetime
-
1993
- 1993-05-13 JP JP5111559A patent/JP2560969B2/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
GB8522977D0 (en) | 1985-10-23 |
JPS6291199A (en) | 1987-04-25 |
JPH0630614B2 (en) | 1994-04-27 |
JPH06319556A (en) | 1994-11-22 |
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