JPS62104584A - Novel plasmid - Google Patents
Novel plasmidInfo
- Publication number
- JPS62104584A JPS62104584A JP60245021A JP24502185A JPS62104584A JP S62104584 A JPS62104584 A JP S62104584A JP 60245021 A JP60245021 A JP 60245021A JP 24502185 A JP24502185 A JP 24502185A JP S62104584 A JPS62104584 A JP S62104584A
- Authority
- JP
- Japan
- Prior art keywords
- hgh
- plasmid
- fragments
- dna
- amino acid
- 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.)
- Pending
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 28
- 235000001014 amino acid Nutrition 0.000 claims abstract description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 10
- 102000002265 Human Growth Hormone Human genes 0.000 claims abstract description 9
- 108010000521 Human Growth Hormone Proteins 0.000 claims abstract description 9
- 239000000854 Human Growth Hormone Substances 0.000 claims abstract description 9
- 229920001184 polypeptide Polymers 0.000 claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 8
- 235000018417 cysteine Nutrition 0.000 claims abstract description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 108020004414 DNA Proteins 0.000 abstract description 32
- 239000012634 fragment Substances 0.000 abstract description 29
- 102000053602 DNA Human genes 0.000 abstract description 17
- 150000001413 amino acids Chemical class 0.000 abstract description 11
- 108020004682 Single-Stranded DNA Proteins 0.000 abstract description 10
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 4
- 230000001131 transforming effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 20
- 238000000034 method Methods 0.000 description 13
- 108091008146 restriction endonucleases Proteins 0.000 description 10
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 8
- 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 7
- 238000005119 centrifugation Methods 0.000 description 7
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- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- -1 for example Chemical compound 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
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- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 3
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 3
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 3
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- 239000000523 sample Substances 0.000 description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 3
- 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 2
- 229920001817 Agar Polymers 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000001384 succinic acid Substances 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- PTDKEUZTUCLKFR-KSAQDQTQSA-N 1-[(2r,4s,5s)-4-hydroxy-5-[1-hydroxy-2,2-bis(4-methoxyphenyl)-2-phenylethyl]oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC(OC)=CC=1)(C=1C=CC=CC=1)C(O)[C@@H]1[C@@H](O)C[C@H](N2C(NC(=O)C(C)=C2)=O)O1 PTDKEUZTUCLKFR-KSAQDQTQSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 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
- VKIGAWAEXPTIOL-UHFFFAOYSA-N 2-hydroxyhexanenitrile Chemical compound CCCCC(O)C#N VKIGAWAEXPTIOL-UHFFFAOYSA-N 0.000 description 1
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- 101000619708 Homo sapiens Peroxiredoxin-6 Proteins 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group 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 1
- 102100022239 Peroxiredoxin-6 Human genes 0.000 description 1
- 241000229231 Phage E Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 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
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101100173585 Schizosaccharomyces pombe (strain 972 / ATCC 24843) fft1 gene Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000000211 autoradiogram Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- MTFJSAGADRTKCI-VMPITWQZSA-N chembl77510 Chemical compound O\N=C\C1=CC=CC=N1 MTFJSAGADRTKCI-VMPITWQZSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 210000001072 colon Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- QRMKTNANRJCRCY-UHFFFAOYSA-N ethylammonium acetate Chemical compound CC[NH3+].CC([O-])=O QRMKTNANRJCRCY-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000002747 voluntary effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 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
- C07K14/61—Growth hormone [GH], i.e. somatotropin
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、ヒト成長ホルモンのN末端から第165位の
アミノ酸が変異された新規なポリペプチドを発現させる
ための新規なプラスミドに関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel plasmid for expressing a novel polypeptide in which the amino acid at position 165 from the N-terminus of human growth hormone is mutated.
ヒト成長ホルモン(以下hGHと示す)は、脳下垂体中
に比較的多く存在し、191 f[illのアミノ酸か
らなる分子−1121500のボ′リペプチドである。Human growth hormone (hereinafter referred to as hGH) is present in a relatively large amount in the pituitary gland, and is a polypeptide of molecule-1121500 consisting of 191 f[ill] amino acids.
その構造中には、N末端から第53位と第165位の間
及び第182位と第189位の間にS−8結合を有して
おり、第165位のアミノ酸はシスティンであることが
知られている。Its structure has S-8 bonds between positions 53 and 165 and between positions 182 and 189 from the N-terminus, and the amino acid at position 165 is cysteine. Are known.
そして、hGHを発現する遺伝子の上記165位に対応
する塩基配列はTGTであることも知られている。It is also known that the base sequence corresponding to the 165th position of the gene expressing hGH is TGT.
しかしながら、hGHの構造の一部を変換せしめて、h
GH様作用を有する新規ポリペプチドを得る試みは未だ
なされていない。However, by partially changing the structure of hGH, h
No attempt has yet been made to obtain a new polypeptide with GH-like activity.
そこで本発明者らは、高活性hG)(様ポリペプチドを
産生ずるための遺伝子を得るべく鋭意研究した結果、h
GHの第165位近辺に対応する遺伝子の塩基配列のう
ち、第163位から第167位に対応する塩基配列CT
G TACTGT TTCCGTの15鎖長(merl
はこの位置だけに存在し、他にこの塩基配列を有する場
所は全くないことを見い出し、更にこの15鎖長の塩基
配列のうち165位の塩基を変異させたものを天然のh
GHプラスミドに組み込むことにより、hGHのアミノ
酸配列のうち、165位のみがシスティンからセリンに
変換された新規ポリペプチドを産生ずるための新規なプ
ラスミドが得られることを見い出し、本発明を完成した
。Therefore, as a result of intensive research to obtain a gene for producing a highly active hG-like polypeptide, the present inventors found that
Base sequence CT corresponding to positions 163 to 167 of the gene base sequence corresponding to around position 165 of GH
G TACTGT 15 chain length of TTCCGT (merl
They discovered that this nucleotide sequence exists only at this position, and that there is no other location that has this nucleotide sequence. Furthermore, they mutated the base at position 165 of this 15-strand long nucleotide sequence and created the natural h
The present invention was completed based on the discovery that a novel plasmid for producing a novel polypeptide in which only position 165 of the amino acid sequence of hGH is converted from cysteine to serine can be obtained by integrating the present invention into a GH plasmid.
すなわち、本発明はhGHのN末端から第165位のア
ミノ酸であるシスティンがセリンに変換された新規ポリ
ペプチドを発現させるための遺伝子であって、該第16
5位のアミノ酸に対応する塩基配列がTCTであるプラ
スミドを提供するものである。That is, the present invention provides a gene for expressing a novel polypeptide in which the 165th amino acid cysteine from the N-terminus of hGH is converted to serine, and the 16th amino acid cysteine is converted to serine.
The present invention provides a plasmid in which the base sequence corresponding to the amino acid at position 5 is TCT.
本発明の新規プラスミドは例えば次の如く製造される。The novel plasmid of the present invention is produced, for example, as follows.
(1)天然hGH発現用のプラスミドであるpGI(−
L9を大腸菌HBIOIに形質転換させ、これを特定の
制限酵素を用いて切断して0.62Kbの断片を得る。(1) pGI(-), a plasmid for expressing natural hGH
L9 is transformed into E. coli HBIOI, which is cut using a specific restriction enzyme to obtain a 0.62 Kb fragment.
(2)大腸菌M13mpHファージベクターを特定の制
限酵素を用いて切断して7.24 Kb の断片を得る
。(2) E. coli M13mpH phage vector is cleaved using a specific restriction enzyme to obtain a 7.24 Kb fragment.
(3) 次いで、上記(1)の0.62Kb断片と(
2)の7.24Kb断片を結合せしめて、鋳型1本鎖D
NAを得る。(3) Next, the 0.62 Kb fragment of (1) above and (
The 7.24 Kb fragment of 2) was ligated to create template single-stranded D
Get NA.
(41)りエステル法の固相合成法によって得られた塩
基配列CTG TACTCT TTCCGTを有する5
1−リン酸化5鎖長オリゴヌクレオタイドと鋳型1本鎖
DNAとをアニーリングさせ、組換1本鎖DNAを得る
。(41) 5 having the base sequence CTG TACTCT TTCCGT obtained by the solid-phase synthesis method of the ester method
The 1-phosphorylated 5-strand oligonucleotide and the template single-stranded DNA are annealed to obtain recombinant single-stranded DNA.
(5)組換1本鎖DNAを通常のポリメラーゼを用いる
方法で塩基を延ばした後組換2本鎖DNAに配列させ、
特定の制限酵素で切断し、0.17Kb断片を得る。(5) extending the bases of the recombinant single-stranded DNA by a method using a normal polymerase, and then arranging it into a recombinant double-stranded DNA;
Cut with a specific restriction enzyme to obtain a 0.17 Kb fragment.
(6) pGH−L9 ′Jt組換2本鎖DNAを切
断したのと同じ制限酵素で切断し、大きな断片を得る。(6) Cut the pGH-L9'Jt recombinant double-stranded DNA with the same restriction enzyme used to cut it to obtain a large fragment.
(7) 0.17 Kb断片と大きな断片とを結合さ
せ、目的とする新規プラスミドを得る。(7) Combine the 0.17 Kb fragment and the large fragment to obtain the desired new plasmid.
以下、上記方法を詳細に説明する。The above method will be explained in detail below.
0、62 Kb断片を得るには、天然hGl(発現用の
プラスミド(pGH−L9 )を大腸菌HB 101細
胞で形質転換した後、その細胞をアンピシリンを含む培
地で培養し、常法の手段例えばアルカリ法〔ラピッド
アイソレーション オプ プラスミドオア バクテリオ
ファージ λDNAイン 1モレキユラー クローニン
グ、ア ラボラトリ−マニュアル”、コールド スフリ
ンクバー バーラボラトリ−、コールド スプリング
ハーバ−。To obtain the 0.62 Kb fragment, a plasmid for expression of natural hGl (pGH-L9) is transformed into Escherichia coli HB 101 cells, the cells are cultured in a medium containing ampicillin, and the cells are treated using conventional methods such as alkaline. Law [Rapid]
Isolation Op Plasmid Or Bacteriophage λDNA In 1 Molecular Cloning, Laboratory Manual”, Cold Spring Bar Laboratory, Cold Spring
Harbor.
ニューヨーク(Rapid tsolation of
plasmid orbacteriophageλ
DNA in″Mo1ecular Cloning、
ALaboratory mnual”、 Co1d
Spring Harber Labora−tor
y、 Co1d Spring Harber、 Ne
w York ) 355頁、1982年〕で形質転換
プラスミドを単離し、これを制限酵素1(pa l 、
Sa召[で切断し、低融点ゲル電気泳動にて回収する
ことにより行なわれる。なお、天然hG)(発現用プラ
スミドpGl(−L9 ハ、プロシーディング オブ
ナショナル アカデミーザ
オブ サイエンス オブ ユナイテッド ステイト オ
ブ アメリカ(Proc、 Nat4. Acad、
Sci。New York (Rapid solution of
plasmid orbacteriophageλ
DNA in″Molar Cloning,
A Laboratory mnual”, Co1d
Spring Harbor Laborator
y, Co1d Spring Harbor, Ne
York), p. 355, 1982], and this was digested with restriction enzyme 1 (pal,
This is carried out by cutting with saline and collecting with low melting point gel electrophoresis. In addition, natural hG) (expression plasmid pGl(-L9),
National Academy of Sciences of the United States of America (Proc, Nat4. Acad,
Sci.
USAI 、 81巻、 5956頁、 1984年に
記載の方法によって得られる。USAI, Vol. 81, p. 5956, 1984.
7、24 Kb断片は、M13mpHのファージベクタ
ーを制限酵素Sma i及びSad [で切断し、ゲル
成気泳動によって回収することによって得られる。The 7,24 Kb fragment is obtained by cleaving the M13mpH phage vector with the restriction enzymes Sma i and Sad [ and recovering it by gel electrophoresis.
鋳型1本鎖DNAを得るには、0.62 Kb断片と7
.24 Kb断片をDNAリガーゼ、例えばT4ファー
ジ大腸菌から得られたT 41Jガーゼを用いて結合さ
せた後、大腸菌JMIOIに感染させて培養する。この
培養したファージDNAをM13mp 11にhGHD
NAが組込まれているか否かを調べる為にX−ga/(
5−ブロモ−4−クロロ−3−インドリル−β−ガラク
トシダーゼ)を含むYT培地(バクトートリプトンと酵
母粗抽出液を含んだ寒天)にファージDNAをまき、3
4℃〜37℃、好ましくは37℃で1晩培養した。その
結果hGHDNAが組込まれた証拠として無色のプラー
クが現われた。このプラークに0.62 Kbの2本鎖
DNAが含まれていることをさらに確認するために、次
の操作を行つ“た。To obtain template single-stranded DNA, a 0.62 Kb fragment and 7
.. The 24 Kb fragment is ligated using DNA ligase, such as T41J gauze obtained from T4 phage E. coli, and then infected and cultured in E. coli JMIOI. This cultured phage DNA was transferred to M13mp11 using hGHD.
X-ga/(
5-Bromo-4-chloro-3-indolyl-β-galactosidase) containing YT medium (agar containing Bacto tryptone and crude yeast extract), 3
The cells were cultured overnight at 4°C to 37°C, preferably at 37°C. As a result, colorless plaques appeared as evidence that hGHDNA had been integrated. To further confirm that this plaque contained 0.62 Kb double-stranded DNA, the following operation was performed.
無色プラークの1部を取り、JM101m胞と一緒に2
倍濃度のYT培地(以下2xYTと表す)中で培養する
。培養後の培養液を取り、遠心分離操作を行い上清部分
と沈澱物を分離した。この沈澱物をアルカリ法で処理し
組換2本鎖7アージDNAを単離した後、制限酵素Ec
oR(及びSan Iで切断し、ゲル電気泳動を行い0
.62 Kbの位置にバンドを確認した。Take one part of the colorless plaque and add it together with JM101m cells.
Culture in double concentration YT medium (hereinafter referred to as 2xYT). After culturing, the culture solution was taken and centrifuged to separate the supernatant and precipitate. This precipitate was treated with an alkaline method to isolate recombinant double-stranded 7age DNA, and then the restriction enzyme Ec
Cut with oR (and San I, perform gel electrophoresis and remove 0
.. A band was confirmed at the 62 Kb position.
確認後、上記上清部分にポリエチレングリコールを加え
、冷所好ましくは4℃付近で数十分放置した後、遠心分
離を行い、鋳型1本鎖DNAを沈澱させる。After confirmation, polyethylene glycol is added to the supernatant and left to stand for several minutes in a cold place, preferably around 4° C., followed by centrifugation to precipitate the template single-stranded DNA.
この沈澱物をEDTAを含む緩衝液、例えばトリス塩酸
緩衝液(pH8)に溶解し、フェノール処理後、さらに
遠心分離操作を行い21−に分離させる。水層部分を分
取し、酢酸ナトリウム、エチルアルコールを加え精製鋳
型1本鎖ファージDNAを沈澱させる。This precipitate is dissolved in a buffer containing EDTA, for example, Tris-HCl buffer (pH 8), treated with phenol, and then centrifuged to separate 21-. The aqueous layer is separated, and sodium acetate and ethyl alcohol are added to precipitate purified template single-stranded phage DNA.
15鎖長オリゴヌクレオタイドは、固体支持体としてポ
リスチレンにコハク酸を介して5’−(4゜4′−ジメ
トキシトリチル)チミジンを結合させた化合物を用い、
各ヌクレオチドダイマーを該支持体上に核酸固相合成器
を使用して順次縮合させ、次いで該支持体から脱離せし
めることによって得られる。The 15-chain oligonucleotide used a compound in which 5'-(4°4'-dimethoxytrityl)thymidine was bonded to polystyrene via succinic acid as a solid support.
It is obtained by sequentially condensing each nucleotide dimer onto the support using a solid phase nucleic acid synthesizer and then releasing it from the support.
この15鎖長オリゴヌクレオタイドの5′末端のリン酸
化は、常法によって、例えばT4ポリヌクレオタイドキ
ナーゼを用いることによって行なわれる。Phosphorylation of the 5' end of this 15-chain oligonucleotide is carried out by a conventional method, for example, by using T4 polynucleotide kinase.
鋳型1本鎖ファージDNAと57−リン酸化15鎖長オ
リゴヌクレオタイドとのアニーリングは、60〜70℃
、好ましくは65℃で数時間反応させた後、ゆっくりと
室温まで戻すことにより行なわれ、これによって相補的
に配列したDNAが得られる。これを4種類のヌクレオ
タイド、即ちdATP、dGTP、 dCTP、 dT
TPを用いて鎖長を順次延ばし、D N A IJガー
ゼで結合し組換2本鎖ファージDNAを得る。この2本
鎖DNAをCaCも処理後、JMlolに感染させ1晩
放置する。この1晩培養した細胞をYT培地上にまいて
プラークを精製させしてフィルターに固定する。その後
ハイプリダイゼーショ/の前処理を行い、次いでハイブ
リダイゼーション処理を行う。そしてフィルター上のフ
ァージに5′末端を4p でラベルした15鎖長のオ
リゴヌクレオタイドを加え、室温に数十時間放置した後
温度を順時上ばて行き、ガイガーカウンターでチェック
しなから相補的に配列したDNAを探索する。Annealing of the template single-stranded phage DNA and the 57-phosphorylated 15-strand oligonucleotide was carried out at 60-70°C.
This is preferably carried out by reacting at 65° C. for several hours and then slowly returning the temperature to room temperature, thereby obtaining complementary sequenced DNA. This is divided into four types of nucleotides: dATP, dGTP, dCTP, and dT.
The chain length is sequentially extended using TP and ligated with DNA IJ gauze to obtain recombinant double-stranded phage DNA. This double-stranded DNA is also treated with CaC, infected with JMlol, and left overnight. The cells cultured overnight are plated on YT medium to purify plaques and fixed on a filter. Thereafter, a pretreatment for hybridization is performed, and then a hybridization treatment is performed. Then, a 15-chain oligonucleotide labeled with 4p at the 5' end was added to the phage on the filter, and after being left at room temperature for several tens of hours, the temperature was gradually increased. Search for DNA arranged in
この結果をもとにして相補的に配列しているプラークを
見つけYT培地から各々取出す。各々のプラークはJM
IOlを1晩発育させた細胞と共に2XYTで各々別々
に培養し、培養後の培養液を遠心分離で各々上清部分と
細胞の沈澱部分とに分ける。各々の上清にポリエチレン
グリコール6000を加え、冷所で数十時間放電した後
、遠心分離操作を行い組換1本鎖ファージDNAの沈澱
を得る。Based on this result, plaques with complementary sequences are found and each is taken out from the YT medium. Each plaque is JM
IOl was cultured separately in 2XYT together with cells grown overnight, and the culture solution after culture was separated into a supernatant portion and a cell precipitate portion by centrifugation. Polyethylene glycol 6000 is added to each supernatant, and after discharge in a cold place for several tens of hours, centrifugation is performed to obtain a precipitate of recombinant single-stranded phage DNA.
この沈澱を各々El:DTAが含まれた緩衝液に溶解し
、フェノール処理後さらに遠心分離し2層に分離させる
。分離した水層部分を各々分取し、酢酸ナトリウム、エ
チルアルコールで精製組換1本鎖ファージDNAを各々
単離させる。この単離した1本鎖ファージDNAの塩基
配列の確認はジデオキシ法で行う。Each of the precipitates is dissolved in a buffer solution containing El:DTA, treated with phenol, and further centrifuged to separate into two layers. The separated aqueous layer portions are separated, and purified recombinant single-stranded phage DNA is isolated using sodium acetate and ethyl alcohol. The base sequence of the isolated single-stranded phage DNA is confirmed by the dideoxy method.
に塩基配列が変異した部分が含まれている。contains a portion with a mutated base sequence.
一方、PGH−L9 グラスミドをBal I及びS&
t lで切断しhGHのC末端に対応する0、17Kb
を欠除した断片をグル電気泳動で回収する。この断片に
ld、 pBR322由来のベクタ一部分1発現用のト
リf (trp) fロモーター領域及びhGHのN末
端部位を含んでいる。On the other hand, PGH-L9 Grasmid was mixed with Bal I and S&
0.17 Kb corresponding to the C-terminus of hGH cut with tl
The fragment lacking the is recovered by gel electrophoresis. This fragment contains ld, the avian f (trp) f promoter region for expression of vector portion 1 derived from pBR322, and the N-terminal region of hGH.
この断片に前記の塩基配列が変異した部分を含む0.1
7 icbの断片1DNAIJガーゼを使用し組込んだ
後常法の手段で処理し、HBIOIに形質転換させる。This fragment contains the mutated portion of the above base sequence.
After incorporating the 7 icb fragment using DNAIJ gauze, it is treated by conventional methods and transformed into HBIOI.
この細胞をアンピシリンを含む培地で培養した後、アル
カリ法で処理しhGHの第165位のアミノ酸に対応す
る塩基配列が変異したプラスミド(pG)I−L9/c
a 3−1 )を単離する。After culturing these cells in a medium containing ampicillin, they were treated with an alkaline method to produce a plasmid (pG) I-L9/c in which the base sequence corresponding to the amino acid at position 165 of hGH was mutated.
a3-1) is isolated.
なお、本発明のグラスミドを製造するために使用する)
(Bl 01、JMIOI、制限#累、M 13 rn
p117アージベクター、DNAリガーゼ、X−gtJ
、YT培地、dATP 、 dGTP 、 dCTP、
dTTP、ポリメラーゼは全て市販されており、容易
に入手することができる。(used for producing Grasmid of the present invention)
(Bl 01, JMIOI, limited #cum, M 13 rn
p117age vector, DNA ligase, X-gtJ
, YT medium, dATP, dGTP, dCTP,
dTTP and polymerase are all commercially available and can be easily obtained.
本発明の新規プラスミドは、大腸菌等に通常の形質転換
法で取シ込ませ、培養することによって、hGHの16
5位のCysのみがSerに変換された新規ペプチドを
得ることができる。この新規ペプチドは、165位がS
erであるために53位と165位の間にS−8結合を
有せず、hG)lとは構造的に大きく異なるものであり
、その活性も天然hGHに比べ高まっている。The novel plasmid of the present invention can be introduced into Escherichia coli etc. by a normal transformation method and cultured to produce hGH 16
A novel peptide in which only Cys at position 5 is converted to Ser can be obtained. This new peptide has S at position 165.
Since it is an er, it does not have an S-8 bond between positions 53 and 165, and is structurally very different from hG)l, and its activity is also higher than that of natural hGH.
次に実施例を挙げて本発明を説明する。 Next, the present invention will be explained with reference to Examples.
実施例1
hGH構造遺伝子の単離
hGH発現用のプラスミド、 pGH−L9をトラン
スフオームしたHB 101細胞を1.51nlのアン
ピシリンを含むLB培地で1晩培養し、アルカリ法でプ
ラスミドを単離した。単離したプラスミドから制限酵素
Hpa I 、 5aI3I処理で得られる0、62K
bの断片を低融点アガロースゲル電気泳動で単離、回収
した。Example 1 Isolation of hGH structural gene HB 101 cells transformed with pGH-L9, a plasmid for hGH expression, were cultured overnight in LB medium containing 1.51 nl of ampicillin, and the plasmid was isolated by the alkaline method. 0.62K obtained from the isolated plasmid by treatment with restriction enzymes Hpa I and 5aI3I
The fragment b was isolated and recovered by low melting point agarose gel electrophoresis.
一ニング
1 pP 0M13 mpH(へfスfi−リサーチラ
ボラトリ−イ/り、 g (Bethesda Re5
earchLab、 Inc、 l )の2本鎖DN
Aを制限酵素Sma[。1 pP 0M13 mpH (Bethesda Re5
double-stranded DN of searchLab, Inc, l)
A is the restriction enzyme Sma[.
Sag lで処理した後、低融点アガロースゲル電気泳
動を行い、7.24Kbの断片を単離回収した。After treatment with Sag I, low melting point agarose gel electrophoresis was performed and a 7.24 Kb fragment was isolated and recovered.
このベクター0.1μmと先に単離したhGHの構造遺
伝子0.62 Kb断片のV3量を50 mM トリス
塩酸、 pH8,0、10mM Mg(J、 、 1
mM EDTA。This vector (0.1 μm) and the amount of V3 of the previously isolated hGH structural gene 0.62 Kb fragment were mixed with 50 mM Tris-HCl, pH 8.0, and 10 mM Mg (J, , 1).
mM EDTA.
6 mMβ−メルカプトエタノール(β−EtSI(l
。6 mM β-mercaptoethanol (β-EtSI (l
.
0、 s mM ATP、 0.11n97−ゼラチン
、200ユニツトT4DNAリガーゼ(全酒造製)、2
5μ2中で16時間反応させて結合した。この反応混合
物から8μkをサンプリングし、CaC4処理した大腸
IMJMIO1に感染させ、JMIQII晩培養細胞Q
、 ’l ml、100 mMイングロピルチオーβ−
ガラクトシド(IPTG l 10μ2.2チ5−ブロ
モ−4−クロロ−3−インドリル−β−D−ガラクト/
ド(X−ga−g l 50 pE、0.8%ソ7トア
ガール3tnlを加えてYT培地上にまいた。37℃で
1晩培養して生じた無色のプラークから8111i1を
とり、JMIO11晩培養細胞25μkを加えて2倍濃
度のYT培地1.5d中で培養した。培養液を6000
rpm5分間遠心し、沈澱から組W82本鎖DNAをア
ルカリ法で単離した。これをEcoRl 、 Sad
rで処理し、アガロースゲル電気泳動によって、株数し
た8個のファージすべてに目的とするhGHの0、62
Kbの断片が組み入れられていることを確認した。0, smM ATP, 0.11n97-gelatin, 200 units T4 DNA ligase (manufactured by Zenshuzo), 2
Binding was carried out by reacting in 5μ2 for 16 hours. 8 μk was sampled from this reaction mixture and infected into CaC4-treated colon IMJMIO1, JMIQII late culture cells Q
, 'l ml, 100 mM ingropyrthio β-
Galactoside (IPTG l 10 μ2.2 5-bromo-4-chloro-3-indolyl-β-D-galacto/
8111i1 was taken from the colorless plaques produced by culturing it at 37°C overnight and cultured in JMIO11 overnight. 25 μk of cells were added and cultured in 1.5 d of double concentration YT medium.
After centrifugation at rpm for 5 minutes, the W8 double-stranded DNA set was isolated from the precipitate using an alkaline method. EcoRl, Sad
All eight phages treated with r and counted by agarose gel electrophoresis were treated with 0,62 phage of the target hGH.
It was confirmed that the Kb fragment was incorporated.
鋳型1本鎖DNAの調製
単離したファージのうち1つを選び、それから得られた
培養液の上清を用いて組換1本鎖DNAを単離した。上
fft1 rnlに20%ポリエチレングリコール60
00(半井化学薬品■発売) 2.、5MNa(420
0μ2を加えて49C30分間放置した後、1ス000
rpm、10分間の遠心で7アージを沈澱させた。沈
澱を10mMトリ、z、 H(J pH8,0、1mM
EDTA 100μ2に溶かし、50μpのフェノ−#
(10mM TrisH(J pH8,0,1mM
EDTA テ飽和したもの)で処理した後遠心で2層を
分離して水層を分取した。これに76量の3MNa0A
c 、 2.5倍量のEtOHを加えてDNAを沈澱さ
せた。DNAは50 μiの10 mM TrisH(
J pH8,0、1rnMEDTAに溶かして一20℃
で保存した。Preparation of Template Single-Stranded DNA One of the isolated phages was selected, and the supernatant of the culture solution obtained from it was used to isolate recombinant single-stranded DNA. 20% polyethylene glycol 60 on top fft1 rnl
00 (Hani Chemicals ■ Released) 2. , 5MNa (420
After adding 0μ2 and leaving it for 30 minutes at 49C, 1s 000
The 7age was precipitated by centrifugation at rpm for 10 minutes. The precipitate was diluted with 10mM Tri, Z, H (J pH 8,0, 1mM
Dissolved in 100μ2 of EDTA, 50μp of pheno-#
(10mM TrisH(J pH8,0,1mM
After treatment with EDTA (saturated), the two layers were separated by centrifugation and the aqueous layer was collected. Add to this 76 amounts of 3MNa0A
c, DNA was precipitated by adding 2.5 times the volume of EtOH. DNA was prepared in 50 μi of 10 mM TrisH (
J pH 8.0, 1rn Dissolved in MEDTA and heated to -20°C.
Saved with.
トリエステル法の固相合成法により合成した。It was synthesized by solid-phase synthesis using the triester method.
固体支持体(樹脂)として、ポリスチレンにスペーサー
であるコハク酸を介して5’−(4、4’−ジメトキシ
トリチル)チミジ/を結合させた化合物(和光純薬玉業
■)を用いた。各ヌクレオチド(ダイマー)(和光紬薬
工業(掬)としては、下記表1記載のものを用いた。As a solid support (resin), a compound (Wako Pure Chemical Industries Ltd.) in which 5'-(4,4'-dimethoxytrityl) thymidine/ was bonded to polystyrene via a succinic acid spacer was used. As each nucleotide (dimer) (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd. (Kikki)), those listed in Table 1 below were used.
表 1
DMTr ;ジメトキシトリチル、 bz ;ベンゾイ
ル基。Table 1 DMTr: dimethoxytrityl, bz: benzoyl group.
1buHイソブチリル基
A;アデノシン、C;シチジン、G;グアノシン、T;
チミジン(1)縮合反応
核酸固相合成器を使用し、樹脂3μmoJ3と縮合剤(
2,4,6−ドリメチルベンゼンスルフオニルー3−ニ
トロトリアゾール(MSNTIIを用い、結合させるべ
きダイマーの添加ごとに表2の操作を繰り返した。1buH isobutyryl group A; adenosine, C; cytidine, G; guanosine, T;
Thymidine (1) condensation reaction Using a nucleic acid solid phase synthesizer, resin 3μmoJ3 and condensation agent (
Using 2,4,6-drimethylbenzenesulfonyl-3-nitrotriazole (MSNTII), the operations in Table 2 were repeated for each addition of dimer to be coupled.
(11)樹脂からの15marオリゴヌクレオタイドの
分離
■ 0.5 M 2−ピリジンアルドオキシム、1゜1
.3.3−テトラメチルグアニジン−1−ジオキサン:
ピリジン:水=5:4:1 1d中に(1)より得られ
たダイマー縮金物20mgを加え室温で24時間攪拌す
る。(11) Separation of 15mar oligonucleotide from resin ■ 0.5 M 2-pyridine aldoxime, 1°1
.. 3.3-Tetramethylguanidine-1-dioxane:
Pyridine:water=5:4:1 20 mg of the dimer condensate obtained from (1) was added to 1d and stirred at room temperature for 24 hours.
■ ■の溶液を留去後、残渣に濃アンモニア水(arn
llを別えて55℃、5時間加熱した後、■ ■の溶液
を酢酸エチル(6dX 31で洗い、水層を濃縮する。■ After distilling off the solution in ■, the residue contains concentrated ammonia water (arn
After heating in separate portions at 55°C for 5 hours, the solution of (1) and (2) was washed with ethyl acetate (6dX31), and the aqueous layer was concentrated.
残渣を逆相(C+slシリカゲルカラム(ウオータース
社55〜105μ)(φ0.7 X 5 cT!11に
かけ、0.1 M )リエチルアンモニウム酢酸塩pH
7中10チから30チのアセトニトリル(合計100m
1)の濃度勾配で溶出す、る。アセトニトリルの20%
濃度付近で溶出されるジメトキシトリチル体のフラクシ
ョ/を集めて濃縮する。The residue was applied to a reverse phase (C+sl silica gel column (Waters Co., Ltd. 55-105 μ) (φ0.7 x 5 cT!11, 0.1 M) ethyl ammonium acetate pH
7 in 10 to 30 degrees of acetonitrile (total 100 m
Elute with the concentration gradient of 1). 20% of acetonitrile
A fraction of the dimethoxytrityl compound eluted around the concentration is collected and concentrated.
■ 残直に80%酢酸水(5rnl lを加え、室温で
15分放置する。反応液をエーテル(5rnlX3)で
洗い、濃縮する。1200D26Qの完全脱保護オリゴ
マーを得る。(2) Add 80% aqueous acetic acid (5 rnl 1) to the residue and let stand at room temperature for 15 minutes. Wash the reaction solution with ether (5 rnl x 3) and concentrate. Completely deprotected oligomer of 1200D26Q is obtained.
■ 上記のオリゴマーの一部を逆相” I8 + 5μ
)シリカゲル高速液体クロマトグラフィーにかけ、精製
した。溶出は0.1 M トリエチルアンモニウム酢酸
塩(1)f(7)中、10.6 %から14チのアセト
ニトリルの濃度勾配を流速1rnl 7分で30分で行
う。ピークの中央部を集め、$41後、水−エタノール
で共沸し脱塩した。■ Part of the above oligomer is reverse phased” I8 + 5μ
) It was purified by silica gel high performance liquid chromatography. Elution is carried out with a concentration gradient of 10.6% to 14% acetonitrile in 0.1 M triethylammonium acetate (1)f (7) at a flow rate of 1 rnl in 7 minutes over 30 minutes. The central part of the peak was collected, and after $41, it was azeotropically distilled with water-ethanol to desalt it.
性状:水溶性白色粉末
UVλH2O260f t、 10,000/base
1ax
高速液体クロマトグラフィ R,T、 22分鋳型1
本領組pDNAと1ケ所組み換えし、Cys TGT
165 f< Ser TCT 185に変異させる様
にデザインした15merのオリゴヌクレオタイドCT
G TACTCT TTCCGT 100 pmo、、
g ’e 50mM TrisH(J p)(8,0、
10mMMgC4、1mMEDTA、6 mMβEtS
H,0,1q/−ゼラチン。Properties: Water-soluble white powder UVλH2O260ft, 10,000/base
1ax high performance liquid chromatography R, T, 22 minute mold 1
Cys TGT was recombined with the original pDNA at one site.
165 f< Ser TCT 15mer oligonucleotide CT designed to mutate to 185
G TACTCT TTCCGT 100 pmo,,
g'e 50mM TrisH (Jp) (8,0,
10mM MgC4, 1mM EDTA, 6mM βEtS
H,0,1q/-gelatin.
500 pmoI3ATP、 lニー” )T4ホl
J 5Zりvオタイドキナーゼ(宝酒造発売)、25μ
p中で37’C:、1.5時間反応させて5′リン酸化
した。500 pmoI3ATP, lny”) T4hol
J5Z Riv Otide Kinase (released by Takara Shuzo), 25μ
5' phosphorylation was carried out by reacting for 1.5 hours at 37'C: in p.p.
またハイブリダイゼーション用には、20 pmo4の
15 marの5′位を30 /j Ci (D (r
−”pl ATP(3000CiAnmoに3 )を唯
一のATP源として、500 pmo4 ATPを含ま
ない上記と同じ条件で高ラベルした。For hybridization, the 5' position of 15 mar of 20 pmo4 is 30 /j Ci (D (r
-''pl ATP (3 in 3000 CiAnmo) was highly labeled under the same conditions as above without 500 pmo4 ATP as the sole ATP source.
鋳型1本鎖組換DNA 2μpに5′リン酸化した1
5 mer 6 pmoAを加え、l Q mM T
risH(J pH7、5、10mM Mg(J2.5
0 mM Na(J 10 pl中で65℃、1時間加
熱後室温に30分間おいてアニーリングさせた。Template single-stranded recombinant DNA 5' phosphorylated 1 to 2μp
Add 5 mer 6 pmoA, l Q mM T
risH(J pH7, 5, 10mM Mg(J2.5
After heating at 65° C. for 1 hour in 0 mM Na (J 10 pl), annealing was performed at room temperature for 30 minutes.
コノ溶液を最終濃度が10 mM TrisH(J p
H7,5゜10 mM MgC4、20mM NaC[
、6mMβEtSH。The Kono solution was diluted with a final concentration of 10 mM TrisH (J p
H7,5゜10mM MgC4, 20mM NaC[
, 6mM βEtSH.
0.6 mM ATP 、 0.25 mM dATP
、 0.25 mM dGTP。0.6mM ATP, 0.25mM dATP
, 0.25 mM dGTP.
0.25 mM dCTP、 0.25 mM dTT
P (全てファルマシア社発売)、全量25μpになる
様に調製し、さらに2ユニツトの大腸菌DNAポリメラ
ーゼ■Klenow断片(ペーリンガーマンハイム社発
売)。0.25mM dCTP, 0.25mM dTT
P (all sold by Pharmacia), prepared to a total volume of 25 μp, and 2 units of Escherichia coli DNA polymerase Klenow fragment (sold by Pehringer Mannheim).
200ユニツトのT4 DNAリガーゼを加えて14
℃で16時間直接プライマーに順次結合を行った。6μ
2をサンプリングしてCa(J、処理したJMIolに
感染し、JMIOII晩培養細胞0.2d。Add 200 units of T4 DNA ligase to 14
Sequential ligation was performed to direct primers for 16 hours at °C. 6μ
Sample 2 and infect Ca(J, treated JMIol, JMIOII late culture cells 0.2 d.
0.8%ソフト アガール3 mlとともにYT培地に
まいて37℃で1晩培養した。The mixture was spread on YT medium together with 3 ml of 0.8% soft agar and cultured at 37°C overnight.
感染した培地を4℃に2時間程度おいた後、ニトロセル
ロースフィルター(ミリポア HAo、45μm )を
培地上のプラークの表面に接触させてファージをフィル
ターに吸着させた。80℃、2時間加熱処理してファー
ジをフィルターに固定した後、20m1の5倍濃度ss
c (5Xsscl [: 1倍ssc = 0.15
M NaC(3,0,015Mクエン酸ナトリウム、
1 mM EDTA ]、5倍濃度のデンハート溶液(
5x Denhardt’s 5olution l
[l Q Q倍=2%牛血清アルブミン、2%ポリビニ
ルピロリドン、2%フィコニー]0.1%ピロリン酸ナ
トリウム中で65℃、1時間プレノ・イブリダイゼーシ
ョンの処理をした。After the infected medium was kept at 4° C. for about 2 hours, a nitrocellulose filter (Millipore HAo, 45 μm) was brought into contact with the surface of the plaque on the medium, and the phages were adsorbed onto the filter. After fixing the phages on the filter by heating at 80°C for 2 hours, 20ml of 5x concentration ss
c (5Xsscl [: 1xssc = 0.15
M NaC (3,0,015M sodium citrate,
1 mM EDTA], 5x Denhardt's solution (
5x Denhardt's 5solution l
[l Q Q times = 2% bovine serum albumin, 2% polyvinylpyrrolidone, 2% ficony] Preno-hybridization was performed in 0.1% sodium pyrophosphate at 65° C. for 1 hour.
20rLlの5Xssc、5Xデ/ハート溶液、0.1
チビロリン酸ナトリウムに5′位を高ラベルして得た1
5merのプローブ10μ2を加えて、室温で15時間
ハイブリダイゼーションを行った。フィルターをガイガ
ーカウンターでチェックしながら、20mの5 X 9
80で30°0137℃、45℃、55℃と順次温度を
上げて洗った後、オートラジオグラムをとり、目的とす
る変異の起った陽性のプラークを選んだ。20 rLl of 5X ssc, 5X De/Hart solution, 0.1
1 obtained by highly labeling the 5' position of sodium tibirophosphate
10μ2 of a 5mer probe was added and hybridization was performed at room temperature for 15 hours. While checking the filter with a Geiger counter, check the 20m 5 x 9
After washing at 80° C. at 30° C., 45° C., and 55° C., an autoradiogram was taken, and positive plaques containing the desired mutation were selected.
プラークハイブリダイゼーションで陽性の場所に対応す
る7アージを16個採取してJMIOI 1晩培養細胞
25μpとともにl、 5 mlの2倍濃度YT培地で
12時間培養した。6,000 rpm 、 5分の遠
心で細胞を沈澱させ、上清から組換1本鎖DNAを単離
した。ジデオキシ法で配列を行い、採取した16個のう
ち、15個が目的の組換体であることを確認した。目的
の組換体を含んでいたファージから得られた沈澱をアル
カリ法で処理して組換2本鎖DNAを得た。制限酵素B
g、e II、Sa[[で切った後、組換部位を含むh
GHのC末端に対応する0、17Kbilfr片をゲル
電気泳動で分離し、回収した。Sixteen 7-age samples corresponding to positive plaque hybridization sites were collected and cultured for 12 hours in 5 ml of double-concentration YT medium with 25 μp of JMIOI overnight cultured cells. Cells were pelleted by centrifugation at 6,000 rpm for 5 minutes, and recombinant single-stranded DNA was isolated from the supernatant. Sequencing was performed using the dideoxy method, and it was confirmed that 15 of the 16 samples collected were the desired recombinants. The precipitate obtained from the phage containing the desired recombinant was treated with an alkaline method to obtain recombinant double-stranded DNA. Restriction enzyme B
g, e II, Sa[[h containing the recombination site after cutting with
A 0.17K bilfr piece corresponding to the C-terminus of GH was separated by gel electrophoresis and recovered.
変異hGH発現用のプラスミドへのりクローニングhG
](の全構造遺伝子を含む発現用のプラスミドpGH−
L9をBg−g If、 Sad [テ処理して、hG
H)c末箋に対応する0、 17 Kbを欠除した大き
な断片をアガロースゲル電気泳動で分離、回収した。Cloning hG into a plasmid for expressing mutant hGH
] (The expression plasmid pGH-
L9 was treated with Bg-g If, Sad [te, hG
H) A large fragment lacking 0.17 Kb corresponding to the c-terminus was separated and recovered by agarose gel electrophoresis.
pBR322由来のベクタ一部分9発現用のtrpプロ
モーター領域及びhGHのN末端部位を含むこの大きな
断片と先に単離した組換部位を含むhG)lのC末端に
対応する0、 17 Kb断片をT4 DNAリガー
ゼで結合させた後、CaCも処理したHBIOIに形質
転換した。得られた形質転換体からアルカリ法でプラス
ミドを単離し、8g6 [、Sad I処理して正しい
長さの挿入があることを確認した。This large fragment containing the trp promoter region for expression of the vector part 9 derived from pBR322 and the N-terminal site of hGH and the 0,17 Kb fragment corresponding to the C-terminus of hG) containing the previously isolated recombination site was transformed into T4. After ligation with DNA ligase, it was transformed into HBIOI which had also been treated with CaC. A plasmid was isolated from the resulting transformant by an alkaline method, and treated with 8g6 [, Sad I to confirm that the insertion had the correct length.
さらに組換体のSer TCT 165のプラスミド及
びオリジナルのCys TGT 165であるpGH−
L9プラスミドを等壇ツつニトロセルロースフィルター
にスポットして、80℃2時間加熱処理、65°C1時
間プレハイブリダイゼーションの後、先のプラークハイ
ブリダイゼーションと同じ条件で5erTCT165に
対応する15rnerをプローブに用いてハイブリダイ
ゼーションを行った。37℃、5Xsscでフィルター
を洗った後オートラジオグラフィーを行うと組換体のS
er TCT 165のプラスミドは非常に強い陽性の
スポットを与えたのに対し、CysTGT 165のp
GH−L9 は非常に弱いスポットしか与えなかった。Additionally, the recombinant Ser TCT 165 plasmid and the original Cys TGT 165 pGH-
The L9 plasmid was spotted on a nitrocellulose filter, heated at 80°C for 2 hours, prehybridized at 65°C for 1 hour, and then subjected to the same conditions as the previous plaque hybridization using 15rner corresponding to 5erTCT165 as a probe. Hybridization was performed. After washing the filter with 5Xssc at 37°C, autoradiography revealed that the recombinant S
The plasmid of er TCT 165 gave a very strong positive spot, whereas the plasmid of CysTGT 165 gave a very strong positive spot.
GH-L9 gave only a very weak spot.
このことから、Ser TCT 165の組換体の断片
がpGH−L9にリフローンされていることが確認され
た。This confirmed that the recombinant Ser TCT 165 fragment was reflown into pGH-L9.
このプラスミドをpGH−L9 / C83−1と命名
し、オリジナルのpGH−L9はそれに対してpGH−
L9 /CCl−1とも呼ぶことにする。This plasmid was named pGH-L9/C83-1, whereas the original pGH-L9 was named pGH-L9/C83-1.
It will also be referred to as L9/CCl-1.
実施例2
hGH組換プラスミドの大腸菌での発現pG)l−L9
/C83−1又はpGH−L9 /CC1−1を形質
転換したI(B 101をアンビシリ/を含むM9カザ
ミノ酸培地で発現させた。1晩培養した細胞50μ6e
新しいアンピシリン−M9−カザミノ咳培地5 mlに
加えて37℃、3時間、培養した後IAA(インドリル
−3−アクリル酸)を最終濃度40μト包になる様に加
えてインダクションをかけ、さらに12時間培養を続け
た。この時コントロールとしてIAAでインダクション
をかけていないものも培養した。IAAでインダクショ
ンをかけた細胞、かけていない細胞それぞれ0.2 m
l全遠心して集め62.5 mM TrisHC[pH
6,8、2Z SDS 、 5 mM EDTA、 1
0 %グリセロール、5チμEtSH,0,1チBFB
20μ2に′f@濁して1000C15分間処理して
細胞をこわした。1ス000rpm、 10分間の遠心
で不溶物を除去した全細胞抽出物をSDSを含む15%
ポリアクリルアミドゲル電気泳妨全行い、分子成約21
,000のhGHに対応するタンパクが高発現している
ことを認めた。Example 2 Expression of hGH recombinant plasmid in E. coli pG)l-L9
I(B101) transformed with /C83-1 or pGH-L9 /CC1-1 was expressed in M9 casamino acid medium containing Ambicily/. 50μ6e of cells cultured overnight
Add 5 ml of fresh ampicillin-M9-casamino cough medium and incubate at 37°C for 3 hours. Add IAA (indolyl-3-acrylic acid) to a final concentration of 40 μg and apply induction for another 12 hours. Cultivation continued. At this time, as a control, cells that were not induced with IAA were also cultured. 0.2 m each for cells with and without IAA induction
Centrifuge and collect 62.5 mM TrisHC [pH
6,8,2Z SDS, 5mM EDTA, 1
0% glycerol, 5 μEtSH, 0.1 μBFB
The cells were suspended at 20μ2 and treated at 1000C for 15 minutes to disrupt the cells. Centrifuge the whole cell extract at 1000 rpm for 10 minutes to remove insoluble matter and dilute it with 15% SDS.
Complete polyacrylamide gel electrophoresis, molecular agreement 21
,000, and a protein corresponding to hGH was found to be highly expressed.
以上 手続補正帯 (自発) 昭和60年11月25日that's all Procedural correction band (voluntary) November 25, 1985
Claims (1)
酸であるシステインがセリンに変換された新規ポリペプ
チドを発現させるための遺伝子であつて、該第165位
のアミノ酸に対応する塩基配列がTCTであるプラスミ
ド。1. A gene for expressing a novel polypeptide in which cysteine, an amino acid at position 165 from the N-terminus of human growth hormone, is converted to serine, and the base sequence corresponding to the amino acid at position 165 is TCT. A plasmid.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60245021A JPS62104584A (en) | 1985-10-31 | 1985-10-31 | Novel plasmid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60245021A JPS62104584A (en) | 1985-10-31 | 1985-10-31 | Novel plasmid |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS62104584A true JPS62104584A (en) | 1987-05-15 |
Family
ID=17127395
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP60245021A Pending JPS62104584A (en) | 1985-10-31 | 1985-10-31 | Novel plasmid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS62104584A (en) |
-
1985
- 1985-10-31 JP JP60245021A patent/JPS62104584A/en active Pending
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