JP5785686B2 - 複数の性質をエンジニアリングするための部位評価ライブラリーを用いた、配列と活性との関係の系統的な評価 - Google Patents
複数の性質をエンジニアリングするための部位評価ライブラリーを用いた、配列と活性との関係の系統的な評価 Download PDFInfo
- Publication number
- JP5785686B2 JP5785686B2 JP2009518173A JP2009518173A JP5785686B2 JP 5785686 B2 JP5785686 B2 JP 5785686B2 JP 2009518173 A JP2009518173 A JP 2009518173A JP 2009518173 A JP2009518173 A JP 2009518173A JP 5785686 B2 JP5785686 B2 JP 5785686B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- sequence
- library
- variant
- activity
- 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 - Fee Related
Links
- 238000011156 evaluation Methods 0.000 title claims description 12
- 230000000694 effects Effects 0.000 title description 59
- 230000009897 systematic effect Effects 0.000 title 1
- 108090000623 proteins and genes Proteins 0.000 claims description 239
- 102000004169 proteins and genes Human genes 0.000 claims description 175
- 238000000034 method Methods 0.000 claims description 84
- 108091005804 Peptidases Proteins 0.000 claims description 79
- 239000004365 Protease Substances 0.000 claims description 77
- 102000004190 Enzymes Human genes 0.000 claims description 76
- 108090000790 Enzymes Proteins 0.000 claims description 76
- 229940088598 enzyme Drugs 0.000 claims description 76
- 230000035772 mutation Effects 0.000 claims description 60
- 239000000203 mixture Substances 0.000 claims description 54
- 238000004140 cleaning Methods 0.000 claims description 38
- 238000012360 testing method Methods 0.000 claims description 31
- 239000000758 substrate Substances 0.000 claims description 23
- 230000001976 improved effect Effects 0.000 claims description 22
- 230000027455 binding Effects 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000003599 detergent Substances 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 12
- 230000014509 gene expression Effects 0.000 claims description 11
- 102000013142 Amylases Human genes 0.000 claims description 7
- 108010065511 Amylases Proteins 0.000 claims description 7
- 108090001060 Lipase Proteins 0.000 claims description 7
- 102000004882 Lipase Human genes 0.000 claims description 7
- 239000004367 Lipase Substances 0.000 claims description 7
- 102000005741 Metalloproteases Human genes 0.000 claims description 7
- 108010006035 Metalloproteases Proteins 0.000 claims description 7
- 102000004316 Oxidoreductases Human genes 0.000 claims description 7
- 108090000854 Oxidoreductases Proteins 0.000 claims description 7
- 102000004357 Transferases Human genes 0.000 claims description 7
- 108090000992 Transferases Proteins 0.000 claims description 7
- 235000019418 amylase Nutrition 0.000 claims description 7
- 108010005400 cutinase Proteins 0.000 claims description 7
- 235000019421 lipase Nutrition 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 108090000371 Esterases Proteins 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 230000005764 inhibitory process Effects 0.000 claims description 5
- 239000003102 growth factor Substances 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 239000004382 Amylase Substances 0.000 claims 1
- 108010059892 Cellulase Proteins 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 229940106157 cellulase Drugs 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 155
- 102000035195 Peptidases Human genes 0.000 description 78
- 150000007523 nucleic acids Chemical group 0.000 description 64
- 235000019419 proteases Nutrition 0.000 description 62
- 108020004414 DNA Proteins 0.000 description 52
- 239000013615 primer Substances 0.000 description 46
- 102000039446 nucleic acids Human genes 0.000 description 40
- 108020004707 nucleic acids Proteins 0.000 description 40
- 238000003556 assay Methods 0.000 description 38
- 108090000765 processed proteins & peptides Proteins 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 32
- 108091028043 Nucleic acid sequence Proteins 0.000 description 29
- 150000001413 amino acids Chemical class 0.000 description 27
- 102000040430 polynucleotide Human genes 0.000 description 27
- 108091033319 polynucleotide Proteins 0.000 description 27
- 239000002157 polynucleotide Substances 0.000 description 27
- 229920001184 polypeptide Polymers 0.000 description 27
- 102000004196 processed proteins & peptides Human genes 0.000 description 27
- 239000000047 product Substances 0.000 description 24
- 239000003153 chemical reaction reagent Substances 0.000 description 23
- 108010022999 Serine Proteases Proteins 0.000 description 21
- 102000012479 Serine Proteases Human genes 0.000 description 21
- 125000003275 alpha amino acid group Chemical group 0.000 description 21
- 241000186321 Cellulomonas Species 0.000 description 19
- 238000012216 screening Methods 0.000 description 19
- 102000011782 Keratins Human genes 0.000 description 16
- 108010076876 Keratins Proteins 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 231100000350 mutagenesis Toxicity 0.000 description 16
- 125000003729 nucleotide group Chemical group 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 108091034117 Oligonucleotide Proteins 0.000 description 15
- 238000002835 absorbance Methods 0.000 description 15
- 238000009826 distribution Methods 0.000 description 15
- 239000012634 fragment Substances 0.000 description 15
- 238000002703 mutagenesis Methods 0.000 description 15
- 238000003752 polymerase chain reaction Methods 0.000 description 15
- 229920002477 rna polymer Polymers 0.000 description 15
- 108010076504 Protein Sorting Signals Proteins 0.000 description 14
- 239000007983 Tris buffer Substances 0.000 description 14
- 239000002773 nucleotide Substances 0.000 description 14
- 230000003321 amplification Effects 0.000 description 13
- 238000003199 nucleic acid amplification method Methods 0.000 description 13
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000002585 base Substances 0.000 description 11
- 238000006460 hydrolysis reaction Methods 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 10
- 108010049190 N,N-dimethylcasein Proteins 0.000 description 10
- 230000007062 hydrolysis Effects 0.000 description 10
- 241000193830 Bacillus <bacterium> Species 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 238000011534 incubation Methods 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 102000053602 DNA Human genes 0.000 description 8
- 241001261512 Gracilibacillus Species 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 239000002243 precursor Substances 0.000 description 8
- 230000017854 proteolysis Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 8
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 7
- 108010076119 Caseins Proteins 0.000 description 7
- 102000011632 Caseins Human genes 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 7
- 239000005018 casein Substances 0.000 description 7
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 7
- 235000021240 caseins Nutrition 0.000 description 7
- 230000002349 favourable effect Effects 0.000 description 7
- 238000002887 multiple sequence alignment Methods 0.000 description 7
- 229920000136 polysorbate Polymers 0.000 description 7
- 230000002797 proteolythic effect Effects 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 230000028327 secretion Effects 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 102000005575 Cellulases Human genes 0.000 description 6
- 108010084185 Cellulases Proteins 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 6
- 108091035707 Consensus sequence Proteins 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 229940025131 amylases Drugs 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 description 5
- 239000007990 PIPES buffer Substances 0.000 description 5
- 108090000787 Subtilisin Proteins 0.000 description 5
- 239000000654 additive Substances 0.000 description 5
- 230000000996 additive effect Effects 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 230000002596 correlated effect Effects 0.000 description 5
- 230000002939 deleterious effect Effects 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 239000004744 fabric Substances 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- LKDMKWNDBAVNQZ-WJNSRDFLSA-N 4-[[(2s)-1-[[(2s)-1-[(2s)-2-[[(2s)-1-(4-nitroanilino)-1-oxo-3-phenylpropan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-oxobutanoic acid Chemical compound OC(=O)CCC(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)CC1=CC=CC=C1 LKDMKWNDBAVNQZ-WJNSRDFLSA-N 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- 239000007987 MES buffer Substances 0.000 description 4
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- -1 for example Substances 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 150000004965 peroxy acids Chemical class 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108010082371 succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide Proteins 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 241000193422 Bacillus lentus Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 101100449439 Drosophila melanogaster grass gene Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- LLQPHQFNMLZJMP-UHFFFAOYSA-N Fentrazamide Chemical compound N1=NN(C=2C(=CC=CC=2)Cl)C(=O)N1C(=O)N(CC)C1CCCCC1 LLQPHQFNMLZJMP-UHFFFAOYSA-N 0.000 description 3
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 239000013522 chelant Substances 0.000 description 3
- 239000002738 chelating agent Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000001627 detrimental effect Effects 0.000 description 3
- 238000012407 engineering method Methods 0.000 description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000004349 growth plate Anatomy 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000012846 protein folding Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- VHJLVAABSRFDPM-UHFFFAOYSA-N 1,4-dithiothreitol Chemical compound SCC(O)C(O)CS VHJLVAABSRFDPM-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000193764 Brevibacillus brevis Species 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 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 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 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 2
- 108010053070 Glutathione Disulfide Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 108090000301 Membrane transport proteins Proteins 0.000 description 2
- 102000003939 Membrane transport proteins Human genes 0.000 description 2
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 description 2
- 229910004835 Na2B4O7 Inorganic materials 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical group [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 241001659629 Virgibacillus Species 0.000 description 2
- 230000035508 accumulation Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 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 2
- 230000003302 anti-idiotype Effects 0.000 description 2
- 101150020960 asp gene Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 239000007844 bleaching agent Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000002825 functional assay Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000003999 initiator Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000002898 library design Methods 0.000 description 2
- 238000007169 ligase reaction Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 230000009061 membrane transport Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 230000001323 posttranslational effect Effects 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- RVCKCEDKBVEEHL-UHFFFAOYSA-N 2,3,4,5,6-pentachlorobenzyl alcohol Chemical compound OCC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl RVCKCEDKBVEEHL-UHFFFAOYSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-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
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 1
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- 241001156739 Actinobacteria <phylum> Species 0.000 description 1
- 241000203809 Actinomycetales Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 241001147782 Amphibacillus Species 0.000 description 1
- 241000555286 Aneurinibacillus Species 0.000 description 1
- 241001626813 Anoxybacillus Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000193752 Bacillus circulans Species 0.000 description 1
- 241001328122 Bacillus clausii Species 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 241000006382 Bacillus halodurans Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 101000851056 Bos taurus Elastin Proteins 0.000 description 1
- 241000555281 Brevibacillus Species 0.000 description 1
- 239000008000 CHES buffer Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241001655317 Cellulomonadaceae Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000321606 Filobacillus Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 241000193004 Halobacillus Species 0.000 description 1
- 244000141359 Malus pumila Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001655327 Micrococcales Species 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- YNLCVAQJIKOXER-UHFFFAOYSA-N N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid Chemical compound OCC(CO)(CO)NCCCS(O)(=O)=O YNLCVAQJIKOXER-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000179039 Paenibacillus Species 0.000 description 1
- 241000194109 Paenibacillus lautus Species 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010078762 Protein Precursors Proteins 0.000 description 1
- 102000014961 Protein Precursors Human genes 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 239000007997 Tricine buffer Substances 0.000 description 1
- 108091061763 Triple-stranded DNA Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 1
- 241000321595 Ureibacillus Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000002742 combinatorial mutagenesis Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000011118 depth filtration Methods 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000013024 dilution buffer Substances 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
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004851 dishwashing Methods 0.000 description 1
- VVSMKOFFCAJOSC-UHFFFAOYSA-L disodium;dodecylbenzene;sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O.CCCCCCCCCCCCC1=CC=CC=C1 VVSMKOFFCAJOSC-UHFFFAOYSA-L 0.000 description 1
- YRIUSKIDOIARQF-UHFFFAOYSA-N dodecyl benzenesulfonate Chemical compound CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 YRIUSKIDOIARQF-UHFFFAOYSA-N 0.000 description 1
- 229940071161 dodecylbenzenesulfonate Drugs 0.000 description 1
- 238000010291 electrical method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- PGBHMTALBVVCIT-VCIWKGPPSA-N framycetin Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CN)O2)N)O[C@@H]1CO PGBHMTALBVVCIT-VCIWKGPPSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000004545 gene duplication Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000011121 hardwood Substances 0.000 description 1
- 238000007825 histological assay Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000011256 inorganic filler Substances 0.000 description 1
- 229910003475 inorganic filler Inorganic materials 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical group OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000004170 methylsulfonyl group Chemical group [H]C([H])([H])S(*)(=O)=O 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 108010020132 microbial serine proteinases Proteins 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229940053050 neomycin sulfate Drugs 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 101150077915 oppA gene Proteins 0.000 description 1
- 238000013021 overheating Methods 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 230000037048 polymerization activity Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000003039 volatile agent Substances 0.000 description 1
- 101150038987 xylR gene Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Detergent Compositions (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
USA, Online Publications,London [1984],pages 181-187; and Wells et al,Gene 34:315-323 [1985])はタンパク質中の幾つかの性質を最適化する変異誘発のために、タンパク質空間を探すにの用いることができる技術のうちのひとつである。幾つ かのグループが飽和突然誘発変異により変化させるべき部位を同定するための発展した方 法を有している。(Reetz et al,Angew. Chem. Int. Edn.,44:4192-4196 [2005]; Kato et al.,J. MoI. Biol.,351 :683-692 [2005]; and Sandberg et al.s Proc. Natl. Acad. Sci.,90:8367-8371 [1993])。しかし、部位を特定するための一般化された方法は提言されていない。
SEL変異体の評価データ
ライブラリーデザイン
ah, Curr. Opin. Struct. Biol.,15:447-452 [2005]; Bloom et ah,Proc.
Natl. Acad. Sci. USA 103:5869-5874 [2006];and Guo et ah, Proc. Natl. Acad. Sci. USA 101 :9205-9210 [2004]参照のこと)、1つの性質においてポジティブな変異体の蓄積は、他の性質について好ましくない結果となる。任意の変異体が任意の性質についてアップである可能性は少なく、任意の変異体がダウンである確立が高く、(>85%)及び活性を高める3以上の蓄積が幾つかの他の性質を減らすことが非常に高いことから、変異体このことは、表2からも容易に理解できる。
定義
Harper Collins Dictionary of Biology,Harper Perennial,NY (1991)は本発明に用いる文言の多くについての一般的な辞書である。本明細書で言及しているものと同じか同等である任意の方法及び物質を本発明の実施に用いることができるが、本明細書では、好適な方法及び物質について記載している。従って、以下で定義される文言は明細書全体を参照することでより深く定義されるものである。本明細書において、単数は、明らかに言及していない限り、複数も含む。別に定義しない限り、核酸配列は左から右に向かって、5’末端から3’末端になるように記載する。アミノ酸配列は左から右に向かって、アミノ末端からカルボキシ末端となるように記載する。本発明をここに言及する特定の方法、手順、及び試薬に限定することは意図していない。それらは当業者により用いられる文脈上の意味に依存して変化することを理解されたい。
et al, (eds.), Bacillus, Plenum Publishing Corp., pages 57-72,
[1989]参照)。
al., Meth. Enzymol., 266:460-480 [1996]参照)、WU−BLAST−2は大部分がデフォルトの値である幾つかのサーチパラメーターを用いる。調節可能なパラメーターは以下の値に設定されている:オーバーラップスパン=1、オーバーラップフラクション=0.125、ワードスレッシュホールド(T)=11。HSP S及びHSPSパラメーターは動的値であり、特定の配列のコンポシシヨン及び所望の配列をサーチされる所望の配列に対する特定のデータベースのコンポジションに依存してプログラム自身により確立される。しかしながら、この値は感受性を高めるために調節することができる。「より長い」配列は配列された領域において最も実際的な残基を有するものである(アラインメントスコアを最大化するためにWU−Blast−2により導入されたギャップは無視される)。
又は見られる酵素を含む。
MAXACAL(商標),MAXAPEM(商標), PROPERASE(商標) proteases(Genencor; U.S. Pat. No. Re 34,606,and U.S. Pat. Nos.5,700,676;5,955,340;6,312,936;and6,482,628),and B. lentus variant protease products を含むがこれらに限定されない比較可能なスブチリシンプロテアーゼ(例えば、市販のプロテアーゼ)の洗浄活性の少なくとも60%、少なくとも70%、少なくとも80%、少なくとも90%、少なくとも95%の洗浄活性を意味する。例示的なスブチリシンプロテアーゼ変異体は、BPNの76、101、103、104、120、159、167、170、194、195、217、235、236、245、248、及び/又は252の位置に等しい残基の置換又は欠失を含むがこれらに限定されない。洗浄性能は、標準的な洗浄サイクル条件の後に分光光度計、又は他の解析技術により決定され、草、血液、又はミルク等の酵素に感受性のある汚れを考慮した各種洗浄アッセイにおいてこれらのスブチリシンと本発明のプロテアーゼを比較することにより決定される。
RP−HPLC(逆相高圧液体クロマトグラフィー);TLC(薄層クロマトグラフィー);MALDI−TOF(マトリックス支援レーザー脱離イオン化-飛行時間分析);Ts(トシル);Bn(ベンジル);Ph(フェニル);Ms(メシル);Et(エチル)、Me(メチル);Taq(サーマスアクチス(Thermits aquaticus)DNAポリメラーゼ);クレノウ(Klenow)(DNAポリメラーゼIラージ(クレノウ)フラグメント);EGTA(エチレングリコール−ビス(β−アミノエチルエーテル)N,N,N’,N‘−四酢酸);EDTA(エチレンジアミン四酢酸);ブラ(bla) (βガラクトシダーゼ又はアンピシリン耐性遺伝子);HDL(高密度流体);MJリサーチ(MJ Research)(MJリサーチ、レノ、ネバダ);ベースクリア(Baseclear)(ベースクリア・ビーブイ・インク、レイデン、オランダ);パーセプティブ(PerSeptive)(パーセプティブバイオシステムズ、フレーミングハム、マサチューセッツ);サーモフィニガン(ThermoFinnigan)(サーモフィニガン,サンジョーズ,カリフォルニア);アルゴ(Argo)(アルゴバイオアナリティカ,モリスプレインズ,ニュジャージ);セイツEKS(Seitz EKS)(セイツシケン(SeitzSchenk)フィルターシステムズ GmbH, バートクロイツナハ, ドイツ);パル(Pall)(パルグループ(Pall Corp), イーストヒルズ, ニュヨーク);スペクトラム(Spectrum)(スペクトラムラボラトリーズ、ドミングランコ(Dominguez Rancho)、カリフォルニニア);モレキュラートラクチャー(Molecular Structure)(モレキュラーストラクチャーグループ、ウッドランド、テキサス);アセライズ(Accelrys)(アセライズインク、サンディエゴ、カリフォルニア);ケミカルコンピューティング(Chemical Computing)(ケミカルコンピューティンググループ、モントリオール、カナダ);ニューブランクウィシュ(New Brunswick) (ニューブランクウィシュ社、エジソン、ニュージャージ);CFT(建材試験センター(Center for Test Materials),フラールディンゲン,オランダ);プロクターアンドギャンブル(Procter & Gamble)(プロクターアンドギャンブル社、シンシナティー、オハイオ);GEヘルスケア(GE
Healthcare)(GEヘルスケア,チャルフォントストリート、チャイルズ,イギリス);DNA2.0(DNA2.0,メロンパーク,カリフォルニア);OXOID(オキソイド(Oxoid)、ベージングストーク,ハンプシャー,イギリス);メガザイム(Megazyme)(メガザイム・インターナショナル・アイルランド社、ベイビジネスパーク、ウィックロー、アイルランド);フィンザイムズ(Finnzymes)(フィンザイムズ・オイ、エスポー、フィンランド);ケルコ(Kelco)(CPケルコ、ウイルミントン、デラウェア);コーニング(Corning)(コーニングライフサイエンス、コーニング、ニューヨーク);(NEN(NENライフサイエンスプロダクツ、ボストン、マサチューセッツ);ファルマAS(Pharma AS)(ファルマAS,オスロ、ノルウェイ);ダイナル(Dynal)(ダイナル,オスロ,ノルウェイ);バイオシンセシス(Bio−Synthesis)(バイオシンセシス、ルイスビル、テキサス);ATCC(アメリカン・タイプ・カルチャー・kレクション、ロックビル、メリーランド);ギブコ/BRL(Gibco/BRL)(ギブコ/BRL、グランドアイランド、ニュヨーク);シグマ(Sigma)(シグマケミカル社、セントルイス、ミズーリ);ファルマシア(Pharmacia)(ファルマシアバイオテック、ピスカタウェイ、ニュジャージ);NCBI(全米バイオテクノロジー情報センター);アプライドバイオシステムズ(Applied Biosystems)(アプライドバイオシステムズ,フォスターシティー,カリフルニア);BDバイオサイエンス及びクロンテック(BD Biosciences 及び/又はClontech)(BDバイオラボサイエンス クロンテックラボラトリーズ、パルアルト、カリフォルニア);オペロンテクノロギーズ(Operon Technologies)(オペロンテクノロジーズ社、アラメダ、カリフォルニア;MWGバイオテック(MWG Biotech)(MWGバイオテック、ハイポイント、ノースカロライナ);オリゴズEtc(Oligos Etc)(オリゴズEtc社、ウィルソンビル,オレゴン);バケム(Bachem)(バケムバイオサイエンス社、キングオブプルシア、ペンシルバニア);ディフコ(Difco)(ディフコラボラトリーズ、デトロイト、ミシガン州);メディアテック(Mediatech) (メディアテック、ハーンドン、バージニア);サンタクルーズ(Santa Cruz)(サンタクルーズバイオテクノロジー社、サンタクルーズ、カリフォルニア);オキソイド(Oxoid)(オキソイド社、オグデンスバーグ、ニューヨーク);ワージントン(Worthington)(ワージントンバイオケミカルグループ、フリーホルド、ニュジャージ);ギブコBRL(ライフテクノロジー社、ゲイサーズバーグ、メリーランド);ミリポア(Millipore)(ミリポア、ビルリカ、マサチューセッツ);バイオラド(Bio−Rad)(バイオラド、ハーロキューズ、カリフォルニア);インビトロジェン(Invitrogen)(インビトロジェングループ、サンディエゴ、カリフォルニア);NEB(ニューイングランドバイオラボズ(New England Biolabs)、ベバリー、マサチューセッツ);シグマ(Sigma)(シグマケミカル社、セントルイス、ミズリー);ピアス(Pierce)(ピアスバイオテクノロジー、ロックフォード、イリノイ);タカラ(Takara)(タカラ・バイオ・インク、大津、日本);ロシュ(Roche)(ホフマン・ロシュ,バーゼル,スイス);EMサイエンス(EM Science)(EMサイエンス、ギボスタウン、ニュジャージー);キアゲン(Qiagen)(キアゲン社、ベレニカ,カリフォルニア);バイオデザイン(Biodesign)(バイオデザイン社、ソーコ、メイン);アプタゲン(Aptagen)(アプタゲン社、ヘンドン、バージニア);ソーバル(Sorvall)(ケンドロラブラトリーによるソーバルブランド製品、アッシュビル、ノーソカロライナ);モレキュラーデバイス(Molecular Devices)(モレキュラーデバイスグループ、サニーベル、カリフォルニア);R&Dシステムズ(R&D Systems)(R&Dシステムズ、ミメアポリス, ミネソタ);ストラタジェン(Stratagene)(ストラタジェンクロニングシステム,ラホーヤ,カリフォルニア);マーシュ(Marsh)(マーシュバイオサイエンス、ロチェスター、ニューヨーク);ジーンアート(Geneart)(ジーンアートGmbH、レーゲンスブルク,ドイツ);ババイオテック(Bio−Tek)(バイオテックインスツルメンツ、ウィヌースキー、バーモント);ビアコア(Biacore)(ビアコア社,ピスカタウェイ、ニュージャージ);ペプロテック(PeproTech)(ペプロテック,ロッキーヒル,ニュージャージ);シンペップ(SynPep)(シンペップ,ダブリン,アイルランド);ニューオブジェクティブ(New Objective)(ニューオブジェクティブブランド;サイエンティフィックインスツルメンツサービス社、リンゴーズ,ニュージャージ);ウォーターズ(Waters)(ウォーターズ社、ミルフォールド、マサチューセッツ);マトリックスサイエンス(Matrix Science)(マトリックスサイエンス、ボストン、マサチューセッツ);ディオネックス(Dionex)(ディオネックスグループ、サニーベル、カリフォルニア);モンサント(Monsanto)(モンサント社、セントルイス
、ミズリー);ウインターシェル(Wintershall)(ウインターシェルAG、カッセル,ドイツ);BASF(BASF社、フローハムパーク,ニュージャージー);ハンツマン(Huntsman)(ハンツマンファーマスーティカル社、ソルトレイクシティー、ユタ);エニケム(Enichem)(エニケムイベリカ,バルセロナ、スペイン);フルカケムAG(Fluka Chemie AG)(フルカケムAG、ブッシュ,スイス);ギストブロカデス(Gist−Brocades)(ギストブロカデス,NV,デルト,オランダ);ダウコーニング(Dow Corning) (ダウコーニンググループ、ミッドランド、ミネソタ)、及びマイクロソフト(Microsoft)(マイクオソフト社、レドモンド、ワシントン)。
実施例1
アッセイ
A.96ウェルマイクロタイタープレートにおけるタンパク質含量決定のためのTCAアッセイ
B.96ウェルマイクロタイタープレートにおけるプロテアーゼのsuc−AAPF−pNAアッセイ
1.0.005%TWEEN(商標)―80(トリス緩衝液)を含む100mMトリス/HCl、pH8.6、
2.10mMCaCl2及び0.005%TWEEN(商標)−80(トリス緩衝液)を含む100mMトリス、pH8.6、
3.DMSO中160mMsuc−AAPF−pNA(suc−AAPF−pNAストック溶液)(シグマ:S−7388)
C.ケラチン加水分解活性
ケラチン:INC902111
洗剤:1.6gの洗剤を1000mlの水に溶解する(pH=8.2)、1190mgのHEPESの他に0.6mlのCaCl2/MgCl2の10,000gpgを添加して、硬度及びバッファー強度をそれぞれ、6gpg及び5mMになるようにする。pHはNaOHで8.2になるように調整する。ピルリルスルホン酸(TNBS)、シグマP−2297(水中5%溶液)
試薬A:45.4gのNa2B4O7・10H 2 O(メルク6308)及び15mlの4N NaOHを最終容量が1000mlになるように溶解する(必要に応じて加熱する)
試薬B:35.2gのNaH2PO41H2O(メルク6346)及び0.6gMa 2 SO 3 (メルク6657)を最終容量が1000mlになるように溶解する。
方法
ケラチン加水分解活性の計算
このアッセイシステムに用いた試薬を以下に示す;
ジメチルカゼイン(DMC):シグマC−9801
TWEEN(商標)−80:シグマP−8074
PIPES緩衝液(遊離酸):15.1gのシグマP−1851を960mlの水に溶かす、4N NaOHを用いてpHを7.0に調節する、1mlの5%TWEEN(商標)−80を添加して容量を1000mlにする。PIPES及びTWEEN(商標)−80の最終濃度はそれぞれ50mM及び0.005%である
ピクリルスルホン酸(TNBS):シグマP−2297(水中5%溶液)
試薬A:45.4gのNa2B4O7 10H 2 O(メルク6308)及び15mlの4N NaOHを最終容量が1000mlになるように溶解する(必要に応じて加熱する)
試薬B:35.2gのNaH2PO41H2O(メルク6346)及び0.6gMa 2 SO 3 (メルク6657)を最終容量が1000mlになるように溶解する
方法
ジメチルカゼイン加水分解活性の計算
E.熱安定性アッセイ
方法
熱安定性の計算
F.LAS安定性
試薬
ドデシルベンゼンスルホネート、硫酸ナトリウム(=LAS):シグマD−2525
TWEEN(商標)−80:シグマP−8074
TRIS緩衝液(遊離酸):6.35gのシグマT−1378を960mlの水に溶解した、pHを4N HClで8.2に調節した。TRISの最終濃度は52.5mMである
LASストック溶液:MQ水中10.5%LAS溶液(=10.5g/100mlMQ)
TRIS緩衝液:100mM/pH8.6(1000mMTris/0.005%Tween80)
TRIS−Ca緩衝液:pH8.6(100mM Tris/10mMCaCl2/0.005%Tween80)
設備機器
フラットボトムMTP:Constar(#9017)
バイオメリックFX
ASYSマルチピペッター
スペクトラマックスMTPリーダー
iEMSインキュベーター/シェーカー
イノバ4330インキュベーター/シェーカー
バイオヒットマルチチャンネルピペッター
BMGサーモスターシェーカー
方法
残余活性(%)=[t=60の値]*100/[t=10の値]
実施例2
グラム陰性アルカリ性バクテリア69B4から69B4プロテアーゼの生成
グルコース(メルク1.08342):10
ペプトン(ディフコ0118):5
イースト抽出物(ディフコ0127):5
K2HPO4:1
MgSO4・7H2O:0.2
NaCl:40
Na2CO3:10
カゼイン:20
アガー:20。
グラントアルカリ性培地(GMA)溶液A(gL −1 )
グルコース(メルク1.08342):10
ペプトン(ディフコ0118):5
イースト抽出物(ディフコ0127):5
K2HPO4:1
MgSO4・7H2O:0.2
800mlの脱イオン水に溶解し、オートクレーブで滅菌した
GMA溶液B(gL 1 )
NaCl:40
Na2CO3:10
育成条件
培養ブロスを発酵槽より回収し、10℃、500×gで30分間遠心分離して、細胞を除去した。得られた上清を、SeitzEKS(SeitzEKSフィルターシステム)上でデプス(depth)ろ過により精製した。得られた滅菌図にの培養物の上清を10kDaをカットオフする(パルオメガ10kDaミニカセット(Pall)を用いたウルトラフィルトレーションにより、約10倍に濃縮した。得られた濃縮粗69B4サンプルを更なる使用に供するまで−20℃で貯蔵した。
精製
濃縮器を用いて)バッファー交換を行った。この物質を酵素の特徴づけを行うために用いた。
実施例3
バチルススブチリス(B.subtilis)におけるASPプロテアーゼ生産
合成ASP遺伝子の発現
実施例4
組合せ変異及び複数変異ライブラリーの生成
組合せ変異の構築
複数変異ライブラリー構築物
キットに付属の5μLのサンプル緩衝液と混合して、95℃で3分間加熱して、DNAを変性させた。この反応物を氷上に2分間置き、その後、遠沈させた。次に、5μLの反応緩衝液及びTempliPhi キットに付属の0.2μLのphi29ポリメラーゼを加えて、反応物をMJリサーチPCRマシーン内で30℃、4時間、インキュベートした。phi29酵素は前記PCRマシーンで、65℃で10分間、インキュベートすることにより、加熱させて失活させた。
実施例5
複数の性質についての有害な変異の相関関係
Claims (7)
- 親タンパク質の変異体のタンパク質エンジニアリングのための方法であって、
a)親タンパク質と前記親タンパク質のタンパク質変異体の部位評価ライブラリーとを提供する工程であって、前記部位評価ライブラリーが所望の1つの位置において修飾された親タンパク質の変異体からなることを特徴とする工程、
b)所望のそれぞれの試験における、少なくとも2つの所望の性質について、前記タンパク質変異体のライブラリーと親タンパク質とを試験する工程、
c)前記所望の試験において、前記変異体について得られた値を前記親タンパク質について得られた値で割って、前記少なくとも2つの所望の性質のパフォーマンスインデックスの値を決定して、前記親タンパク質と比較した前記タンパク質変異体についての見かけの自由エネルギーの差(△△Gapp)を提供する、工程、
d)2つ以上の所望の位置における変異を組み合わせてなるタンパク質変異体について、前記少なくとも2つの所望の性質のパフォーマンスインデックスの予測値を決定する工程であって、前記2つ以上の所望の位置が、前記少なくとも2つの所望の性質のいずれかにおけるアンプロダクティブ部位を含まないことを特徴とし、前記2つ以上の位置における変異を組み合わせてなる変異体についての予測されるパフォーマンスインデックスの予測値を、前記c)において提供された前記2つ以上の所望の位置におけるそれぞれの変異の△△G app の値を加えた値に基づいて得ることを特徴とする工程、及び
e)工程d)で決定されたパフォーマンスインデックスの予測値に基づき、2つ以上の変異を組み合わせてなり、前記親タンパク質と比較して改善された第一の性質と親タンパク質と比較して少なくとも90%である第二の性質とを有することが予測されるタンパク質変異体を同定し、それゆえ少なくとも1つの所望の性質を有するメンバーが豊富なタンパク質変異体ライブラリーを提供する工程、を含む方法。 - 前記所望の性質が、電荷、洗浄性能、硬表面洗浄性能、熱安定性、貯蔵安定性、洗剤安定性、基質結合性、酵素抑制、発現レベル、反応速度、及び基質分解性から選択される、請求項1の方法。
- 前記親タンパク質が酵素である、請求項1の方法。
- 前記酵素がプロテアーゼ、トランスフェラーゼ、メタロプロテアーゼ、エステラーゼ、アミラーゼ、セルラーゼ、オキシダーゼ、クチナーゼ、及びリパーゼである、請求項1の方法。
- 前記親タンパク質が抗体及び成長因子から選択される請求項1の方法。
- 前記親タンパク質及びタンパク質変異体が少なくとも1つの洗剤成分の成分である、請求項1の方法。
- 前記洗浄性能がpH5乃至12.0のpHを有する粉末又は液体洗剤に製剤化された洗浄組成物中で試験される、請求項2の方法。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US81620206P | 2006-06-23 | 2006-06-23 | |
US60/816,202 | 2006-06-23 | ||
US93331207P | 2007-06-06 | 2007-06-06 | |
US60/933,312 | 2007-06-06 | ||
PCT/US2007/014556 WO2008002472A2 (en) | 2006-06-23 | 2007-06-22 | Systematic evaluation of sequence and activity relationships using site evaluation libraries for engineering multiple properties |
Publications (3)
Publication Number | Publication Date |
---|---|
JP2009540862A JP2009540862A (ja) | 2009-11-26 |
JP2009540862A5 JP2009540862A5 (ja) | 2015-03-12 |
JP5785686B2 true JP5785686B2 (ja) | 2015-09-30 |
Family
ID=38659837
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2009518173A Expired - Fee Related JP5785686B2 (ja) | 2006-06-23 | 2007-06-22 | 複数の性質をエンジニアリングするための部位評価ライブラリーを用いた、配列と活性との関係の系統的な評価 |
Country Status (12)
Country | Link |
---|---|
US (3) | US20080004186A1 (ja) |
EP (1) | EP2032698B1 (ja) |
JP (1) | JP5785686B2 (ja) |
KR (1) | KR101486087B1 (ja) |
CN (1) | CN101473036B (ja) |
CA (1) | CA2654269C (ja) |
DK (1) | DK2032698T3 (ja) |
ES (1) | ES2388753T3 (ja) |
HK (1) | HK1128934A1 (ja) |
PL (1) | PL2032698T3 (ja) |
PT (1) | PT2032698E (ja) |
WO (1) | WO2008002472A2 (ja) |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2264160A3 (en) | 2001-05-15 | 2011-08-31 | Novozymes A/S | Alpha-amylase variant with altered properties |
US7985569B2 (en) * | 2003-11-19 | 2011-07-26 | Danisco Us Inc. | Cellulomonas 69B4 serine protease variants |
EP2171057B1 (en) * | 2007-06-06 | 2015-09-30 | Danisco US Inc. | Methods for improving protein properties |
BRPI0911667A2 (pt) | 2008-04-23 | 2019-04-24 | Danisco Us Inc | variante de sintase de isopreno para produção microbiana aprimorada de isopreno |
PL2297313T3 (pl) | 2008-06-06 | 2015-08-31 | Danisco Us Inc | Warianty alfa-amylazy z Bacillus subtilis i sposoby ich zastosowania |
EP2623591B1 (en) * | 2008-06-06 | 2016-08-24 | Danisco US Inc. | Geobacillus stearothermophilus alpha-amylase (AMYS) variants with improved properties |
US8852912B2 (en) * | 2009-04-01 | 2014-10-07 | Danisco Us Inc. | Compositions and methods comprising alpha-amylase variants with altered properties |
SG10201501879PA (en) | 2009-04-23 | 2015-05-28 | Danisco Us Inc | Three-dimensional structure of isoprene synthase and its use thereof for generating variants |
EP4159833A3 (en) * | 2009-12-09 | 2023-07-26 | The Procter & Gamble Company | Fabric and home care products |
WO2012010406A1 (en) | 2010-07-22 | 2012-01-26 | Unilever Plc | Combinations of rhamnolipids and enzymes for improved cleaning |
BR112013010551A8 (pt) * | 2010-10-27 | 2018-07-03 | Danisco Us Inc | variantes de isopreno sintase para produção aprimorada de isopreno |
US20120157448A1 (en) | 2010-11-22 | 2012-06-21 | Adam Cook | Compounds for treating neurodegenerative diseases |
MX2013009177A (es) * | 2011-02-16 | 2013-08-29 | Novozymes As | Composiciones detergentes que comprenden metaloproteasas de m7 o m35. |
WO2013001078A1 (en) | 2011-06-30 | 2013-01-03 | Novozymes A/S | Alpha-amylase variants |
DE102011118027A1 (de) * | 2011-09-12 | 2013-03-14 | Henkel Ag & Co. Kgaa | Verfahren zur Anpassung eines Hydrolytischen Enzyms an eine das hydrolytische Enzym stabilisierende Komponente |
ES2644007T3 (es) | 2012-01-26 | 2017-11-27 | Novozymes A/S | Uso de polipéptidos con actividad de proteasa en piensos para animales y en detergentes |
JP6067409B2 (ja) | 2012-04-10 | 2017-01-25 | 花王株式会社 | アルカリプロテアーゼの溶解性向上方法 |
US9163263B2 (en) | 2012-05-02 | 2015-10-20 | The Goodyear Tire & Rubber Company | Identification of isoprene synthase variants with improved properties for the production of isoprene |
CN104394708A (zh) * | 2012-06-20 | 2015-03-04 | 诺维信公司 | 具有蛋白酶活性的多肽在动物饲料和洗涤剂中的用途 |
US9441215B2 (en) | 2013-02-06 | 2016-09-13 | Novozymes A/S | Polypeptides having protease activity |
Family Cites Families (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4760025A (en) * | 1984-05-29 | 1988-07-26 | Genencor, Inc. | Modified enzymes and methods for making same |
US5972682A (en) * | 1984-05-29 | 1999-10-26 | Genencor International, Inc. | Enzymatically active modified subtilisins |
US5700676A (en) * | 1984-05-29 | 1997-12-23 | Genencor International Inc. | Modified subtilisins having amino acid alterations |
US4965188A (en) * | 1986-08-22 | 1990-10-23 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme |
US4683195A (en) * | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4683202A (en) * | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US5322770A (en) * | 1989-12-22 | 1994-06-21 | Hoffman-Laroche Inc. | Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription |
US5340735A (en) | 1991-05-29 | 1994-08-23 | Cognis, Inc. | Bacillus lentus alkaline protease variants with increased stability |
US5830837A (en) * | 1994-11-22 | 1998-11-03 | Novo Nordisk A/S | Amylase variants |
ES2302330T3 (es) | 1994-02-24 | 2008-07-01 | Henkel Kommanditgesellschaft Auf Aktien | Enzimas mejoradas y detergentes que las contienen. |
US6682924B1 (en) * | 1995-05-05 | 2004-01-27 | Novozymes A/S | Protease variants and compositions |
DE19530816A1 (de) | 1995-08-23 | 1997-02-27 | Cognis Bio Umwelt | Verwendung von mutierter Subtilisin-Protease in kosmetischen Produkten |
US6355255B1 (en) * | 1998-12-07 | 2002-03-12 | Regents Of The University Of Minnesota | Streptococcal C5a peptidase vaccine |
MA24811A1 (fr) * | 1997-10-23 | 1999-12-31 | Procter & Gamble | Compositions de lavage contenant des variantes de proteases multisubstituees |
CN101440360A (zh) * | 1997-11-21 | 2009-05-27 | 诺沃奇梅兹有限公司 | 蛋白酶变体及组合物 |
DK1042501T3 (da) | 1997-12-24 | 2007-08-13 | Genencor Int | Fremgangsmåde til analyse af vaskeevnen af et enzym |
US6376450B1 (en) * | 1998-10-23 | 2002-04-23 | Chanchal Kumar Ghosh | Cleaning compositions containing multiply-substituted protease variants |
US6933140B1 (en) * | 1999-11-05 | 2005-08-23 | Genencor International, Inc. | Enzymes useful for changing the properties of polyester |
US6582914B1 (en) * | 2000-10-26 | 2003-06-24 | Genencor International, Inc. | Method for generating a library of oligonucleotides comprising a controlled distribution of mutations |
DE10129187A1 (de) * | 2001-06-19 | 2003-01-02 | Studiengesellschaft Kohle Mbh | Optimierung von synthetischen Katalysatoren durch gerichtete Evolution |
BRPI0306955B1 (pt) | 2002-01-16 | 2016-03-22 | Danisco Us Inc | variante de protease subtilisina de bacillus lentus, dna, vetor de expressão, célula hospedeira procariótica, bem como composição de limpeza |
CA2531494A1 (en) * | 2003-07-11 | 2005-01-20 | Novozymes A/S | Method of screening for improved specific activity of enzymes |
CA2544076A1 (en) * | 2003-10-22 | 2005-05-06 | Nascacell Ip Gmbh | Method for the identification of enzymes having desired properties by immobilisation of the reaction product on the surface of enzyme-presenting organisms |
US7985569B2 (en) * | 2003-11-19 | 2011-07-26 | Danisco Us Inc. | Cellulomonas 69B4 serine protease variants |
DK1694847T3 (da) | 2003-11-19 | 2012-09-03 | Danisco Us Inc | Serinproteaser, nucleinsyrer kodende for serinenzymer og vektorer og værtsceller omfattende disse. |
EP2295554B1 (en) * | 2003-12-03 | 2012-11-28 | Danisco US Inc. | Perhydrolase |
US20060040327A1 (en) * | 2004-08-18 | 2006-02-23 | Terry Amiss | Methods of screening proteins |
AR055444A1 (es) * | 2005-10-12 | 2007-08-22 | Procter & Gamble | Uso y produccion de metalopretasa neutra estable al almacenamiento |
-
2007
- 2007-06-22 PL PL07796357T patent/PL2032698T3/pl unknown
- 2007-06-22 CN CN2007800232384A patent/CN101473036B/zh active Active
- 2007-06-22 ES ES07796357T patent/ES2388753T3/es active Active
- 2007-06-22 DK DK07796357.7T patent/DK2032698T3/da active
- 2007-06-22 EP EP07796357A patent/EP2032698B1/en not_active Revoked
- 2007-06-22 KR KR1020087031336A patent/KR101486087B1/ko not_active IP Right Cessation
- 2007-06-22 PT PT07796357T patent/PT2032698E/pt unknown
- 2007-06-22 US US11/821,468 patent/US20080004186A1/en not_active Abandoned
- 2007-06-22 WO PCT/US2007/014556 patent/WO2008002472A2/en active Application Filing
- 2007-06-22 JP JP2009518173A patent/JP5785686B2/ja not_active Expired - Fee Related
- 2007-06-22 CA CA2654269A patent/CA2654269C/en active Active
-
2009
- 2009-09-11 HK HK09108365.1A patent/HK1128934A1/xx unknown
-
2010
- 2010-05-12 US US12/778,915 patent/US8648015B2/en active Active
-
2013
- 2013-12-20 US US14/137,539 patent/US20140187440A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
CN101473036A (zh) | 2009-07-01 |
KR20090023408A (ko) | 2009-03-04 |
WO2008002472A3 (en) | 2008-03-13 |
EP2032698B1 (en) | 2012-06-27 |
US20140187440A1 (en) | 2014-07-03 |
WO2008002472A2 (en) | 2008-01-03 |
ES2388753T3 (es) | 2012-10-18 |
EP2032698A2 (en) | 2009-03-11 |
US20110082048A1 (en) | 2011-04-07 |
CA2654269A1 (en) | 2008-12-03 |
CA2654269C (en) | 2015-09-22 |
US8648015B2 (en) | 2014-02-11 |
PL2032698T3 (pl) | 2012-11-30 |
JP2009540862A (ja) | 2009-11-26 |
PT2032698E (pt) | 2012-09-24 |
CN101473036B (zh) | 2012-12-12 |
US20080004186A1 (en) | 2008-01-03 |
DK2032698T3 (da) | 2012-09-03 |
KR101486087B1 (ko) | 2015-01-26 |
HK1128934A1 (en) | 2009-11-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5785686B2 (ja) | 複数の性質をエンジニアリングするための部位評価ライブラリーを用いた、配列と活性との関係の系統的な評価 | |
JP2009540862A5 (ja) | ||
EP2578680B1 (en) | Compositions and methods comprising variant microbial proteases | |
JP5674462B2 (ja) | タンパク質の特性の改良方法 | |
US20180094216A1 (en) | Use and production of citrate-stable neutral metalloproteases | |
KR20100075985A (ko) | 세린 프로테아제가 없는 배경에서의 중성 메탈로프로테아제의 제조 및 용도 | |
RU2520808C2 (ru) | Способ идентификации улучшенных вариантов белка |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20100521 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20100524 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20121225 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130321 |
|
A524 | Written submission of copy of amendment under article 19 pct |
Free format text: JAPANESE INTERMEDIATE CODE: A524 Effective date: 20130321 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20131203 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20140303 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20140310 |
|
A524 | Written submission of copy of amendment under article 19 pct |
Free format text: JAPANESE INTERMEDIATE CODE: A524 Effective date: 20140403 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20140729 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20141201 |
|
A524 | Written submission of copy of amendment under article 19 pct |
Free format text: JAPANESE INTERMEDIATE CODE: A524 Effective date: 20141201 |
|
A524 | Written submission of copy of amendment under article 19 pct |
Free format text: JAPANESE INTERMEDIATE CODE: A524 Effective date: 20141201 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20150122 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20150331 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20150513 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20150630 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20150727 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5785686 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
LAPS | Cancellation because of no payment of annual fees |