JP2022130501A - エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 - Google Patents
エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 Download PDFInfo
- Publication number
- JP2022130501A JP2022130501A JP2022097799A JP2022097799A JP2022130501A JP 2022130501 A JP2022130501 A JP 2022130501A JP 2022097799 A JP2022097799 A JP 2022097799A JP 2022097799 A JP2022097799 A JP 2022097799A JP 2022130501 A JP2022130501 A JP 2022130501A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- yeast
- library
- dna
- electroporation
- 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
- 210000004027 cell Anatomy 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 46
- 210000005253 yeast cell Anatomy 0.000 title claims abstract description 44
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 63
- 238000004520 electroporation Methods 0.000 claims abstract description 38
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 21
- 239000000600 sorbitol Substances 0.000 claims abstract description 21
- 238000005406 washing Methods 0.000 claims abstract description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 239000001110 calcium chloride Substances 0.000 claims abstract description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 6
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 108091008874 T cell receptors Proteins 0.000 claims description 48
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 48
- 239000013598 vector Substances 0.000 claims description 38
- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 230000009466 transformation Effects 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 14
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 235000011148 calcium chloride Nutrition 0.000 claims description 5
- 238000004364 calculation method Methods 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound 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 SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- 229910000073 phosphorus hydride Inorganic materials 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000010899 nucleation Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 abstract description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 56
- 108020004414 DNA Proteins 0.000 description 37
- 239000000427 antigen Substances 0.000 description 20
- 108091007433 antigens Proteins 0.000 description 18
- 102000036639 antigens Human genes 0.000 description 18
- 239000000047 product Substances 0.000 description 16
- 230000014509 gene expression Effects 0.000 description 11
- 230000005684 electric field Effects 0.000 description 10
- 239000013604 expression vector Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 8
- 238000002818 protein evolution Methods 0.000 description 8
- 230000010076 replication Effects 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 239000013611 chromosomal DNA Substances 0.000 description 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 239000007222 ypd medium Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 210000002230 centromere Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001902 propagating effect Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 2
- 239000005517 L01XE01 - Imatinib Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229960002303 citric acid monohydrate Drugs 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003783 haploid cell Anatomy 0.000 description 2
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- OJHZNMVJJKMFGX-RNWHKREASA-N (4r,4ar,7ar,12bs)-9-methoxy-3-methyl-1,2,4,4a,5,6,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one;2,3-dihydroxybutanedioic acid Chemical compound OC(=O)C(O)C(O)C(O)=O.O=C([C@@H]1O2)CC[C@H]3[C@]4([H])N(C)CC[C@]13C1=C2C(OC)=CC=C1C4 OJHZNMVJJKMFGX-RNWHKREASA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- -1 0.01 Chemical compound 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108010023063 Bacto-peptone Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 241000235036 Debaryomyces hansenii Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 101150007280 LEU2 gene Proteins 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000235346 Schizosaccharomyces Species 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000307523 Xenostegia media Species 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 229940080856 gleevec Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940022353 herceptin Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229960002411 imatinib Drugs 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound 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
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 229920000867 polyelectrolyte Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 229940124606 potential therapeutic agent Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000013605 shuttle vector 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
- 239000001509 sodium citrate Substances 0.000 description 1
- 229960000999 sodium citrate dihydrate Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000010399 three-hybrid screening Methods 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- 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
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
1.YPD培地
酵母抽出物 10g
バクトペプトン 20g
デキストロース 20g
H2Oで容積を1Lにする(滅菌グルコースを高圧蒸気滅菌溶液に添加する)
クエン酸ナトリウム二水和物 14.8g(50mM最終)
クエン酸一水和物 4.2g(20mM最終)
800mlのH2O中、高圧蒸気滅菌。
カザミノ酸 5.0g
酵母窒素原礎培地(アミノ酸なし) 6.7g
グルコース 20g
H2Oで容積を1Lにして滅菌濾過する
ソルビトール 182.2g
寒天 15g
クエン酸ナトリウム 14.8g
クエン酸一水和物 4.2g
800mlのH2O中、高圧蒸気滅菌し約55°Cに冷却する。
カザミノ酸 5.0g
酵母窒素原礎培地(アミノ酸なし) 6.7g
グルコース 20g
カナマイシン硫酸塩 35mg
200mlのH2O中、滅菌濾過、冷却した高圧蒸気滅菌溶液に添加する
-80℃から新たに解凍した20μlの酵母原液をYPD寒天プレート上に画線塗抹し、30℃で2日間培養した。単一コロニー(コロニー全体を採取する)をYPD寒天プレートから15mlのYPD培地に採取して、30℃で一晩振盪した。翌朝、10mlの培養物を100mlの新鮮なYPD培地に移し入れ、振盪を30℃で7時間継続した。OD600を測定して、1Lの冷YPD培地をOD600が0.2になるように接種した。振盪フラスコを予冷した(4℃)振盪機に入れた。振盪機は、就業日が始まる5時間前に加熱(30℃)および振盪(250rpm)が開始するようにプログラムした。OD600が1.5に達するまで培養を実施したが、これは通常、振盪開始後6時間であった。
400μlの細胞をH2O中の5〜10μlのDNA(ベクター)と混合し、3分間氷上に保ち、予冷した0.2cm電気穿孔キュベットに移し入れた。BioRad MicroPulser電気穿孔システムを用いて、細胞を2.5kVで電気穿孔した。典型的な時定数は約4ミリ秒であり、好ましくは4ミリ秒であった。電気穿孔した細胞を、振盪せずに30℃で1時間、10mlの1Mソルビトール:YPD培地の1:1混合物に移し入れた。細胞を室温で3分間2,000gで収集し、室温で10mlのSD−CAAに再懸濁した。多様性の計算のために希釈を実施した(1:105~1:107)。カナマイシンを含有するSD−CAAプレート上で、希釈液を30℃で1日間℃、そして室温で3日間培養した。ライブラリーを100mlのSD-CAAに移し入れ(好ましくは電気穿孔毎に)、振盪を30℃および160rpmで24時間継続した。拡大されたライブラリーは、誘導のために直接使用し得て、または4℃で2週間保存し得る。長期保存は、-80℃で30%のグリセロール中で凍結することによって実施し得る。
Claims (9)
- a)酵母細胞を1.0~2のOD600に培養するステップと;
b)前記細胞を冷水で洗浄するステップと;
c)前記細胞をソルビトールとCaCl2とを含んでなる冷溶液で洗浄するステップと;
d)前記細胞を酢酸リチウムとトリス2-カルボキシエチル)ホスフィン(TCEP)とを含んでなる溶液中でインキュベートするステップと;
e)前記細胞をソルビトールとCaCl2とを含んでなる冷溶液で洗浄するステップと;
f)前記細胞をソルビトールを含んでなる溶液に再懸濁するステップと;
g)任意選択的に、前記細胞を適切に保存するステップと
を含んでなる、エレクトロコンピテント酵母細胞を調製する方法。 - a)請求項1に記載の方法によるエレクトロコンピテント酵母細胞を提供するステップと;
b)前記細胞をソルビトールを含んでなる冷溶液で洗浄するステップと;
c)前記細胞を形質移入されるDNAと混合し、電気穿孔前混合物を形成するステップと;
d)前記電気穿孔前混合物を適切な電気穿孔キュベットに移し入れるステップと;
e)前記細胞を2.5kV/cm~12.5kV/cmで2~5ミリ秒間電気穿孔するステップと
を含んでなる、エレクトロコンピテント酵母細胞を形質移入する方法。 - 前記DNAが線状または環状である、請求項2に記載の方法。
- 前記DNAが、例えば酵母表面ディスプレイライブラリーの形態である、目的のタンパク質のライブラリーをコードするDNA断片のライブラリーを含んでなる、請求項2または3に記載の方法。
- 前記ディスプレイライブラリーが、T細胞受容体(TCR)ライブラリーである、請求項4に記載の方法。
- 前記形質転換効率が、1×108酵母形質転換体/μgベクターDNAよりも高く、好ましくは2×108酵母形質転換体/μgベクターDNAよりも高い、請求項2~5のいずれか一項に記載の方法
- a)請求項2~6のいずれか一項に記載の方法による形質移入された酵母細胞を提供するステップと;
b)前記形質移入細胞を増殖培地中でソルビトール溶液の1:1混合物に希釈するステップと;
c)細胞を適切な増殖培地中に再懸濁するステップと;
d)任意選択的に、多様性の計算のために希釈を実施して、前記希釈液をカナマイシンを含有するSD-CAAプレート上播に種するステップと;
e)前記ライブラリーを適切な増殖培地に移し入れ、前記ライブラリーを電気穿孔毎に拡大するステップと;
f)任意選択的に、前記拡大されたイブラリーを適切に保存するステップと
を含んでなる、例えば酵母表面ディスプレイライブラリーの形態である、目的のタンパク質の改善された酵母ライブラリーを生成する方法。 - 前記ディスプレイライブラリーが、T細胞受容体ライブラリーである、請求項7に記載の方法。
- 前記ライブラリーの多様性が、1012よりも高い、請求項7または8に記載の方法。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102016121899.5A DE102016121899A1 (de) | 2016-11-15 | 2016-11-15 | Verfahren zur Herstellung von elektrokompetenten Hefezellen und Verfahren zur Verwendung dieser Zellen |
DE102016121899.5 | 2016-11-15 | ||
PCT/EP2017/079011 WO2018091396A1 (en) | 2016-11-15 | 2017-11-13 | Method for preparing electrocompetent yeast cells, and method for using said cells |
JP2019521754A JP7133226B2 (ja) | 2016-11-15 | 2017-11-13 | エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019521754A Division JP7133226B2 (ja) | 2016-11-15 | 2017-11-13 | エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
JP2022130501A true JP2022130501A (ja) | 2022-09-06 |
Family
ID=60484335
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019521754A Active JP7133226B2 (ja) | 2016-11-15 | 2017-11-13 | エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 |
JP2022097799A Pending JP2022130501A (ja) | 2016-11-15 | 2022-06-17 | エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019521754A Active JP7133226B2 (ja) | 2016-11-15 | 2017-11-13 | エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 |
Country Status (23)
Country | Link |
---|---|
US (3) | US10683495B2 (ja) |
EP (1) | EP3541947B1 (ja) |
JP (2) | JP7133226B2 (ja) |
KR (1) | KR102194520B1 (ja) |
CN (1) | CN109983130B (ja) |
AR (1) | AR110087A1 (ja) |
AU (1) | AU2017360666C1 (ja) |
BR (1) | BR112019008385A2 (ja) |
CA (1) | CA3043953A1 (ja) |
DE (1) | DE102016121899A1 (ja) |
DK (1) | DK3541947T3 (ja) |
EA (1) | EA201990758A1 (ja) |
ES (1) | ES2952039T3 (ja) |
IL (1) | IL266538A (ja) |
MA (1) | MA46835A (ja) |
MX (1) | MX2019005622A (ja) |
MY (1) | MY191776A (ja) |
NZ (1) | NZ752651A (ja) |
PH (1) | PH12019501001A1 (ja) |
SG (1) | SG11201903329WA (ja) |
TW (1) | TWI720268B (ja) |
UA (1) | UA125076C2 (ja) |
WO (1) | WO2018091396A1 (ja) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102017125888A1 (de) | 2017-11-06 | 2019-05-23 | Immatics Biotechnologies Gmbh | Neue konstruierte T-Zell-Rezeptoren und deren Verwendung in Immuntherapie |
IL274505B2 (en) | 2017-11-06 | 2024-04-01 | Immatics Biotechnologies Gmbh | Transgenic T-cell receptors and immunotherapy using them |
DE102019108125B4 (de) | 2019-03-28 | 2022-02-03 | Immatics US, Inc. | Cd28 t-zellkulturen, zusammensetzungen und verfahren zu deren verwendung |
WO2020191172A1 (en) | 2019-03-19 | 2020-09-24 | Immatics US, Inc. | Cd28 t cell cultures, compositions, and methods of using thereof |
DE102019121007A1 (de) | 2019-08-02 | 2021-02-04 | Immatics Biotechnologies Gmbh | Antigenbindende Proteine, die spezifisch an MAGE-A binden |
EP4090365A1 (en) | 2020-01-15 | 2022-11-23 | Immatics Biotechnologies GmbH | Antigen binding proteins specifically binding prame |
JP2024518378A (ja) | 2021-05-05 | 2024-05-01 | イマティクス バイオテクノロジーズ ゲーエムベーハー | Prameに特異的に結合する抗原結合タンパク質 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007222179A (ja) * | 1991-04-10 | 2007-09-06 | Scripps Res Inst:The | ファージミドを使用するヘテロ二量体受容体ライブラリー |
JP2010536359A (ja) * | 2007-08-20 | 2010-12-02 | ネクステラ エーエス | pVIIIファージディスプレイ |
JP2011512841A (ja) * | 2008-03-03 | 2011-04-28 | アボット・ラボラトリーズ | 酵母を形質転換するための方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009114096A1 (en) | 2008-03-13 | 2009-09-17 | Cardiac Pacemakers, Inc. | Systems and devices for modulating autonomic tone |
EP2598528A1 (en) | 2010-07-28 | 2013-06-05 | Immunocore Ltd. | T cell receptors |
AU2015371083B2 (en) * | 2014-12-22 | 2022-01-27 | Universität Zürich | Directed evolution of membrane proteins in eukaryotic cells with a cell wall |
-
2016
- 2016-11-15 DE DE102016121899.5A patent/DE102016121899A1/de active Pending
-
2017
- 2017-11-13 NZ NZ752651A patent/NZ752651A/en unknown
- 2017-11-13 MA MA046835A patent/MA46835A/fr unknown
- 2017-11-13 EP EP17805126.4A patent/EP3541947B1/en active Active
- 2017-11-13 EA EA201990758A patent/EA201990758A1/ru unknown
- 2017-11-13 JP JP2019521754A patent/JP7133226B2/ja active Active
- 2017-11-13 WO PCT/EP2017/079011 patent/WO2018091396A1/en unknown
- 2017-11-13 SG SG11201903329WA patent/SG11201903329WA/en unknown
- 2017-11-13 ES ES17805126T patent/ES2952039T3/es active Active
- 2017-11-13 AU AU2017360666A patent/AU2017360666C1/en active Active
- 2017-11-13 MX MX2019005622A patent/MX2019005622A/es unknown
- 2017-11-13 CA CA3043953A patent/CA3043953A1/en active Pending
- 2017-11-13 KR KR1020197013641A patent/KR102194520B1/ko active IP Right Grant
- 2017-11-13 CN CN201780069926.8A patent/CN109983130B/zh active Active
- 2017-11-13 DK DK17805126.4T patent/DK3541947T3/da active
- 2017-11-13 MY MYPI2019002307A patent/MY191776A/en unknown
- 2017-11-13 UA UAA201904554A patent/UA125076C2/uk unknown
- 2017-11-13 BR BR112019008385A patent/BR112019008385A2/pt unknown
- 2017-11-15 US US15/813,879 patent/US10683495B2/en active Active
- 2017-11-15 TW TW106139472A patent/TWI720268B/zh active
- 2017-11-15 AR ARP170103182A patent/AR110087A1/es unknown
-
2019
- 2019-05-07 PH PH12019501001A patent/PH12019501001A1/en unknown
- 2019-05-08 IL IL266538A patent/IL266538A/en unknown
-
2020
- 2020-05-08 US US16/869,784 patent/US11104894B2/en active Active
-
2021
- 2021-07-29 US US17/389,061 patent/US20210355478A1/en active Pending
-
2022
- 2022-06-17 JP JP2022097799A patent/JP2022130501A/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007222179A (ja) * | 1991-04-10 | 2007-09-06 | Scripps Res Inst:The | ファージミドを使用するヘテロ二量体受容体ライブラリー |
JP2010536359A (ja) * | 2007-08-20 | 2010-12-02 | ネクステラ エーエス | pVIIIファージディスプレイ |
JP2011512841A (ja) * | 2008-03-03 | 2011-04-28 | アボット・ラボラトリーズ | 酵母を形質転換するための方法 |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7133226B2 (ja) | エレクトロコンピテント酵母細胞を調製する方法および前記細胞を使用する方法 | |
JP5647009B2 (ja) | 酵母を形質転換するための方法 | |
SA94150015B1 (ar) | إنتاج وتنقية الليمفوكين lymphokine | |
WO2022052877A1 (zh) | 对细胞中的靶位点进行基因编辑的方法 | |
EA043912B1 (ru) | Способ получения электрокомпетентных дрожжевых клеток и способ применения указанных клеток | |
CA2730741A1 (en) | Self-replicating vector lacking an antibiotic-resistance gene | |
WO2019237391A1 (zh) | CRISPR/Cas9 靶向敲除人 TXGP1 基因及其特异性 gRNA | |
CN113073099B (zh) | sgRNA库、敲低基因文库及敲低基因文库的构建方法和应用 | |
CN115197968A (zh) | 抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用 | |
CN115992175A (zh) | 一种将目的基因定点整合至免疫细胞特定位点的方法及其应用 | |
Bobik et al. | Genetic Engineering of Native Chain Combinations of B-Cell Repertoires on the Surface of Methylotrophic Yeasts Pichia pastoris | |
CN112891523A (zh) | 猪带绦虫Ts14-3-3.3 DNA疫苗制备及鉴定方法 | |
WO2019237372A1 (zh) | 定点插入act35基因的cho细胞株的构建方法及其用途 | |
WO2019237387A1 (zh) | 用于敲除人ACT35基因的gRNA序列及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220715 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220715 |
|
RD03 | Notification of appointment of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7423 Effective date: 20230227 |
|
RD07 | Notification of extinguishment of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7427 Effective date: 20230302 |
|
RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20230303 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230704 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20231002 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231201 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20240305 |
|
RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20240502 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20240604 |