JP4477923B2 - 無細胞タンパク質合成用抽出液の製造方法及びその方法により製造される細胞抽出液 - Google Patents
無細胞タンパク質合成用抽出液の製造方法及びその方法により製造される細胞抽出液 Download PDFInfo
- Publication number
- JP4477923B2 JP4477923B2 JP2004105251A JP2004105251A JP4477923B2 JP 4477923 B2 JP4477923 B2 JP 4477923B2 JP 2004105251 A JP2004105251 A JP 2004105251A JP 2004105251 A JP2004105251 A JP 2004105251A JP 4477923 B2 JP4477923 B2 JP 4477923B2
- Authority
- JP
- Japan
- Prior art keywords
- extract
- cell
- cells
- protein synthesis
- coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000000284 extract Substances 0.000 title claims abstract description 98
- 230000014616 translation Effects 0.000 title claims abstract description 83
- 238000001243 protein synthesis Methods 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 45
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 241000588724 Escherichia coli Species 0.000 claims abstract description 76
- 230000012010 growth Effects 0.000 claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 35
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 33
- 210000004027 cell Anatomy 0.000 claims description 86
- 230000000694 effects Effects 0.000 claims description 26
- 238000000502 dialysis Methods 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 7
- 241000672609 Escherichia coli BL21 Species 0.000 claims description 5
- 102000002278 Ribosomal Proteins Human genes 0.000 claims description 2
- 108010000605 Ribosomal Proteins Proteins 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 40
- 238000006243 chemical reaction Methods 0.000 description 29
- 230000015572 biosynthetic process Effects 0.000 description 20
- 230000003698 anagen phase Effects 0.000 description 19
- 238000003786 synthesis reaction Methods 0.000 description 19
- 239000000243 solution Substances 0.000 description 16
- 238000013519 translation Methods 0.000 description 13
- 239000000203 mixture Substances 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 238000013518 transcription Methods 0.000 description 9
- 230000035897 transcription Effects 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 210000003705 ribosome Anatomy 0.000 description 7
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 6
- 108020004566 Transfer RNA Proteins 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 241000209140 Triticum Species 0.000 description 5
- 235000021307 Triticum Nutrition 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 210000001995 reticulocyte Anatomy 0.000 description 5
- 102000013009 Pyruvate Kinase Human genes 0.000 description 4
- 108020005115 Pyruvate Kinase Proteins 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000004420 Creatine Kinase Human genes 0.000 description 3
- 108010042126 Creatine kinase Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 2
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 2
- 102000005877 Peptide Initiation Factors Human genes 0.000 description 2
- 108010044843 Peptide Initiation Factors Proteins 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000004671 cell-free system Anatomy 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000385 dialysis solution Substances 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 229960000304 folic acid Drugs 0.000 description 2
- 235000019152 folic acid Nutrition 0.000 description 2
- 239000011724 folic acid Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000001293 nucleolytic effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- XQDQRCRASHAZBA-UHFFFAOYSA-N 2,4-dinitro-1-thiocyanatobenzene Chemical compound [O-][N+](=O)C1=CC=C(SC#N)C([N+]([O-])=O)=C1 XQDQRCRASHAZBA-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 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 1
- 108020005075 5S Ribosomal RNA Proteins 0.000 description 1
- 102000052866 Amino Acyl-tRNA Synthetases Human genes 0.000 description 1
- 108700028939 Amino Acyl-tRNA Synthetases Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 230000004568 DNA-binding 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
- 208000003468 Ehrlich Tumor Carcinoma Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 102000009097 Phosphorylases Human genes 0.000 description 1
- 108010073135 Phosphorylases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108090000829 Ribosome Inactivating Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000235343 Saccharomycetales Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 1
- 229940100228 acetyl coenzyme a Drugs 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 125000000266 alpha-aminoacyl group Chemical group 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- -1 cAMP Substances 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
- 239000005516 coenzyme A Substances 0.000 description 1
- 229940093530 coenzyme a Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002298 density-gradient ultracentrifugation Methods 0.000 description 1
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- WDRWZVWLVBXVOI-QTNFYWBSSA-L dipotassium;(2s)-2-aminopentanedioate Chemical compound [K+].[K+].[O-]C(=O)[C@@H](N)CCC([O-])=O WDRWZVWLVBXVOI-QTNFYWBSSA-L 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 101150023479 hsdS gene Proteins 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Chemical class 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Substances 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011707 mineral Chemical class 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 235000013919 monopotassium glutamate Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000004708 ribosome subunit Anatomy 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
また、本発明は、上記方法により製造された大腸菌抽出液と、タンパク質をコードする直鎖状鋳型DNAとを含む無細胞タンパク質合成系を用いるタンパク質の製造方法に関する。
系であることが好ましい。
大腸菌BL21株(F−,ompT,hsdS B)及びA19株(rna,met)を通常の液体培地を用いて一晩培養し、種培養を行った。これらの種培養液を7l(リットル)の2xYT培地(16g/lのバクトトリプトン、10g/lの酵母エキス、及び5g/lのNaCl)を含むファーメンターに接種し、十分な通気を行いながら400rpmの攪拌速度にて、26℃、30℃、及び37℃のそれぞれの温度で培養を行った。24時間まで経時的にサンプリングを行って試料の濁度(600nmの吸光度)を測定して菌体量を推定した。その結果を図1に示した。図1に示した結果より、培養温度が低くなるにつれて誘導期から対数増殖期後期までの時間は長くなるが、培養開始10〜24時間経過後にはほぼ一定の濁度を示す定常期に達することが分かる。また、図2は図1に示した増殖曲線の濁度を菌体数に換算し、縦軸を対数にしてプロットしたものである。図2に示した結果より、菌体の対数増殖期では右上がりの直線を示し、菌体数が対数的に増殖していることが分かる。また、低温になるにつれて対数増殖期の時間が顕著に長くなっている。
大腸菌S30抽出液は、Zubayら(前掲)の方法に従って、実施例1で培養したそれぞれの菌株から、対数増殖期後期(OD600=3、菌体数では約109個/mlのとき)、及び定常期(24時間経過後)の細胞を用いて調製した。タンパク質合成反応は下記の表1に示した組成の溶液に、pK7−CAT(CAT発現ベクター;Kim et al.,Eur. J. Biochem. 239, 881-886, 1996参照)を120ng(4ng/μlx30μl)添加し、上記各大腸菌のS30抽出液7.2μlを加えて全量を30μlとした反応溶液中で、37℃、1時間バッチ法により行った。合成されたCATタンパク質の定量はShawらの方法(Methods Enzymol. 735-755, 1975参照)に従った。すなわち、アセチルコエンザイムAとクロラムフェニコールを基質としてCATによるクロラムフェニコールのアセチル化反応を行い、その結果生じた還元型コエンザイムAを5,5’−ジチオビス−2−ニトロ安息香酸(DNTB)を用いて発色定量した。37℃、412nmにおける吸光度の単位時間当たりの増加量よりCATの活性を定量し、これを指標としてCATタンパク質量を決定した。
実施例1において、37℃及び26℃で培養した大腸菌BL21株から実施例2の方法に従ってS30抽出液を調製し、これらの抽出液を6〜38%のショ糖を含む緩衝液(20mM HEPES,5mM MgCl2,100mM NH4Cl,4.5mM 2−メルカプトエタノール)の上にのせ、ベックマンSW28ロータを用いて17000rpm、17時間超遠心分離を行った後、0.8mlずつのフラクションに分画した。縦軸は260nmの吸光度を測定してリボソームの存在画分を推定した。その結果を図4に示した。
次に、CATタンパク質をコードする直鎖状二本鎖DNAを鋳型として、透析系での無細胞タンパク質合成反応によりCATタンパク質の合成を行った。
直鎖状二本鎖DNAは、実施例2で使用したCAT発現ベクターpK7-CATを鋳型とし、5’プライマーM13-45 Fw:5'-CCAGGGTTTTCCCAGTCACGAC-3'(配列番号1)及び3’プライマーM13 Rev:5'-AATTTCACACAGGAAACAGCTATGAC-3'(配列番号2)を用い、通常のPCRにより得た。PCRの反応液組成は以下の表6に示したとおりである。また、ポリメラーゼ連鎖反応(PCR)は、初期変性を94℃で2分間行ったあと、94℃で30秒間、53℃で30秒間、72℃で2分間のサイクルを10回、続いて94℃で30秒間、53℃で30秒間、72℃で(2分+5秒/サイクル)間のサイクルを20回繰り返し、最後に72℃で5分間の伸長反応を1回行った。
Claims (8)
- 抑制された生育条件として26℃以上30℃以下の培養温度で大腸菌を培養し、前記培養の定常期における細胞を回収し、前記回収された細胞を破砕する無細胞タンパク質合成用抽出液の製造方法において、前記大腸菌は大腸菌BL21株であることを特徴とする無細胞タンパク質合成用抽出液の製造方法。
- 抑制された生育条件として26℃以上30℃以下の培養温度で大腸菌を培養する工程、
前記培養された大腸菌の定常期における細胞を回収する工程、及び
前記回収された大腸菌細胞からS30抽出液を調製する工程、
を含む無細胞タンパク質合成用抽出液の製造方法において、
前記大腸菌は大腸菌BL21株であることを特徴とする無細胞タンパク質合成用抽出液の製造方法。 - 請求項2に記載の方法により製造されることを特徴とする無細胞タンパク質合成用の大腸菌抽出液。
- 単位リボソーム量当たりのタンパク質合成活性が向上したリボソームを含むことを特徴とする請求項3に記載の大腸菌抽出液。
- 請求項2に記載の方法により製造された大腸菌抽出液と、タンパク質をコードする直鎖状鋳型DNAとを含む無細胞タンパク質合成系を用いることを特徴とするタンパク質の製造方法。
- 前記無細胞タンパク質合成系が透析系である請求項5に記載の方法。
- 請求項3又は4に記載の大腸菌抽出液を含むことを特徴とする無細胞タンパク質合成用キット。
- 前記無細胞タンパク質合成が直鎖状鋳型DNAを用いて行われる請求項7に記載のキット。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004105251A JP4477923B2 (ja) | 2003-05-22 | 2004-03-31 | 無細胞タンパク質合成用抽出液の製造方法及びその方法により製造される細胞抽出液 |
AT04734357T ATE396275T1 (de) | 2003-05-22 | 2004-05-21 | Verfahren zur hestellung eines extrakts für zellfreie proteinsynthese und damit hergestellter zellextrakt |
PCT/JP2004/006948 WO2004104209A1 (ja) | 2003-05-22 | 2004-05-21 | 無細胞タンパク質合成用抽出液の製造方法及びその方法により製造される細胞抽出液 |
DE602004013986T DE602004013986D1 (de) | 2003-05-22 | 2004-05-21 | Verfahren zur hestellung eines extrakts für zellfreie proteinsynthese und damit hergestellter zellextrakt |
EP04734357A EP1655375B1 (en) | 2003-05-22 | 2004-05-21 | Process for producing extract for cell-free protein synthesis and cell extract produced thereby |
US11/282,613 US7615344B2 (en) | 2003-05-22 | 2005-11-21 | Process for producing extract for cell-free protein synthesis and cell extract produced thereby |
US12/565,638 US20100168389A1 (en) | 2003-05-22 | 2009-09-23 | Process for producing extract for cell-free protein synthesis and cell extract produced thereby |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003145221 | 2003-05-22 | ||
JP2004105251A JP4477923B2 (ja) | 2003-05-22 | 2004-03-31 | 無細胞タンパク質合成用抽出液の製造方法及びその方法により製造される細胞抽出液 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2005006646A JP2005006646A (ja) | 2005-01-13 |
JP4477923B2 true JP4477923B2 (ja) | 2010-06-09 |
Family
ID=33478976
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2004105251A Expired - Lifetime JP4477923B2 (ja) | 2003-05-22 | 2004-03-31 | 無細胞タンパク質合成用抽出液の製造方法及びその方法により製造される細胞抽出液 |
Country Status (6)
Country | Link |
---|---|
US (2) | US7615344B2 (ja) |
EP (1) | EP1655375B1 (ja) |
JP (1) | JP4477923B2 (ja) |
AT (1) | ATE396275T1 (ja) |
DE (1) | DE602004013986D1 (ja) |
WO (1) | WO2004104209A1 (ja) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4441170B2 (ja) | 2002-11-28 | 2010-03-31 | 独立行政法人理化学研究所 | S12リボソームタンパク質に変異を有する大腸菌細胞抽出液及びそれを用いる無細胞系によるタンパク質の製造方法 |
JP4868731B2 (ja) | 2004-11-17 | 2012-02-01 | 独立行政法人理化学研究所 | 哺乳動物培養細胞由来の無細胞タンパク質合成システム |
JP4787488B2 (ja) | 2004-11-19 | 2011-10-05 | 独立行政法人理化学研究所 | 直鎖状鋳型dnaを用いた無細胞タンパク質合成方法及びそのための細胞抽出液 |
DK2213747T3 (da) | 2007-11-05 | 2021-08-09 | Riken | Fremgangsmåde til fremstilling af membranprotein |
WO2017022696A1 (ja) | 2015-07-31 | 2017-02-09 | 国立研究開発法人理化学研究所 | 膜タンパク質の製造方法およびその利用 |
WO2021015095A1 (ja) * | 2019-07-19 | 2021-01-28 | 大陽日酸株式会社 | タンパク質の製造方法及び無細胞タンパク質合成系キット |
CN110938644A (zh) * | 2019-11-20 | 2020-03-31 | 华南理工大学 | 一种可视化的非细胞体外显色体系及其制备方法和应用 |
US20240309391A1 (en) * | 2021-07-15 | 2024-09-19 | North Carolina State University | Compositions, methods, and systems for enhanced dna transformation in bacteria |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SU1618761A1 (ru) | 1987-04-29 | 1991-01-07 | Институт Белка Ан Ссср | Способ получени пептидов и белков в бесклеточной системе трансл ции |
JPH04200390A (ja) | 1990-11-30 | 1992-07-21 | Shigeyuki Yokoyama | 無細胞ポリペプチド合成系によるポリペプチドの製造方法 |
CA2118508A1 (en) * | 1992-04-24 | 1993-11-11 | Elizabeth S. Ward | Recombinant production of immunoglobulin-like domains in prokaryotic cells |
JP2501081B2 (ja) | 1993-10-13 | 1996-05-29 | 株式会社ソキア | 基準平面設定装置 |
KR0131166B1 (ko) * | 1994-05-04 | 1998-04-11 | 최차용 | 무세포시스템에서 단백질을 제조하는 방법 |
ATE286140T1 (de) | 2000-05-03 | 2005-01-15 | Emd Biosciences Inc | E. coli extrakt zur synthese von proteinen |
JP4590249B2 (ja) * | 2004-11-17 | 2010-12-01 | 独立行政法人理化学研究所 | 糖タンパク質合成用の無細胞タンパク質合成システム |
-
2004
- 2004-03-31 JP JP2004105251A patent/JP4477923B2/ja not_active Expired - Lifetime
- 2004-05-21 WO PCT/JP2004/006948 patent/WO2004104209A1/ja active IP Right Grant
- 2004-05-21 DE DE602004013986T patent/DE602004013986D1/de not_active Expired - Lifetime
- 2004-05-21 AT AT04734357T patent/ATE396275T1/de not_active IP Right Cessation
- 2004-05-21 EP EP04734357A patent/EP1655375B1/en not_active Expired - Lifetime
-
2005
- 2005-11-21 US US11/282,613 patent/US7615344B2/en active Active
-
2009
- 2009-09-23 US US12/565,638 patent/US20100168389A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
JP2005006646A (ja) | 2005-01-13 |
DE602004013986D1 (de) | 2008-07-03 |
US20100168389A1 (en) | 2010-07-01 |
EP1655375A4 (en) | 2006-08-02 |
EP1655375B1 (en) | 2008-05-21 |
US7615344B2 (en) | 2009-11-10 |
WO2004104209A1 (ja) | 2004-12-02 |
ATE396275T1 (de) | 2008-06-15 |
EP1655375A1 (en) | 2006-05-10 |
US20060134735A1 (en) | 2006-06-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9528137B2 (en) | Methods for cell-free protein synthesis | |
US6994986B2 (en) | In vitro synthesis of polypeptides by optimizing amino acid metabolism | |
US20100168389A1 (en) | Process for producing extract for cell-free protein synthesis and cell extract produced thereby | |
WO2000055353A1 (en) | In vitro macromolecule biosynthesis methods using exogenous amino acids and a novel atp regeneration system | |
Wilmes et al. | Fed-batch process for the psychrotolerant marine bacterium Pseudoalteromonas haloplanktis | |
EP3564376A1 (en) | Gene which encodes alanyl-glutamine dipeptide biosynthetic enzyme and application thereof | |
US11673921B2 (en) | Cell-free protein synthesis platform derived from cellular extracts of Vibrio natriegens | |
EP1278883B1 (en) | E. coli extract for protein synthesis | |
CN106148296A (zh) | 一种重组谷氨酰胺转氨酶的生产方法 | |
US20100216184A1 (en) | Preparation of cell extract and its application for cell-free protein systhesis | |
JP2020505951A (ja) | 無細胞タンパク質合成のためのプロモーター構築物 | |
Brüsehaber et al. | Production of pig liver esterase in batch fermentation of E. coli Origami | |
US20230117150A1 (en) | Fully orthogonal system for protein synthisis in bacterial cells | |
EP1816207B1 (en) | Cell-free protein synthesis method with the use of linear template dna and cell extract therefor | |
Pasini et al. | Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures | |
JP2022535651A (ja) | 好熱性タンパク質を利用した組換えインビトロ転写及び翻訳のための系、方法及び組成物 | |
US20210355434A1 (en) | Methods of Producing Cannabinoids | |
Hiller et al. | The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes | |
KR101572547B1 (ko) | 재조합 코리네박테리움 글루타미쿰 및 그의 용도 | |
EP4001415A1 (en) | Protein production method and cell-free protein synthesis kit | |
JP2020000071A (ja) | L−システインの製造方法 | |
JP2020000070A (ja) | L−システインの分解抑制 | |
KR101123070B1 (ko) | 세포내 에너지 농도가 높은 세포 및 그의 용도 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20070330 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20091104 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20091224 |
|
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: 20100302 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20100312 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 4477923 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20130319 Year of fee payment: 3 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20160319 Year of fee payment: 6 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
EXPY | Cancellation because of completion of term |