JPH0333315B2 - - Google Patents
Info
- Publication number
- JPH0333315B2 JPH0333315B2 JP24701787A JP24701787A JPH0333315B2 JP H0333315 B2 JPH0333315 B2 JP H0333315B2 JP 24701787 A JP24701787 A JP 24701787A JP 24701787 A JP24701787 A JP 24701787A JP H0333315 B2 JPH0333315 B2 JP H0333315B2
- Authority
- JP
- Japan
- Prior art keywords
- dhfr
- purification
- bacillus subtilis
- dihydrofolate reductase
- enzyme
- 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
Links
- 238000000746 purification Methods 0.000 claims description 20
- 238000004440 column chromatography Methods 0.000 claims description 15
- 102000037865 fusion proteins Human genes 0.000 claims description 15
- 108020001507 fusion proteins Proteins 0.000 claims description 15
- 241000588724 Escherichia coli Species 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 229960000553 somatostatin Drugs 0.000 claims description 6
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 claims description 5
- 101900343733 Bacillus subtilis Dihydrofolate reductase Proteins 0.000 claims description 5
- 229960002385 streptomycin sulfate Drugs 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 108010022337 Leucine Enkephalin Proteins 0.000 claims description 3
- URLZCHNOLZSCCA-UHFFFAOYSA-N leu-enkephalin Chemical compound C=1C=C(O)C=CC=1CC(N)C(=O)NCC(=O)NCC(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 URLZCHNOLZSCCA-UHFFFAOYSA-N 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 238000005194 fractionation Methods 0.000 claims description 2
- 101800004538 Bradykinin Proteins 0.000 claims 2
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 claims 2
- 102100035792 Kininogen-1 Human genes 0.000 claims 2
- 102000005157 Somatostatin Human genes 0.000 claims 2
- 108010056088 Somatostatin Proteins 0.000 claims 2
- 108010022394 Threonine synthase Proteins 0.000 claims 2
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 claims 2
- 102000004419 dihydrofolate reductase Human genes 0.000 claims 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 claims 2
- 108010092674 Enkephalins Proteins 0.000 claims 1
- URLZCHNOLZSCCA-VABKMULXSA-N Leu-enkephalin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 URLZCHNOLZSCCA-VABKMULXSA-N 0.000 claims 1
- 238000005571 anion exchange chromatography Methods 0.000 claims 1
- 238000001641 gel filtration chromatography Methods 0.000 claims 1
- 230000002209 hydrophobic effect Effects 0.000 claims 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 22
- 108020001096 dihydrofolate reductase Proteins 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 18
- 229940088598 enzyme Drugs 0.000 description 18
- 230000001580 bacterial effect Effects 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 14
- 244000063299 Bacillus subtilis Species 0.000 description 10
- 235000014469 Bacillus subtilis Nutrition 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 239000000284 extract Substances 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 238000010306 acid treatment Methods 0.000 description 4
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 239000000969 carrier Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229960001931 ampicillin sodium Drugs 0.000 description 2
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].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 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 230000005526 G1 to G0 transition Effects 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
- 101000926713 Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) Dihydrofolate reductase HdrA Proteins 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24701787A JPS6486871A (en) | 1987-09-30 | 1987-09-30 | Method for separating and purifying recombinant protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24701787A JPS6486871A (en) | 1987-09-30 | 1987-09-30 | Method for separating and purifying recombinant protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6486871A JPS6486871A (en) | 1989-03-31 |
| JPH0333315B2 true JPH0333315B2 (enrdf_load_stackoverflow) | 1991-05-16 |
Family
ID=17157159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24701787A Granted JPS6486871A (en) | 1987-09-30 | 1987-09-30 | Method for separating and purifying recombinant protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6486871A (enrdf_load_stackoverflow) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002007220A1 (fr) | 2000-07-19 | 2002-01-24 | Shindo Company, Ltd. | Dispositif a semi-conducteurs |
-
1987
- 1987-09-30 JP JP24701787A patent/JPS6486871A/ja active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002007220A1 (fr) | 2000-07-19 | 2002-01-24 | Shindo Company, Ltd. | Dispositif a semi-conducteurs |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6486871A (en) | 1989-03-31 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |