CY1341A - Human tissue plasminogen activator pharmaceutical compositions containing it processes for making it and dna and transformed cell intermediates therefor - Google Patents

Human tissue plasminogen activator pharmaceutical compositions containing it processes for making it and dna and transformed cell intermediates therefor Download PDF

Info

Publication number: CY1341A
Authority: CY; Cyprus
Prior art keywords: plasminogen activator; tissue plasminogen; human; dna; human tissue
Prior art date: 1982-05-05

Application number

CY1341A

Other languages

English (en)

Original Assignee

Genentech Inc

Priority date (The priority date 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 date listed.)

1982-05-05

Filing date

1983-05-04

Publication date

1987-01-16

Family has litigation

First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=27409206&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=CY1341(A) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.

1983-04-07 Priority claimed from US06/483,052 external-priority patent/US4766075A/en

1983-05-04 Application filed by Genentech Inc filed Critical Genentech Inc

1987-01-16 Publication of CY1341A publication Critical patent/CY1341A/en

Links

108090000373 Tissue Plasminogen Activator Proteins 0.000 title claims description 159
102000003978 Tissue Plasminogen Activator Human genes 0.000 title claims description 154
229960000187 tissue plasminogen activator Drugs 0.000 title claims description 151
241000282414 Homo sapiens Species 0.000 title claims description 66
238000000034 method Methods 0.000 title claims description 59
230000008569 process Effects 0.000 title claims description 9
239000008194 pharmaceutical composition Substances 0.000 title claims description 5
239000000543 intermediate Substances 0.000 title description 2
108090000623 proteins and genes Proteins 0.000 claims description 69
239000013612 plasmid Substances 0.000 claims description 65
108020004414 DNA Proteins 0.000 claims description 46
102000004169 proteins and genes Human genes 0.000 claims description 44
230000014509 gene expression Effects 0.000 claims description 36
101000801481 Homo sapiens Tissue-type plasminogen activator Proteins 0.000 claims description 24
150000001413 amino acids Chemical class 0.000 claims description 24
108090000765 processed proteins & peptides Proteins 0.000 claims description 24
238000004113 cell culture Methods 0.000 claims description 22
102000047823 human PLAT Human genes 0.000 claims description 22
102000001938 Plasminogen Activators Human genes 0.000 claims description 19
229940127126 plasminogen activator Drugs 0.000 claims description 19
239000000203 mixture Substances 0.000 claims description 18
108091028043 Nucleic acid sequence Proteins 0.000 claims description 16
102000004196 processed proteins & peptides Human genes 0.000 claims description 16
108010001014 Plasminogen Activators Proteins 0.000 claims description 15
239000013604 expression vector Substances 0.000 claims description 12
229920001184 polypeptide Polymers 0.000 claims description 12
244000005700 microbiome Species 0.000 claims description 9
230000001131 transforming effect Effects 0.000 claims description 4
230000013595 glycosylation Effects 0.000 claims description 3
238000006206 glycosylation reaction Methods 0.000 claims description 3
208000019553 vascular disease Diseases 0.000 claims description 3
108050006955 Tissue-type plasminogen activator Proteins 0.000 claims 1
238000012258 culturing Methods 0.000 claims 1
238000007911 parenteral administration Methods 0.000 claims 1
210000004027 cell Anatomy 0.000 description 116
239000012634 fragment Substances 0.000 description 68
FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 58
229960000485 methotrexate Drugs 0.000 description 58
108010022394 Threonine synthase Proteins 0.000 description 52
102000004419 dihydrofolate reductase Human genes 0.000 description 51
235000018102 proteins Nutrition 0.000 description 42
239000000499 gel Substances 0.000 description 34
108020004635 Complementary DNA Proteins 0.000 description 31
238000010804 cDNA synthesis Methods 0.000 description 31
239000002299 complementary DNA Substances 0.000 description 31
LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 30
239000000523 sample Substances 0.000 description 29
BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 28
102000013566 Plasminogen Human genes 0.000 description 28
108010051456 Plasminogen Proteins 0.000 description 28
230000000694 effects Effects 0.000 description 28
229950003499 fibrin Drugs 0.000 description 28
239000000047 product Substances 0.000 description 28
239000013598 vector Substances 0.000 description 27
102000009123 Fibrin Human genes 0.000 description 26
108010073385 Fibrin Proteins 0.000 description 26
229940024606 amino acid Drugs 0.000 description 25
235000001014 amino acid Nutrition 0.000 description 24
239000000284 extract Substances 0.000 description 23
229940012957 plasmin Drugs 0.000 description 23
238000003556 assay Methods 0.000 description 22
210000001519 tissue Anatomy 0.000 description 22
HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 20
ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 20
238000006243 chemical reaction Methods 0.000 description 20
239000002609 medium Substances 0.000 description 20
241000588724 Escherichia coli Species 0.000 description 16
201000001441 melanoma Diseases 0.000 description 16
108020004999 messenger RNA Proteins 0.000 description 15
238000010276 construction Methods 0.000 description 14
241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 13
238000007792 addition Methods 0.000 description 13
230000003321 amplification Effects 0.000 description 13
238000003199 nucleic acid amplification method Methods 0.000 description 13
239000000243 solution Substances 0.000 description 13
108091032973 (ribonucleotides)n+m Proteins 0.000 description 12
108091026890 Coding region Proteins 0.000 description 12
230000003480 fibrinolytic effect Effects 0.000 description 12
238000004519 manufacturing process Methods 0.000 description 12
229920002401 polyacrylamide Polymers 0.000 description 12
238000002360 preparation method Methods 0.000 description 12
238000013519 translation Methods 0.000 description 12
230000014616 translation Effects 0.000 description 12
102000004190 Enzymes Human genes 0.000 description 11
108090000790 Enzymes Proteins 0.000 description 11
102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 11
108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 11
125000003275 alpha amino acid group Chemical group 0.000 description 11
229940088598 enzyme Drugs 0.000 description 11
230000010076 replication Effects 0.000 description 11
229960005356 urokinase Drugs 0.000 description 11
108020004511 Recombinant DNA Proteins 0.000 description 10
230000029087 digestion Effects 0.000 description 10
150000007523 nucleic acids Chemical class 0.000 description 10
210000002966 serum Anatomy 0.000 description 10
DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 9
239000000020 Nitrocellulose Substances 0.000 description 9
238000001962 electrophoresis Methods 0.000 description 9
239000000463 material Substances 0.000 description 9
229920001220 nitrocellulos Polymers 0.000 description 9
108020004707 nucleic acids Proteins 0.000 description 9
102000039446 nucleic acids Human genes 0.000 description 9
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 8
XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
239000012190 activator Substances 0.000 description 8
230000012010 growth Effects 0.000 description 8
FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 7
QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 7
240000004808 Saccharomyces cerevisiae Species 0.000 description 7
235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 7
230000015572 biosynthetic process Effects 0.000 description 7
230000000875 corresponding effect Effects 0.000 description 7
238000005516 engineering process Methods 0.000 description 7
239000003527 fibrinolytic agent Substances 0.000 description 7
238000011534 incubation Methods 0.000 description 7
230000007246 mechanism Effects 0.000 description 7
230000000813 microbial effect Effects 0.000 description 7
108091008146 restriction endonucleases Proteins 0.000 description 7
WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
108010076504 Protein Sorting Signals Proteins 0.000 description 6
102000012479 Serine Proteases Human genes 0.000 description 6
108010022999 Serine Proteases Proteins 0.000 description 6
IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
108090000631 Trypsin Proteins 0.000 description 6
102000004142 Trypsin Human genes 0.000 description 6
QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 6
239000011543 agarose gel Substances 0.000 description 6
230000001580 bacterial effect Effects 0.000 description 6
239000000872 buffer Substances 0.000 description 6
238000003776 cleavage reaction Methods 0.000 description 6
230000009089 cytolysis Effects 0.000 description 6
239000013613 expression plasmid Substances 0.000 description 6
238000000605 extraction Methods 0.000 description 6
238000009396 hybridization Methods 0.000 description 6
FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 6
238000000338 in vitro Methods 0.000 description 6
239000002773 nucleotide Substances 0.000 description 6
125000003729 nucleotide group Chemical group 0.000 description 6
230000007017 scission Effects 0.000 description 6
239000012588 trypsin Substances 0.000 description 6
QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
108020004705 Codon Proteins 0.000 description 5
108010049003 Fibrinogen Proteins 0.000 description 5
102000008946 Fibrinogen Human genes 0.000 description 5
241000283973 Oryctolagus cuniculus Species 0.000 description 5
238000012300 Sequence Analysis Methods 0.000 description 5
MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
229960000723 ampicillin Drugs 0.000 description 5
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 5
239000004202 carbamide Substances 0.000 description 5
238000005119 centrifugation Methods 0.000 description 5
230000002950 deficient Effects 0.000 description 5
238000001514 detection method Methods 0.000 description 5
201000010099 disease Diseases 0.000 description 5
208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
229930182817 methionine Natural products 0.000 description 5
230000037452 priming Effects 0.000 description 5
IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
241001646716 Escherichia coli K-12 Species 0.000 description 4
ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
239000004471 Glycine Substances 0.000 description 4
101000605403 Homo sapiens Plasminogen Proteins 0.000 description 4
DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
241001465754 Metazoa Species 0.000 description 4
108091034117 Oligonucleotide Proteins 0.000 description 4
229940124158 Protease/peptidase inhibitor Drugs 0.000 description 4
239000004098 Tetracycline Substances 0.000 description 4
208000007536 Thrombosis Diseases 0.000 description 4
239000007983 Tris buffer Substances 0.000 description 4
238000002835 absorbance Methods 0.000 description 4
239000002253 acid Substances 0.000 description 4
239000000427 antigen Substances 0.000 description 4
150000001875 compounds Chemical class 0.000 description 4
230000006870 function Effects 0.000 description 4
210000001589 microsome Anatomy 0.000 description 4
210000000496 pancreas Anatomy 0.000 description 4
239000000137 peptide hydrolase inhibitor Substances 0.000 description 4
235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
229920000053 polysorbate 80 Polymers 0.000 description 4
238000001556 precipitation Methods 0.000 description 4
238000011160 research Methods 0.000 description 4
238000012216 screening Methods 0.000 description 4
238000010561 standard procedure Methods 0.000 description 4
239000006228 supernatant Substances 0.000 description 4
229960002180 tetracycline Drugs 0.000 description 4
229930101283 tetracycline Natural products 0.000 description 4
235000019364 tetracycline Nutrition 0.000 description 4
150000003522 tetracyclines Chemical class 0.000 description 4
238000001890 transfection Methods 0.000 description 4
LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
230000003612 virological effect Effects 0.000 description 4
XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 3
229920000936 Agarose Polymers 0.000 description 3
241000894006 Bacteria Species 0.000 description 3
DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 3
101150074155 DHFR gene Proteins 0.000 description 3
102000004594 DNA Polymerase I Human genes 0.000 description 3
108010017826 DNA Polymerase I Proteins 0.000 description 3
108020003215 DNA Probes Proteins 0.000 description 3
239000003298 DNA probe Substances 0.000 description 3
IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 3
108700026244 Open Reading Frames Proteins 0.000 description 3
102000035195 Peptidases Human genes 0.000 description 3
108091005804 Peptidases Proteins 0.000 description 3
108091000080 Phosphotransferase Proteins 0.000 description 3
239000004365 Protease Substances 0.000 description 3
102100027378 Prothrombin Human genes 0.000 description 3
108010094028 Prothrombin Proteins 0.000 description 3
FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
108010023197 Streptokinase Proteins 0.000 description 3
241000251539 Vertebrata <Metazoa> Species 0.000 description 3
108091007433 antigens Proteins 0.000 description 3
102000036639 antigens Human genes 0.000 description 3
IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 3
210000004369 blood Anatomy 0.000 description 3
239000008280 blood Substances 0.000 description 3
210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
238000010367 cloning Methods 0.000 description 3
238000012217 deletion Methods 0.000 description 3
230000037430 deletion Effects 0.000 description 3
238000013461 design Methods 0.000 description 3
238000010790 dilution Methods 0.000 description 3
239000012895 dilution Substances 0.000 description 3
230000002526 effect on cardiovascular system Effects 0.000 description 3
229940012952 fibrinogen Drugs 0.000 description 3
230000023597 hemostasis Effects 0.000 description 3
238000004128 high performance liquid chromatography Methods 0.000 description 3
238000001114 immunoprecipitation Methods 0.000 description 3
238000003780 insertion Methods 0.000 description 3
230000037431 insertion Effects 0.000 description 3
238000001990 intravenous administration Methods 0.000 description 3
238000002955 isolation Methods 0.000 description 3
108010087750 lysyl-plasminogen Proteins 0.000 description 3
239000008188 pellet Substances 0.000 description 3
102000020233 phosphotransferase Human genes 0.000 description 3
239000013600 plasmid vector Substances 0.000 description 3
125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
229940039716 prothrombin Drugs 0.000 description 3
239000011541 reaction mixture Substances 0.000 description 3
230000002829 reductive effect Effects 0.000 description 3
238000012163 sequencing technique Methods 0.000 description 3
229960005202 streptokinase Drugs 0.000 description 3
238000012360 testing method Methods 0.000 description 3
229960000103 thrombolytic agent Drugs 0.000 description 3
229940104230 thymidine Drugs 0.000 description 3
230000009466 transformation Effects 0.000 description 3
PLVPPLCLBIEYEA-WAYWQWQTSA-N (z)-3-(1h-indol-3-yl)prop-2-enoic acid Chemical compound C1=CC=C2C(\C=C/C(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-WAYWQWQTSA-N 0.000 description 2
OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 2
229920001817 Agar Polymers 0.000 description 2
102000002260 Alkaline Phosphatase Human genes 0.000 description 2
108020004774 Alkaline Phosphatase Proteins 0.000 description 2
108010039627 Aprotinin Proteins 0.000 description 2
241000972773 Aulopiformes Species 0.000 description 2
108091003079 Bovine Serum Albumin Proteins 0.000 description 2
UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
241000699802 Cricetulus griseus Species 0.000 description 2
102000053602 DNA Human genes 0.000 description 2
102000012410 DNA Ligases Human genes 0.000 description 2
108010061982 DNA Ligases Proteins 0.000 description 2
108050009160 DNA polymerase 1 Proteins 0.000 description 2
102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 2
108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 2
WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
102000003839 Human Proteins Human genes 0.000 description 2
108090000144 Human Proteins Proteins 0.000 description 2
DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
108010047761 Interferon-alpha Proteins 0.000 description 2
102000006992 Interferon-alpha Human genes 0.000 description 2
CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 2
WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
208000010378 Pulmonary Embolism Diseases 0.000 description 2
PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
238000002105 Southern blotting Methods 0.000 description 2
108091081024 Start codon Proteins 0.000 description 2
KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
206010047249 Venous thrombosis Diseases 0.000 description 2
241000700605 Viruses Species 0.000 description 2
230000009102 absorption Effects 0.000 description 2
238000010521 absorption reaction Methods 0.000 description 2
229960000583 acetic acid Drugs 0.000 description 2
230000004913 activation Effects 0.000 description 2
239000008272 agar Substances 0.000 description 2
239000000783 alginic acid Substances 0.000 description 2
238000004458 analytical method Methods 0.000 description 2
229960004405 aprotinin Drugs 0.000 description 2
238000000211 autoradiogram Methods 0.000 description 2
230000008901 benefit Effects 0.000 description 2
230000000975 bioactive effect Effects 0.000 description 2
239000001110 calcium chloride Substances 0.000 description 2
229910001628 calcium chloride Inorganic materials 0.000 description 2
239000001506 calcium phosphate Substances 0.000 description 2
229910000389 calcium phosphate Inorganic materials 0.000 description 2
235000011010 calcium phosphates Nutrition 0.000 description 2
230000015556 catabolic process Effects 0.000 description 2
210000002421 cell wall Anatomy 0.000 description 2
230000001413 cellular effect Effects 0.000 description 2
210000000349 chromosome Anatomy 0.000 description 2
230000015271 coagulation Effects 0.000 description 2
238000005345 coagulation Methods 0.000 description 2
230000000295 complement effect Effects 0.000 description 2
230000002596 correlated effect Effects 0.000 description 2
238000006731 degradation reaction Methods 0.000 description 2
230000001419 dependent effect Effects 0.000 description 2
238000010586 diagram Methods 0.000 description 2
238000000502 dialysis Methods 0.000 description 2
VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
229940079593 drug Drugs 0.000 description 2
239000003814 drug Substances 0.000 description 2
ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
229960005542 ethidium bromide Drugs 0.000 description 2
210000003527 eukaryotic cell Anatomy 0.000 description 2
238000000855 fermentation Methods 0.000 description 2
230000004151 fermentation Effects 0.000 description 2
238000005194 fractionation Methods 0.000 description 2
ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
239000001963 growth medium Substances 0.000 description 2
230000002439 hemostatic effect Effects 0.000 description 2
230000001900 immune effect Effects 0.000 description 2
238000001727 in vivo Methods 0.000 description 2
PLVPPLCLBIEYEA-UHFFFAOYSA-N indoleacrylic acid Natural products C1=CC=C2C(C=CC(=O)O)=CNC2=C1 PLVPPLCLBIEYEA-UHFFFAOYSA-N 0.000 description 2
239000003112 inhibitor Substances 0.000 description 2
ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 2
238000011835 investigation Methods 0.000 description 2
239000007788 liquid Substances 0.000 description 2
239000006166 lysate Substances 0.000 description 2
239000003550 marker Substances 0.000 description 2
239000012528 membrane Substances 0.000 description 2
238000009629 microbiological culture Methods 0.000 description 2
238000013508 migration Methods 0.000 description 2
230000005012 migration Effects 0.000 description 2
238000012544 monitoring process Methods 0.000 description 2
229910052757 nitrogen Inorganic materials 0.000 description 2
230000036961 partial effect Effects 0.000 description 2
QKFJKGMPGYROCL-UHFFFAOYSA-N phenyl isothiocyanate Chemical compound S=C=NC1=CC=CC=C1 QKFJKGMPGYROCL-UHFFFAOYSA-N 0.000 description 2
239000002953 phosphate buffered saline Substances 0.000 description 2
230000033885 plasminogen activation Effects 0.000 description 2
230000008488 polyadenylation Effects 0.000 description 2
210000001236 prokaryotic cell Anatomy 0.000 description 2
230000000644 propagated effect Effects 0.000 description 2
230000002797 proteolythic effect Effects 0.000 description 2
230000006337 proteolytic cleavage Effects 0.000 description 2
239000000700 radioactive tracer Substances 0.000 description 2
210000001995 reticulocyte Anatomy 0.000 description 2
235000019515 salmon Nutrition 0.000 description 2
239000006152 selective media Substances 0.000 description 2
238000000926 separation method Methods 0.000 description 2
239000011734 sodium Substances 0.000 description 2
229910052708 sodium Inorganic materials 0.000 description 2
239000011780 sodium chloride Substances 0.000 description 2
239000001488 sodium phosphate Substances 0.000 description 2
229910000162 sodium phosphate Inorganic materials 0.000 description 2
241000894007 species Species 0.000 description 2
239000000126 substance Substances 0.000 description 2
238000006467 substitution reaction Methods 0.000 description 2
239000000758 substrate Substances 0.000 description 2
FRGKKTITADJNOE-UHFFFAOYSA-N sulfanyloxyethane Chemical compound CCOS FRGKKTITADJNOE-UHFFFAOYSA-N 0.000 description 2
239000000725 suspension Substances 0.000 description 2
230000001225 therapeutic effect Effects 0.000 description 2
238000002560 therapeutic procedure Methods 0.000 description 2
230000002537 thrombolytic effect Effects 0.000 description 2
238000013518 transcription Methods 0.000 description 2
230000035897 transcription Effects 0.000 description 2
QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
238000011144 upstream manufacturing Methods 0.000 description 2
230000002792 vascular Effects 0.000 description 2
CAJXYXPLLJDEOB-SLFFLAALSA-N (2s)-6-amino-2-[[(2s)-2-[[(2r)-2-amino-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-n-(4-nitrophenyl)hexanamide Chemical compound CC(C)[C@@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NC1=CC=C([N+]([O-])=O)C=C1 CAJXYXPLLJDEOB-SLFFLAALSA-N 0.000 description 1
VFWCMGCRMGJXDK-UHFFFAOYSA-N 1-chlorobutane Chemical compound CCCCCl VFWCMGCRMGJXDK-UHFFFAOYSA-N 0.000 description 1
GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-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
HRTNVZOWEFVIGJ-UHFFFAOYSA-N 2-anilino-4h-1,3-thiazol-5-one Chemical class S1C(=O)CN=C1NC1=CC=CC=C1 HRTNVZOWEFVIGJ-UHFFFAOYSA-N 0.000 description 1
GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
ZZRIQDWDJVLELF-UHFFFAOYSA-N 3-phenyl-2-sulfanylideneimidazolidin-4-one Chemical compound O=C1CNC(=S)N1C1=CC=CC=C1 ZZRIQDWDJVLELF-UHFFFAOYSA-N 0.000 description 1
102000013563 Acid Phosphatase Human genes 0.000 description 1
108010051457 Acid Phosphatase Proteins 0.000 description 1
101710187573 Alcohol dehydrogenase 2 Proteins 0.000 description 1
101710133776 Alcohol dehydrogenase class-3 Proteins 0.000 description 1
239000004475 Arginine Substances 0.000 description 1
208000031104 Arterial Occlusive disease Diseases 0.000 description 1
DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
244000063299 Bacillus subtilis Species 0.000 description 1
108020000946 Bacterial DNA Proteins 0.000 description 1
102000015081 Blood Coagulation Factors Human genes 0.000 description 1
108010039209 Blood Coagulation Factors Proteins 0.000 description 1
102000004506 Blood Proteins Human genes 0.000 description 1
108010017384 Blood Proteins Proteins 0.000 description 1
OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
241000283707 Capra Species 0.000 description 1
208000024172 Cardiovascular disease Diseases 0.000 description 1
241000282693 Cercopithecidae Species 0.000 description 1
102000004420 Creatine Kinase Human genes 0.000 description 1
108010042126 Creatine kinase Proteins 0.000 description 1
FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
238000012270 DNA recombination Methods 0.000 description 1
206010051055 Deep vein thrombosis Diseases 0.000 description 1
108010042407 Endonucleases Proteins 0.000 description 1
102000004533 Endonucleases Human genes 0.000 description 1
241000588921 Enterobacteriaceae Species 0.000 description 1
241001522878 Escherichia coli B Species 0.000 description 1
241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
108010014172 Factor V Proteins 0.000 description 1
108010054218 Factor VIII Proteins 0.000 description 1
102000001690 Factor VIII Human genes 0.000 description 1
108700039691 Genetic Promoter Regions Proteins 0.000 description 1
108700007698 Genetic Terminator Regions Proteins 0.000 description 1
102000030595 Glucokinase Human genes 0.000 description 1
108010021582 Glucokinase Proteins 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
HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
102000005548 Hexokinase Human genes 0.000 description 1
108700040460 Hexokinases Proteins 0.000 description 1
108091006905 Human Serum Albumin Proteins 0.000 description 1
102000008100 Human Serum Albumin Human genes 0.000 description 1
241000701109 Human adenovirus 2 Species 0.000 description 1
108010078049 Interferon alpha-2 Proteins 0.000 description 1
108091092195 Intron Proteins 0.000 description 1
QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
102000003960 Ligases Human genes 0.000 description 1
108090000364 Ligases Proteins 0.000 description 1
239000006137 Luria-Bertani broth Substances 0.000 description 1
239000004472 Lysine Substances 0.000 description 1
GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
229930195725 Mannitol Natural products 0.000 description 1
230000004988 N-glycosylation Effects 0.000 description 1
125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 1
101100293593 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) nar-1 gene Proteins 0.000 description 1
101710163270 Nuclease Proteins 0.000 description 1
101710149086 Nuclease S1 Proteins 0.000 description 1
102000004316 Oxidoreductases Human genes 0.000 description 1
108090000854 Oxidoreductases Proteins 0.000 description 1
229910019142 PO4 Inorganic materials 0.000 description 1
108010087702 Penicillinase Proteins 0.000 description 1
102000001105 Phosphofructokinases Human genes 0.000 description 1
108010069341 Phosphofructokinases Proteins 0.000 description 1
102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
108010059712 Pronase Proteins 0.000 description 1
241000589516 Pseudomonas Species 0.000 description 1
108010011939 Pyruvate Decarboxylase Proteins 0.000 description 1
102000013009 Pyruvate Kinase Human genes 0.000 description 1
108020005115 Pyruvate Kinase Proteins 0.000 description 1
108091034057 RNA (poly(A)) Proteins 0.000 description 1
108020005067 RNA Splice Sites Proteins 0.000 description 1
238000002123 RNA extraction Methods 0.000 description 1
102000006382 Ribonucleases Human genes 0.000 description 1
108010083644 Ribonucleases Proteins 0.000 description 1
101150001535 SRC gene Proteins 0.000 description 1
241000235070 Saccharomyces Species 0.000 description 1
241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
241000607720 Serratia Species 0.000 description 1
VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
208000006011 Stroke Diseases 0.000 description 1
101150006914 TRP1 gene Proteins 0.000 description 1
108091036066 Three prime untranslated region Proteins 0.000 description 1
AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
239000004473 Threonine Substances 0.000 description 1
102000004357 Transferases Human genes 0.000 description 1
108090000992 Transferases Proteins 0.000 description 1
102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
101710146079 Xanthine-guanine phosphoribosyltransferase Proteins 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
239000008351 acetate buffer Substances 0.000 description 1
OZZOVSQSDIWNIP-UHFFFAOYSA-N acetic acid;azane Chemical compound [NH4+].[NH4+].CC([O-])=O.CC([O-])=O OZZOVSQSDIWNIP-UHFFFAOYSA-N 0.000 description 1
150000007513 acids Chemical class 0.000 description 1
230000003213 activating effect Effects 0.000 description 1
230000001154 acute effect Effects 0.000 description 1
238000000246 agarose gel electrophoresis Methods 0.000 description 1
235000004279 alanine Nutrition 0.000 description 1
WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
230000004075 alteration Effects 0.000 description 1
239000003146 anticoagulant agent Substances 0.000 description 1
229940127219 anticoagulant drug Drugs 0.000 description 1
230000000890 antigenic effect Effects 0.000 description 1
239000012223 aqueous fraction Substances 0.000 description 1
239000008346 aqueous phase Substances 0.000 description 1
ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
208000021328 arterial occlusion Diseases 0.000 description 1
235000009582 asparagine Nutrition 0.000 description 1
229960001230 asparagine Drugs 0.000 description 1
235000003704 aspartic acid Nutrition 0.000 description 1
244000052616 bacterial pathogen Species 0.000 description 1
230000004888 barrier function Effects 0.000 description 1
239000011324 bead Substances 0.000 description 1
OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
238000004166 bioassay Methods 0.000 description 1
210000004204 blood vessel Anatomy 0.000 description 1
239000001045 blue dye Substances 0.000 description 1
230000037396 body weight Effects 0.000 description 1
229940098773 bovine serum albumin Drugs 0.000 description 1
UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
239000007853 buffer solution Substances 0.000 description 1
239000007975 buffered saline Substances 0.000 description 1
239000011575 calcium Substances 0.000 description 1
229910052791 calcium Inorganic materials 0.000 description 1
125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
230000003197 catalytic effect Effects 0.000 description 1
230000036978 cell physiology Effects 0.000 description 1
230000019522 cellular metabolic process Effects 0.000 description 1
229920002678 cellulose Polymers 0.000 description 1
239000001913 cellulose Substances 0.000 description 1
238000012512 characterization method Methods 0.000 description 1
239000003153 chemical reaction reagent Substances 0.000 description 1
YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
229960001231 choline Drugs 0.000 description 1
OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
238000004587 chromatography analysis Methods 0.000 description 1
239000003593 chromogenic compound Substances 0.000 description 1
239000013611 chromosomal DNA Substances 0.000 description 1
230000002759 chromosomal effect Effects 0.000 description 1
230000002860 competitive effect Effects 0.000 description 1
239000000356 contaminant Substances 0.000 description 1
230000001276 controlling effect Effects 0.000 description 1
235000018417 cysteine Nutrition 0.000 description 1
XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
108091092330 cytoplasmic RNA Proteins 0.000 description 1
SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
230000003247 decreasing effect Effects 0.000 description 1
230000003413 degradative effect Effects 0.000 description 1
230000000593 degrading effect Effects 0.000 description 1
229960000633 dextran sulfate Drugs 0.000 description 1
239000008355 dextrose injection Substances 0.000 description 1
OZRNSSUDZOLUSN-LBPRGKRZSA-N dihydrofolic acid Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-N 0.000 description 1
239000003085 diluting agent 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
238000004090 dissolution Methods 0.000 description 1
230000006334 disulfide bridging Effects 0.000 description 1
125000002228 disulfide group Chemical group 0.000 description 1
150000002019 disulfides Chemical class 0.000 description 1
239000002552 dosage form Substances 0.000 description 1
230000002255 enzymatic effect Effects 0.000 description 1
238000012869 ethanol precipitation Methods 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
PHTXVQQRWJXYPP-UHFFFAOYSA-N ethyltrifluoromethylaminoindane Chemical compound C1=C(C(F)(F)F)C=C2CC(NCC)CC2=C1 PHTXVQQRWJXYPP-UHFFFAOYSA-N 0.000 description 1
238000002474 experimental method Methods 0.000 description 1
229960000301 factor viii Drugs 0.000 description 1
239000012894 fetal calf serum Substances 0.000 description 1
230000020764 fibrinolysis Effects 0.000 description 1
238000011049 filling Methods 0.000 description 1
238000009472 formulation Methods 0.000 description 1
230000008014 freezing Effects 0.000 description 1
238000007710 freezing Methods 0.000 description 1
230000004927 fusion Effects 0.000 description 1
229930182830 galactose Natural products 0.000 description 1
238000001502 gel electrophoresis Methods 0.000 description 1
239000012362 glacial acetic acid Substances 0.000 description 1
239000008103 glucose Substances 0.000 description 1
235000013922 glutamic acid Nutrition 0.000 description 1
239000004220 glutamic acid Substances 0.000 description 1
230000002414 glycolytic effect Effects 0.000 description 1
229960000789 guanidine hydrochloride Drugs 0.000 description 1
PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
239000000383 hazardous chemical Substances 0.000 description 1
231100000206 health hazard Toxicity 0.000 description 1
229940025294 hemin Drugs 0.000 description 1
BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 1
230000002008 hemorrhagic effect Effects 0.000 description 1
229960002897 heparin Drugs 0.000 description 1
229920000669 heparin Polymers 0.000 description 1
208000002672 hepatitis B Diseases 0.000 description 1
HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
230000002209 hydrophobic effect Effects 0.000 description 1
125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
238000001802 infusion Methods 0.000 description 1
230000002401 inhibitory effect Effects 0.000 description 1
239000007924 injection Substances 0.000 description 1
238000002347 injection Methods 0.000 description 1
JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
-1 isocytochrome C Proteins 0.000 description 1
AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
229960000310 isoleucine Drugs 0.000 description 1
238000002372 labelling Methods 0.000 description 1
239000008101 lactose Substances 0.000 description 1
230000003902 lesion Effects 0.000 description 1
239000003446 ligand Substances 0.000 description 1
230000000670 limiting effect Effects 0.000 description 1
210000004698 lymphocyte Anatomy 0.000 description 1
230000002934 lysing effect Effects 0.000 description 1
229910001629 magnesium chloride Inorganic materials 0.000 description 1
230000014759 maintenance of location Effects 0.000 description 1
210000004962 mammalian cell Anatomy 0.000 description 1
239000000594 mannitol Substances 0.000 description 1
235000010355 mannitol Nutrition 0.000 description 1
239000011159 matrix material Substances 0.000 description 1
230000004060 metabolic process Effects 0.000 description 1
230000002906 microbiologic effect Effects 0.000 description 1
230000003228 microsomal effect Effects 0.000 description 1
230000004048 modification Effects 0.000 description 1
238000012986 modification Methods 0.000 description 1
238000002703 mutagenesis Methods 0.000 description 1
231100000350 mutagenesis Toxicity 0.000 description 1
230000035772 mutation Effects 0.000 description 1
208000010125 myocardial infarction Diseases 0.000 description 1
210000004897 n-terminal region Anatomy 0.000 description 1
238000010899 nucleation Methods 0.000 description 1
210000004940 nucleus Anatomy 0.000 description 1
229940046166 oligodeoxynucleotide Drugs 0.000 description 1
229920001542 oligosaccharide Polymers 0.000 description 1
150000002482 oligosaccharides Chemical class 0.000 description 1
210000001672 ovary Anatomy 0.000 description 1
230000037361 pathway Effects 0.000 description 1
229950009506 penicillinase Drugs 0.000 description 1
230000002093 peripheral effect Effects 0.000 description 1
239000012071 phase Substances 0.000 description 1
COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
229940117953 phenylisothiocyanate Drugs 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
230000007180 physiological regulation Effects 0.000 description 1
239000002797 plasminogen activator inhibitor Substances 0.000 description 1
238000006116 polymerization reaction Methods 0.000 description 1
108091033319 polynucleotide Proteins 0.000 description 1
102000040430 polynucleotide Human genes 0.000 description 1
239000002157 polynucleotide Substances 0.000 description 1
239000001103 potassium chloride Substances 0.000 description 1
235000011164 potassium chloride Nutrition 0.000 description 1
230000003389 potentiating effect Effects 0.000 description 1
239000002244 precipitate Substances 0.000 description 1
239000002243 precursor Substances 0.000 description 1
108010067999 preproalbumin Proteins 0.000 description 1
230000001737 promoting effect Effects 0.000 description 1
230000000069 prophylactic effect Effects 0.000 description 1
230000017854 proteolysis Effects 0.000 description 1
210000001938 protoplast Anatomy 0.000 description 1
238000000746 purification Methods 0.000 description 1
230000002285 radioactive effect Effects 0.000 description 1
238000003127 radioimmunoassay Methods 0.000 description 1
230000008439 repair process Effects 0.000 description 1
238000012552 review Methods 0.000 description 1
239000003161 ribonuclease inhibitor Substances 0.000 description 1
108020004418 ribosomal RNA Proteins 0.000 description 1
210000003705 ribosome Anatomy 0.000 description 1
230000028327 secretion Effects 0.000 description 1
230000035945 sensitivity Effects 0.000 description 1
239000001632 sodium acetate Substances 0.000 description 1
235000017281 sodium acetate Nutrition 0.000 description 1
235000017557 sodium bicarbonate Nutrition 0.000 description 1
229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
239000008354 sodium chloride injection Substances 0.000 description 1
239000001509 sodium citrate Substances 0.000 description 1
NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
239000007787 solid Substances 0.000 description 1
239000007790 solid phase Substances 0.000 description 1
238000009987 spinning Methods 0.000 description 1
LXMSZDCAJNLERA-ZHYRCANASA-N spironolactone Chemical compound C([C@@H]1[C@]2(C)CC[C@@H]3[C@@]4(C)CCC(=O)C=C4C[C@H]([C@@H]13)SC(=O)C)C[C@@]21CCC(=O)O1 LXMSZDCAJNLERA-ZHYRCANASA-N 0.000 description 1
238000010186 staining Methods 0.000 description 1
239000008227 sterile water for injection Substances 0.000 description 1
230000000638 stimulation Effects 0.000 description 1
230000004083 survival effect Effects 0.000 description 1
238000003786 synthesis reaction Methods 0.000 description 1
231100000331 toxic Toxicity 0.000 description 1
230000002588 toxic effect Effects 0.000 description 1
230000002103 transcriptional effect Effects 0.000 description 1
238000012546 transfer Methods 0.000 description 1
230000014621 translational initiation Effects 0.000 description 1
PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
210000002700 urine Anatomy 0.000 description 1
239000004474 valine 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)
- C12N9/003—Dihydrofolate reductase [DHFR] (1.5.1.3)
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
- 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21069—Protein C activated (3.4.21.69)
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Chemical & Material Sciences (AREA)
Engineering & Computer Science (AREA)
Organic Chemistry (AREA)
Genetics & Genomics (AREA)
Bioinformatics & Cheminformatics (AREA)
Zoology (AREA)
Wood Science & Technology (AREA)
General Health & Medical Sciences (AREA)
General Engineering & Computer Science (AREA)
Biomedical Technology (AREA)
Biochemistry (AREA)
Biotechnology (AREA)
Medicinal Chemistry (AREA)
Molecular Biology (AREA)
Microbiology (AREA)
Chemical Kinetics & Catalysis (AREA)
Hematology (AREA)
Pharmacology & Pharmacy (AREA)
Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
Veterinary Medicine (AREA)
General Chemical & Material Sciences (AREA)
Public Health (AREA)
Animal Behavior & Ethology (AREA)
Diabetes (AREA)
Physics & Mathematics (AREA)
Biophysics (AREA)
Plant Pathology (AREA)
Enzymes And Modification Thereof (AREA)
Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Micro-Organisms Or Cultivation Processes Thereof (AREA)
Preparation Of Compounds By Using Micro-Organisms (AREA)

CY1341A 1982-05-05 1983-05-04 Human tissue plasminogen activator pharmaceutical compositions containing it processes for making it and dna and transformed cell intermediates therefor CY1341A (en)

Applications Claiming Priority (3)

Application Number	Priority Date	Filing Date	Title
US37486082A	1982-05-05	1982-05-05
US39800382A	1982-07-14	1982-07-14
US06/483,052 US4766075A (en)	1982-07-14	1983-04-07	Human tissue plasminogen activator

Publications (1)

Publication Number	Publication Date
CY1341A true CY1341A (en)	1987-01-16

Family

ID=27409206

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
CY1341A CY1341A (en)	1982-05-05	1983-05-04	Human tissue plasminogen activator pharmaceutical compositions containing it processes for making it and dna and transformed cell intermediates therefor

Country Status (26)

Country	Link
EP (1)	EP0093619B2 (pl)
JP (2)	JPH0216981A (pl)
AR (1)	AR241654A1 (pl)
BG (1)	BG60253B2 (pl)
BR (1)	BR8302318A (pl)
CA (1)	CA1293211C (pl)
CS (1)	CS248705B2 (pl)
CY (1)	CY1341A (pl)
DD (1)	DD210303A5 (pl)
DE (3)	DE93619T1 (pl)
DK (1)	DK174236B1 (pl)
ES (1)	ES522085A0 (pl)
FR (1)	FR2526443B1 (pl)
GB (1)	GB2119804B (pl)
GR (1)	GR79202B (pl)
HK (1)	HK88586A (pl)
IE (1)	IE54975B1 (pl)
IL (1)	IL68561A (pl)
KE (1)	KE3647A (pl)
MY (1)	MY8700119A (pl)
NO (2)	NO831575L (pl)
OA (1)	OA07419A (pl)
PL (1)	PL152438B1 (pl)
PT (1)	PT76636B (pl)
RO (1)	RO90639B (pl)
ZW (1)	ZW10483A1 (pl)

Families Citing this family (106)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US4558010A (en) *	1980-04-03	1985-12-10	Abbott Laboratories	Recombinant deoxyribonucleic acid which codes for plasminogen activator and method of making plasminogen activator protein therefrom
GR79202B (pl) *	1982-05-05	1984-10-22	Genentech Inc
DE3390272C2 (de) *	1982-10-29	1987-09-17	Mitsui Toatsu Chemicals	Plasminogen-Aktivator, Verfahren zu seiner Herstellung und denselben enthaltendes thrombolytisches Mittel
US5010002A (en) *	1983-01-19	1991-04-23	Genentech, Inc.	Human t-PA production using vectors coding DHFR protein
US5011795A (en) *	1983-01-19	1991-04-30	Genentech, Inc.	Human tPA production using vectors coding for DHFR protein
US4713339A (en) *	1983-01-19	1987-12-15	Genentech, Inc.	Polycistronic expression vector construction
AU572108B2 (en) *	1983-01-19	1988-05-05	Genentech Inc.	Human tpa production using vectors coding for dhfr protein
AU2353384A (en) *	1983-01-19	1984-07-26	Genentech Inc.	Amplification in eukaryotic host cells
US5169762A (en) *	1983-03-03	1992-12-08	Genentech, Inc.	Human nerve growth factor by recombinant technology
US4816567A (en)	1983-04-08	1989-03-28	Genentech, Inc.	Recombinant immunoglobin preparations
JPS59196824A (ja) *	1983-04-21	1984-11-08	Kowa Co	吸着防止剤
US5639639A (en) *	1983-11-02	1997-06-17	Genzyme Corporation	Recombinant heterodimeric human fertility hormones, and methods, cells, vectors and DNA for the production thereof
PT79518B (pt) *	1983-11-21	1986-12-12	Ciba Geigy Ag	54). 5 ^ig do plasmídeo pBR 322 /PHO5 Bam Rsa 637 são digeridos com a endonuclease de restrição Xbal. Apos digestão completa o DNA ê extraído com fenol/clorofórmio e precipitado com etanol. 0 DNA ê redissolvido em Tris.HCl 50mM pH8,0 numa concentração de 50 p-g/nd. e ê digerido com 11 unidades de fosfatase alcalina do intestino de vitela (Boehringer), durante 1 hora a 379C. - 97 fORHjf y\| A enzima ê inativada a 65 9C durante 1 hora. 0 DNA ê purificado por cromatografia de troca iõnica em DE 52 ( Whatman) e precipitação com etanol como descrito no Exemplo 9Aa. Dois oligodeoxinucleotídeos sintéticos com a formula 5’-CTAGAAGGGGTATCTTTGGATAAGA - 3' 3’- TTCCCCATAGAAACCTATTCTCTAG -5' são fosforilados individualmente nas suas extremidades 5' como descrito no Exemplo 9Ab. Os oligodeoxinacleotídeos fosforilados são misturados em quantidades equimolares . A mistura ê aquecida durante 10 min. a 759C, depois deixada arrefecer lentamente atê à temperatura ambiente e guardada a -209C. Este adaptador ê designado por adaptador C. 1 ^ig (120 pmoles) do adaptador C emparelhado e fosforilado e 5 pg (1,5 pmoles) de pBR322/ /PH05 Bam-Rsa 637 desfosforilado e cortado com Xba I são ligados em 40^1 de Tris.HCl 60mM pH 7,5, MgCl2 lOmM, DTT 5mM, ATP lmM, 1600 unidades da ligase de DNA do T4 a 159C durante 20 horas. A ligase de DNA ê inactivada durante 10 min. a 859C e o excesso de adaptadores ê removido por precipitação com isopropanol. 0 DNA ê redissolvido em H20 numa concentração de 0,25 mg/ml. Os múltiplos de adaptadores são removidos por digestão com a endonuclease de restrição BglII. 0 DNA ê precipitado com etanol, redissolvido e depois digerido com Bam HI. Os fragmentos de restrição são separados num gel de 0,6¾ de agarose de baixo ponto de fusão em Tris-borato-EDTA pH8,3. 0 fragmento Bam HI-BglII de 662 pb (derivado de fragmento Bam HI-Rsa I de 637 pb) ê localizado e removido do gel. c) Isolamento de um fragmento de vector contendo a sequência codificadora do TPA maduro ( fig. 23) 5 yig do plasmídeo p31R/SS-TPA /\ 2, são digeridos com as endonucleases de restrição Bam HI e BglII. Após digestão completo de DNA e precipitado com etanol e ressuspenso em Tris.HCl 50mM pH 8,0 numa concentração de 75 jxg /ml. Os fragmentos de DNA sao desforilados nas suas extremidades 5' por digestão com 1 unidade de fosfatase alcalina de intestino de vitela ( Boehringer) em 60 de Tris.HCl 50mM pH 8,0 durante 1 hora a 379 C. A fosfatase ê inactivada por incubação a 659C durante 1,5 horas. 0 DNA é purificado por cromatografia de troca iónica em DE-52 (Whatman) como descrito no Exemplo 9Aa. 0 DNA ê precipitado com etanol, redissolvido em Tris-HCl lOmM, pH8,0 e aplicada num gel de 0,6 °'o de agarose de ponto de fusão baixo. 0 fragmento maior Bam HI -BglII de 5,2 kb ê removido do gel. d) Ligação dos fragmentos de DNA isolados e transformação de E.coli HB 101 (cf. figs 23 e 24 ). Dois blocos de gel de agarose de ponto de fusão baixo contendo 0,1 pmoles do fragmento Bam HIBglII de 5,2 kb de p31R/SS-TPA/\ 2 e 0,2 pmoles do fragmen to BamHI -BglII de 662 pb de pBR322/PHO5 Bam-Rsa 637, respectivamente, são misturados, liquefeitos a 659C e diluídos para baixar a concentração de agarose para 0,3¾. A ligação ê feita em 150^1 de Tris.HCl 60mM, pH 7,5; MgCl^ lOmM, 99 jr £ DTT 5mM, ATP ImM e 800 unidades de ligase de DNA do T4 (Biolabs) a 159C durante 16 horas. Amostras de 10 ^il e 30 yjl são misturadas com 100 p.1 de células E.coliHB 101 competentes para a transformação como descrito no Exemplo 4a. - R 6 colonias transformadas amp são crescidas individualmente em meio LB contendo 100 ^g/ml de ampicilina. 0 DNA de plasmídeo ê analisado quanto ao tamanho e orientação da invenção por corte com a endonuclease de restrição Bam Hl. Um único clone com a orientação correcta da inserção é designado por p31/PHO5 637-TPA (cf. fig. 23). 0 plasmídeo pBR 322/PHO5 Bam-Rsa 637 pode também ser substituído por pBR 322/PHO5 Bam-Rsa 595, pBR 322/PHO5 Bam-Rsa 811 e pBR 322/PHO5 Bam-Rsa 1312 . Seguindo o processo descrito atras são isolados clones únicos com inserções do tamanho e orientação esperados e designados por p31/PHO5 595C-TPA, p51/PHO5 811C-TPA e p31/PHO5 1312 C-TPA. 0 adaptador C pode também ser substituído pelo adaptador B com a seguinte férmula 5' -CTAGATAAGAGATCTGC -3’ 3’ TATTCTCTAGACG -5' o qual codifica Lis -Arg na construção final (fig. 24). A fosforilação dos oligodeoxinucleotídeos sintéticos e emparelhamento são como descrito para o adaptador C. Para a adição do adaptador 5 de pBR 322/PHO5 Bam-Rsa 637 cortado com Xba I são digeridos com 2 unidades de nuclease (Sigma) em 50 yjl de acetato de sédio 30mM pH 4,6, NaCI 200mM e ZnSO^ ImM durante 40 min. a 379C para criar extremidades cerses (ver fig.23). Apos extração com fenol clorofórmio o DNA ê precipitado com etanol e redissolvido em H^O numa concentração de 0,25 mg/ml. 0,4 ^g (120 pmoles) de adaptador A fosforilado e emparelhado e 5 ^ig (1,5 pmoles) de pBR 322/PHO5 Bam -Rsa 637 cortado com Xba I /S^ são ligados através das extremidades cerses como descrito para o adaptador C exceptuando a utilização de ATP 3,5 mM. As outras construções são como descrito atras. Os clones resultantes são identificado pela letra A e referidos como p31/PHO5 595A-TPA, p31/PHO5 637A-TPA, p31/PHO5 811A-TPA e p31/PHO5 1312 A-TPA. Exemplo 16: Subclonagem de construção genética no vector de levedura de elevado numero de copias pJDB2O7 e transformação de S. cerevisiae GRF18 As construções descritas nos Exemplos 13-19 contêm o promotor de PH05 com ou sem extensões para a região codificadora do gene de PH05 . A região codificadora do TPA com ou sem prepo-sequência e os sinais de terminação da transcrição de PH05 num arranjo tendem, tudo inserido num vector derivado do pBR 322. Os fragmentos Bam HI-HIND III contendo todo o arranjo são ligados ao fragmento Bam Hl -Hind III de 6,4 kb do pJDB 207 como descrito para uma reacção analoga no Exemplo 10. Células E.coli HB101 competentes são transformadas e vãrias colonias amp^ de cada experiência são crescidas individualmente em meio LB com 100 ^ig/ml de ampicilina. 0 DNA de plasmídio é analisado por corte com as endonucleases de restrição Hind III, Bam Hl e Pst I. Partindo dos plasmídeos p31/PHO5-TPA 18, ρ31 /PHO5-TPA 42, p31R/SS-TPA /\ 2, p31R/SS-TPA/\ 3, Ρ31/ΡΗΟ5 595C-TPA, p31/PHO5 637C-TPA,’ p31/PHO5 811C-TPA, P31/PHO5 1312C-TPA, p31/PHO5 595B-TPA, p31/PHO5 637-TPA, P31/PHO5 811 B-TPA, p31/PHO5 1312 B-TPA, p31/PHO5 595 A-TPA P31/PHO5 637-TPA, p31/PHO5 811-A-TPA e p31/PHO5 1312A-TPA obtem-se os clones que se seguem com as inserções correctas PJDB2O7/PHO5-TPA18 PJDB2O7/PHO5-TPA42 PJDB2O7R/PHO5-SS-TPA Λ 2 PJDB2O7R/PHO5-SS-TPA Λ 3 PJDB2O7/PHO5 595C-TPA PJDB2O7/PHO5 637C-TPA PJDB2O7/PHO5 811C-TPA PJDB2O7/PHO5 1312C-TPA PJDB2O7/PHO5 595B-TPA PJDB207/PH05 637B-TPA pJDB2O7/PHO5 811B-TPA PJDB2O7/PHO5 1312B-TPA pJDB2O7/PHO5 595A-TPA PJDB2O7/PHO5 637A-TPA PJDB2O7/PHO5 811A-TPA PJDB2O7/PHO5 1312A-TPA . Estes plasmídeos são introduzidos individualmente em Saccharomyces cerevisiae estirpe GRF 18 como descrito no Exemplo 11. Uma única colónia de levedura de cada uma das transformações de levedura ê picked. Os clones obtidos são designados por . Saccharomyces cerevisíae GRF18/pJDB2Ò7/PHO5-TPA18 Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5-TPA42 Saccharomyces cerevisíae GRF18/pJDB2O7R/PHO5-SS-TPA/\ 2 Sacchãtúiuycea cerevisíae GRFi8/pJD32O7R/?HO5~SS-TPA /\ 3 Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 595C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 637C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 811C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 1312C-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 595B-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 637B-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 811B-TPA Saccharomyces cerevisíae GRF18/pJDB2O7/PHO5 1312B-TPA As células de levedura são crescidas colhidas e extraídas como descrito nos Exemplos 11 e 12. A actividade de TPA nos extractos celulares é testado como descrito no Exemplo 12. Os resultados estão apresen tados na tabela 3. Tabela 3: Actividade de TPA da estirpe de levedura Saccharomyces cerevisiae GRF18 transformada com diferentes plasmídeos e testada em condições de desrepressão (Pi baixo) : plasmídeo Actividade de TPA(unidades/l de cultura de células de levedura/ OD^qq) PJDB2O7/PHO5-TPA18 750 PJDB2O7/PHO5-TPA42 700 PJDB2O7R/PHO5-SS-TPA_A 2 600 PJDB2O7R/PHO5-SS-TPA 3 650 PJDB2O7/PHO5 595C-TPA 900 Exemplo 17: Substituição cromossómica do gene de PHO5 pelo gene do TPA (ver fig.25) A região codificadora da proteína TPA ê colocada no cromossoma II de levedura na posição da região codificadora da proteína PH05 . Experimentálmente isto ê con seguido por cotransformação. Saccharomyces cerevisiae estirpe GRF 18 ê cotransformada com 25 ^ig do plasmídeo p31/PHO5 -TPA 18, linearizado com BnmHI e Hind III, e 3 ^xg do plasmídeo de levedura Yepl3 (51). De 24 colónias Leu obtidas, uma ê Pho5 e ao mesmo tempo produz aproximadamente 35 unidades de TPA/1 de cultura de células de levedura /0D600. Por crescimento deste transformante num meio contendo leucina (YPD, 10g/l de Bacto Yeast Extract, 20g/l de Bacto Peptone, 20 g/l de glucose) durante 10 gerações, segregantes Leu~são obtidos com uma frequência de 10-20¾ os quais provavelmente perderam o plasmideo Yep31. Por análise ou Southern do DNA total de levedura isolado dos segregantes Leu (52) verifica-se que no nível de DNA que as sequências codificadoras da proteína TPA substituem as sequências codificadores da proteína PH05 deixando inal. teradas as sequências ladeantes do cromossoma II. Seleccionou-se uma estirpe que se designou por Saccharomyces cerevisiae GRF 18 (pho-5-ΤΡΑ)♦ Exemplo 18: Melhoramento de estirpe por mutação A estirpe Saccharomyces cerevisiae GRF18 /pJDB2O7/PHO5-TPA (IA) é ainda desenvolvida para produção de TPA por mutação e selecção de baixa actividade de fosfatase ácida. Saccharomyces cerevisiae H2 4 3/pJDB 20 7/ /PH05 -TPA (IA) um mutante termo-sensível de Saccharomyces cerevisiae GRF18/pJDB2O7/PHO5 TPA (IA) ê obtido por irra_2 diação com UV (0,08 yiWcm , 15 segundos, 99,2¾ de morte). As colónias são crescidas em meio YPD osmc ticamente estabilizado contendo sorbitol 1,2M (10 g/l de Bacto Yeast Extract, 20 g/l de Bacto Peptone, 20g/l de glucose, 20 g/l de agar Bacto) e transferidas para meio YPD sem sorbitol. Saccharomyces cerevisiae H243 ê isolada como colónia de crescimento lento. Cerca de 10$ células de Saccharomyces cerevisiae H243/pJDB2O7/PHO5 -TPA (IA) são semeadas em agar YNB (6,8 1 de yeast Nitrogen Base sem aminoãcidos ; 20 g/1 de glucose, 20 g/1 de agar Bacto, suplementado com 20 mg/1 de histidina) e são irradiadas directamente com UV (0,08 -2 uWcm durante 12 segundos). A morte e de 98¾. As colonias obtidas a partir das células sobreviventes são transferidas para meio de Pi baixo e dois dias depois coradas para a actividade de fosfatase acida por um teste de sobrecamada de agar mole (48). Os mutantes negativos para a fosfatase _4 acida são obtidos com uma frequência de mutaçao de 3,2x10 . A estirpe com o título de TPA mais elevado é seleccionado e referido como Saccharomyces cerevisiae H423/pJDB2O7/PHO5-TPA (IA). As actividades de TPA de tres estirpes diferentes de levedura transformadas com pJDB2O7/PHO5-TPA (IA) estão apresentadas na Tabela 4. Tabela 4: Actividade de TPA de diferentes estirpes de Saccharomyces cerevisiae transformadas em condi ções de desrepressão (Pi baixo) Actividade de TPA (unidades/1 de cultura de células de levedura /OD^qq) GRF18/pJDB2O7/PHO5-TPA(lA) 625 H243/pJDB2O7/PHO5 -TPA(IA) 1560 H423/pJDB2O7/PHO5 -TPA(IA) 7200 • 1 Exemplo 19 : Produção de TPA por uma estirpe recombinante da levedura Saccharomyces cerevisiae numa escala de 30 1. Saccharomyces cerevisiae estirpe GRF 18/pJDB2O7/PHO5-TPA(lA) possui um plasmídeo que inclui uma marca para leucina permitindo manutenção selectiva do plasmídeo no organismo hospedeiro, um gene estrutural para o TPA humano e o promotor da fosfatase acida PH05 que permite a expressão do gene do TPA em meios com quantidades limitativas de fosfato inorgânico (condições de desrepressão). A estirpe ê mantida em culturas de agar inclinado preparadas com um meio definido sem o aminoãcido leucina para assegurar a retenção do plasmídeo. Agar recentemente inoculado e incubado durante 24 horas a 30?C. A cultura â superfície de um dos agares inclinados ê ressuspensa em 3ml de meio de pre-cultura o qual ê então transferido para o primeiro balão de pre-cultura com agitação. 0 balão de 500 ml tem uma unica saída e contem 100 ml de meio I de pre-cultura tendo o seguinte composição ( valoreq em g/1). L-asparagina, 2,0; L-histidina, 0,02 glucose, 10 ; Kt^PO^, 1,0 ; MgSO4· 7H2O, 0,5 ; NaCl 0,1 ; CaCl2~2H2O, 0,1 ; solução de vitaminas 5 jil/1 e solução de micronutriantes 5 ml/1. 0 meio e ajustado a pH7,2 usando NaOH antes da esterilização . A glucose, vitaminas e micronutrientes são esterilizados à parte e adicionados ao meio. As soluções "stock” de vitaminas e micronutrientes têm as seguintes composições (em g/1): Vitaminas - biotina, 0,0002; D-pantotenato de cãlcio , 0,04 ; acido fõlico, 0,0002; acido micotínico, 0,04 ; acido p-aminobenzõico, 0,02 ; hidrocloreto de piridoxina, 0,04; riboflãvina, 0,02 ; hidrocloreto de tiamina, 0,04; e inositol 0,2 em 1 1 de agua desionizada ; micronutrientes - acido bórico 0,05 ; CuSO "^H 0, 0,004 ; Kl, 0,01; FeCl^.óH^O, 0,02 ; MnSO^, 0,04 ; NaMoO^. 2^0, 0,02 e ZnSO^.71^0, 0,04 em 1 1 de agua desionizada. 0 meio ê preparado usando agua desionizada. A primeira pré-cultura é incubada durante 24 horas a 309C num agitador orbitol com 5cm de excentricidade a uma velocidade de 250 rev/min. Os balões com a primeira pré-cultura constituem o inoculo para os balões da segunda pré-cultura. Estes balões recebem um inoculo de 1¾ (v/v). Eles contem 100 ml de meio II de pré-cultura tendo a seguinte composição (mg/1): Extracto de levedura (Difco), 10,0 ; L-asparagina, 6,6; Κ^ΡΟ^ 1.0 ; MgSO^.7H2O, 1,0 ; L-histidina, 0,02 e D-glucose (mono-hidrato) , 33,0. 0 meio que ê preparado usando agua desionizada tem um pH de aproximadamente 6,0. A glucose é esterilizada separadamente. As condições de incubação são idênticas âs da primeira pré-cultura. 0 caldo de cultura de 3 balões ê combinado para dar um inoculo a 1¾ v/v para o fermentador principal de produção. 0 fermentador de produção tem um volume total de aproximadamente 45 1, contem 4 saliências e um agitador de turbina de disco de seis lâminas com um diâmetro de 115 mm. A velocidade de agitação é de 600 rev/min, a sobrepressão de 0,3 bar e uma velocidade de arejamento de 0,5 vol/vol/min. * ,· Η Ο fermentador contem 30 1 de um meio com as composições seguintes: (valores em g/1): L-asparagina, 2,0 ; L-histidina, 0,02 ; Kí^PO^, 0,03; MgSO^. 71^0, 0,5; NaCl, 0,1, CaCl2.2H2O, 0,1 ; KC1, ip ; D-glucose (mono -hidrato) , 20,0 ; solução de vitaminas 5 ml/1 e solução micronutrientes, 5ml/l. 0 meio ê ajustado pH 7,2 usando NaOH antes da esterilização. A glucose, vitaminas e micronutrientes são esterilizados separadamente e adicionados ao meio. As soluções "stock" de vitaminas e micronutrientes têm composição idêntica às descritas para o meio I de prê-cultura. A temperatura de fermentação ê de 309C. 0 pH desce para 5,0 onde ê controlado usando NaOH. Apos fermentação durante cerca de 20 horas atinge-se a produção máxima de TPA. A densidade óptica que atinge 2-3 unidades e a actividade de fosfatase ácida dão indicações uteis àcer ca do progresso da fermentação. 0 calor de fermentação ê arrefecido para 109C antes da colheita das células de levedura. Exemplo 20: Recuperação e purificação do TPA e pro-TPA por cromatografia com anticorpos monoclonais anti-TPA a) Preparação de extractos celulares 0 líquido de cultura (pH4) dos 30 1 de fermentação (cf.Exemplo 19) é arrefecido atê 109C e centrifugado usando uma centrífuga Alfa-Laval BRPX-207. As células separadas são ressuspensas em 21 de tampão de lise A ( NaH2P04 20mM, f^HPO^ 80mM, NaCl 150 mM, 0,01¾ de Tween, Benzamidina lOmM e EDTA lOmM). • 1 ί »» A solução é arrefecida atê 59C e passa da através de um moinho Dyno (tipo KDL-Pilot) com uma velocidade de alimentação de 101/h. 0 moinho estã equipado com 500 ml de pérolas de vidro de 0,5 -0,75 m de diâmetro e ê ligado a 3000 rev/min. Antes da adição das pérolas de vidro às células em suspensão aquelas são lavadas com 300 ml de tampão de lise A contendo 30 mg de BSA. A suspensão de células ê centrifugada ( rotor Sorvall 653, 40 min. 9000 rpm) e o sedimento rejeitado. b) Digestão do DNA^e fraccionamento com sulfato de amonio Adiciona-se 5 mg de DNAse a 2 1 da suspensão clarificada e a mistura ê agitada durante 30 min. a 49C. Subsequentemente a solução é levada a 35¾ de saturação com (NH^^SO^ (p/v) e ê agitada durante 1 hora a 49C. A solução ê centrifugada como descrito atrãs e o sedimento contendo toda a actividade de TPA ê ressuspensa em 1 1 de tampão H contendo 1¾ de Tween e 0,1¾ de SDS. A suspensão ê agitada durante pelo menos 1 hora a 49C. c) Cromatografia de imunoafinidade I) Purificação do anticorpo anti-TPA 5 ml de soro de coelho, anti -TPA (oferecido pelo Dr. E. Reich, Friedrich -Miescher-Institut Basel) são passados por uma coluna de sefarose com lisina para renovar o plasminogenio de soro. A coluna ê lavada com 1-2 volumes de PB5 pH 7,4 contendo NaCl 100 mM, KCI 2,7 mM, Na2HP04 8,lmM e KH2PO4 1,5 mM. $ rf* « « Ο líquido da coluna e de lavagens são juntos. 0 anti-soro sem plasminogênio é precipitado pela adição de (NH^)2SO^ sólido para uma concentração final de 50¾ (p/v). A solução ê mantida durante a noite a 4?C e em seguida centrifugada ( rotor Sorvall GSA,1200 rpm, 20 min.) . 0 precipitado e dissolvido num peque no volume de PB5 e dializado contra PBS contendo 0,1 ¾ de NaNj. A IgG desta solução dializada e purificada numa coluna de proteína A -sefarose (55). II) Preparação de uma coluna com anticorpo anti-TPA 13 mg de anticorpo anti-TPA purificado são dializados contra MOPS 0,1 M pH7,0. 0 anticorpo e ligado a 5 ml de Affigel 10 activado ( Biorad ) segundo as instruções do fabricante. A matrix do gel ó equilibrada com PBS contendo 1¾ de Tween 80, 0,1¾ de SDS, NaCl 0,2M, Benzamidin lOmM e 0,1¾ de NaN^. A matrix e aplicada numa coluna de 5 ml. III) Purificação de TPA por cromatografia de imunoafinidade. 0 sedimento redissolvido do fraccionamento com sulfato de amónio ê centrifugado para remover o material insolúvel ( rotor Sorvall GSA, 30 min. 12000rpm) e subsequentemente aplicado â coluna de afinidade a uma ve locidade de fluxo de cerca de lOml/hr a 49C. Depois de todo o volume ter passado através da coluna esta ê lavada com 10 volumes da coluna de PBS contendo 0,05¾. de Tween 80. A coluna ê eluída usando glicina-ΗΟΙΟ,ΙΜ pH2,5 contendo 0,1 ¾ de Tween. São colhidas fracções de 2 ml e testados para a actividade de TPA segundo o método de Rânby (49). Como substracto usa-se D-Val-Leu-Lis -pNA (Kabi -S-2251). As fracções com actividades mais elevadas são reunidas, neutralizadas usando Tris IM e concentradas por ultrafiltração usando uma membrana Amicon YM10. 0 TPA assim obtido apresenta cerca de 90¾ de pureza e estã predominantemente em forma de cadeia unica ( pro-TPA) como evidenciado por SDS-PAGE em condições de redução. 0 peso molecular aparente em condições não redutoras ê de cerca de 60 000 por SDS -PAGE (ver figura A). Figuras A e B : electroforese em gel de poliacrilamida-SDS do produto do gene TPA em levedura (Figura A) e a sua actividade enzimãtica num gel de actividade (placas com caseína) ( figura B). B:gel de actividade A:gel com SDS ad figura A (SDS-PAGE): Faixas 1 e 4 : proteínas padrão (marcadores de peso molecular) Faixas 2 e 5 : TPA de melanoma Faixas 3 e 6 : TPA de levedura Faixas 1 - 3 : em condições de redução Faixas 4 - 6 : em condições de não-redução As amostras correram num gel de 12,5¾ que se corou para proteínas usando azul Coomassie brilhante. ad. figura B (gel de actividade, cf. Exemplo 23): Faixa 1 : TPA de melanoma Faixa 2 : TPA de levedura Sobre-camada para plasminogénio de agar com caseira (referência 56) . Exemplo 21: Recuperação e purificação de TPA e pro-TPA por cromatografia de afinidade com DE-3 a. Preparação de uma coluna de DE-3 Sepharosé 26 mg do inibidor DE-3 purificado a partir de Erythrina latíssima (16) são acoplados a 5 ml de Sepharose 4b ® activada com brometo de cianogênio (Pharmacia) segundo as instruções do fabricante. A matrix ê equilibrada com tampão fosfato salino ("PBS") pH 7,4 contendo NaCl 0,4M, 0,1¾ de Triton X-100R e 0,02¾ de azida de sódio. A matrix ê então acamada numa coluna de 5 ml. b. Purificação de TPA e pro-TPA por cromatografia em DE-3 R Sepharose 4b Os extractos celulares (cf. Exemplo 20a) são tornados 0,4 M relativamente a NaCl e 0,1¾ para Triton X-lOCr^ e filtrados através de uma membrana de 0,45 ^im (Millipore). A solução ê então aplicada à coluna de DE-3 Sepharose (ver atras) a uma velocidade de fluxo de 45ml/hr â temperatura ambiente e o efluente ê rejeitado. Depois do volume total do extracto ter passado através da coluna esta ê lavada com cerca de 50 ml de PB5 contendo NaCl 0,4 M e 0,1 ¾ de Triton X-lOO*' sendo colhidos fracções de 2 ml a 49C. 0 conteúdo protêico de cada fracção ê determinado por medição da absorvância de UV a 280nm. A proteína adsorvida verificou-se ser eluída como um pico bem definido. As fracções contendo os valores mais elevados de absorvância de UV e de activida125 de fibrinolítica determinada pelo teste da I fibrina (47) são reunidas dando 8 ml de solução que se guardou a -209 C. Isto representa aproximadamente 70-80¾ da actividade total aplicada à coluna. As fracções com as actividades »· mais baixas são reunidas separadamente. A recuperação total de actividade em ambos os conjuntos de fracções monta os 90-100¾. c). Purificação de pro-TPA por cromatografia em DE-3 Sepharose 4b® na presença de um inibidor de proteíases. A cromatografia dos extractos de células de levedura ê feita de modo semelhante ao descrito no Exemplo 21b exceptuando a inclusão neste caso do inibidor de tripsina pancreãtica bãsica (BPTI) a 0,1 KlU/ml usando o teste fluorimétrico como Cbz-Gly-Gly-Arg-AMC como substracto (37) determinou-se o conteúdo em TPA e pro-TPA da solução purificada. 90¾ do TPA estã na forma de pro-enzima e 10¾ na forma activa. Assim, a conversão de pro-TPA em TPA durante o processo de purificação é inibido pelo BPTI. Exemplo 22: Separação de pro-TPA do TPA A solução de TPA e pro-TPA obtida como descrito no Exemplo 21c e ajustada a pH8,0 com Tris.HCl O,1M contendo 0,1¾ de Triton X-lOcP e tornado lmM relativamente ao diisopropilfluorofosfato. Apos incubação a 37?C durante 4 horas, a mistura ê passada através de uma coluna de 5 ml de DE-3 Sepharose® (cf. Exemplo 21a). 0 efluente contendo o TPA irreversivelmente inibido é rejeitado. A coluna e lavada com 6 volumes de coluna de PBS contendo NaCl 0,4 M e 0,1¾ de Triton X-IOC^ e subsequentemente eluída com PB5 contendo K SCN 1,6M, NaCl 0,4M e 0,1¾ de Triton X-10C^ como descrito no Exemplo 21b . As fracções apresentando as absorvân. 1 s· P0RWI « ? » ... Ή , - - - , __. «τ'. cias de UV mais elevadas são reunidas. 0 conjunto de fracções contem pro-TPA numa forma substancialmente pura uma vez que não se detecta actividade amidolítica no teste fluorimêtrico usando Cbz-Gli-Gli-Arg-AMC (37) como substrato. A actividade amidolítica assim como a fibrinolítica pode ser restabelecida por tratamento com plasmina a qual converte pro-TPA em enzima activa. Exemplo 23: Propriedades do produto do gene TPA em levedura a. Propriedades enzimãticas 0 produto do gene TPA em levedura obtido segundo o Exemplo 19 e purificado por eromatografia de afinidade em DE-3 (cf. Exemplo 21b) ê enzimãticamente activo como se mostra por clivagem de plasminogênio zimogênio em plasmina na ausência e na presença de fibrina. A actividade enzimãtica pode ser verificada pelos testes colorimêtricos descritos por Ranby (49) (cf. Exemplo 12) e por Verheijen et al. (60) , em placas de caseína e de fibrina (56) e num teste de fase solida com 12 9 I - fibrinogenio ( 47). A actividade enzimãtica em placas de caseína ê mostrada na figura B ( cf. Exemplo 20 c III). A actividade enzimãtica do TPA obtido ê grandemente estimulada pela fibrina. 0 TPA parcialmente purificado por eromatografia de afinidade em DE-3 ê testado num teste de velocidade parabólica (cf. Ranby (49)). Tomando a velocidade reacção Δ E versus Δ t^ entre e 5 minutos como uma medida da actividade enzimãtica a estimulação por concentração óptimas de fibrina ê de 10-20 vezes ( ca. 100 yig /ml) . 0 efeito pode ser observado com fragmentos de fibrina obtidos por digestão com CnBr
NZ210501A (en) *	1983-12-13	1991-08-27	Kirin Amgen Inc	Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence
US4703008A (en) *	1983-12-13	1987-10-27	Kiren-Amgen, Inc.	DNA sequences encoding erythropoietin
KR850004274A (ko) *	1983-12-13	1985-07-11	원본미기재	에리트로포이에틴의 제조방법
JP2648301B2 (ja) *	1983-12-27	1997-08-27	ジエネテイツクス・インスチチユ−ト・インコ−ポレ−テツド	真核細胞の形質転換のための補助ｄｎａを含むベクター
DE3585097D1 (de) *	1984-02-27	1992-02-20	Green Cross Corp	Herstellung menschlicher urokinase.
EP0156169B1 (en) *	1984-02-29	1991-12-18	Asahi Kasei Kogyo Kabushiki Kaisha	An aqueous solution of a tissue plasminogen activator dissolved therein at an increased concentration and a method
US4758512A (en) *	1984-03-06	1988-07-19	President And Fellows Of Harvard College	Hosts and methods for producing recombinant products in high yields
EP0173552B1 (en) *	1984-08-24	1991-10-09	The Upjohn Company	Recombinant dna compounds and the expression of polypeptides such as tpa
US4753879A (en) *	1984-08-27	1988-06-28	Biogen N.V.	Modified tissue plasminogen activators
EP0174835A1 (en) *	1984-09-11	1986-03-19	The Upjohn Company	Human tissue plasminogen activator and recombinant DNA compounds
UA54363C2 (uk)	1984-09-28	2003-03-17	Кірін-Амген, Інк	Виділена молекула днк, яка кодує людський еритропоетин (варіанти), біологічно функціональний кільцевий плазмідний або вірусний днк-вектор, штам еукаріотичних клітин-хазяїв (варіанти), спосіб одержання поліпептиду, фармацевтична композиція
IE81135B1 (en) *	1984-10-01	2000-03-22	Genzyme Corp	Recombinant DNA techniques and the products thereof
FI90990C (fi) *	1984-12-18	1994-04-25	Boehringer Ingelheim Int	Rekombinantti-DNA-molekyyli, transformoitu isäntäorganismi ja menetelmä interferonin valmistamiseksi
DE3500961A1 (de) *	1985-01-14	1986-07-17	Gesellschaft für Biotechnologische Forschung mbH (GBF), 3300 Braunschweig	Escherichia-coli-staemme, dna-teilsequenzen dieser staemme und herstellungsverfahren
EP0460709B1 (en)	1985-01-28	1997-05-21	Xoma Corporation	AraB promoters and method of producing polypeptides, including cecropins, by microbiological techniques
EP0201153A3 (en) *	1985-02-09	1987-10-07	Beecham Group Plc	Modified enzyme and process for its preparation
GB8508717D0 (en) *	1985-04-03	1985-05-09	Beecham Group Plc	Composition
EP0207589A1 (en) *	1985-04-03	1987-01-07	Beecham Group Plc	Fibrinolytic enzyme
US5736134A (en) *	1985-04-22	1998-04-07	Genentech, In.C	Tissue plasminogen activator variants
FR2581652B1 (fr) *	1985-04-22	1989-12-22	Genentech Inc	Nouveaux mutants de l'activateur tissulaire du plasminogene humain
US5756093A (en) *	1985-04-22	1998-05-26	Genentech, Inc.	Tissue plasminogen activator variants
FR2593393B1 (fr) *	1985-05-28	1989-06-02	Wellcome Found	Solution aqueuse a usage parenteral d'activateur tissulaire du plasminogene, procede pour la preparer et recipient obture la contenant
AU593264B2 (en) *	1985-07-10	1990-02-08	Kanegafuchi Kagaku Kogyo Kabushiki Kaisha	Chromosomal DNA sequence, expression vector for human tissue plasminogen activating factor, cultured cells transfected with same and method of producing said activating factor
US4916071A (en)	1985-08-14	1990-04-10	American Home Products Corporation	Poly-kringle plasminogen activator
US5244806A (en) *	1985-08-26	1993-09-14	Eli Lilly And Company	DNA encoding novel tissue plasminogen activator derivatives having kringles 1 and 2 deleted, vectors and host cells
USH2055H1 (en)	1985-09-20	2002-12-03	Zymogenetics, Inc.	Expression of tissue-type plasminogen activator in bacteria
DE3537708A1 (de)	1985-10-23	1987-04-23	Boehringer Mannheim Gmbh	Verfahren zur aktivierung von t-pa nach expression in prokaryonten
GB8528321D0 (en) *	1985-11-18	1985-12-24	Ciba Geigy Ag	Modified fibrinolytic agents
JP2581668B2 (ja) *	1985-11-27	1997-02-12	三井東圧化学株式会社	ヒト正常細胞由来のヒト組織プラスミノ−ゲン活性化因子をコ−ドする新しいｄｎａ配列とそれを含むベクタ−及び細胞
EP0227400B1 (en) *	1985-12-16	1991-12-04	Ethicon, Inc.	Inhibition of post-surgical adhesion formation by the topical administration of tissue plasminogen activator
US5106741A (en) *	1985-12-20	1992-04-21	The Upjohn Company	Tissue plasminogen activator (TPA) analogs
WO1987003906A1 (en)	1985-12-20	1987-07-02	The Upjohn Company	Tissue plasminogen activator (tpa) analogs
DK623186A (da) *	1985-12-23	1987-08-26	Chiron Corp	Polypeptid med vaevsplasminogen-aktivator-virkning
JPS62149625A (ja) *	1985-12-25	1987-07-03	Green Cross Corp:The	生理活性物質の製造方法
DK43387A (da) *	1986-01-29	1987-07-30	Beecham Group Plc	Fibrinolytisk enzym
US5258298A (en) *	1986-01-31	1993-11-02	Genetics Institute, Inc.	Deletion and glycosylation mutant of human tissue plasminogen activator
US5837518A (en) *	1986-01-31	1998-11-17	Genetics Institute, Inc.	Thrombolytic proteins
US5204255A (en) *	1986-01-31	1993-04-20	Sagami Chemical Research Center	Hybrid tissue plasminogen activator/urokinase polypeptides
US5002887A (en) *	1986-01-31	1991-03-26	Genetics Institute, Inc.	Truncated thrombolytic proteins
DE3781420T2 (de) *	1986-01-31	1993-02-25	Central Glass Co Ltd	Hybrides plasminogenaktivatoraehnliches polypeptid.
EP0234051B1 (en) *	1986-02-17	1991-12-11	Stichting Centraal Laboratorium van de Bloedtransfusiedienst van het Nederlandse Rode Kruis	Tissue-type plasminogen activator mutants; recombinant genetic information coding therefor and process for preparing said mutants; their use and pharmaceutical compositions
FR2594845B1 (fr) *	1986-02-21	1989-12-01	Genetica	Preparation par voie microbiologique de l'activateur tissulaire humain du plasminogene (t-pa) et conversion de l'enzyme ainsi obtenue en sa forme active
US4751084A (en) *	1986-02-26	1988-06-14	Monsanto Company	Tissue plasminogen activator from normal human colon cells
US4927630A (en) *	1986-02-26	1990-05-22	Monsanto Company	Tissue plasminogen activator from normal human colon cells
US4851517A (en) *	1986-02-26	1989-07-25	Monsanto Company	Tissue plasminogen activator oligosaccharide from normal human colon cells
US5132214A (en) *	1986-04-09	1992-07-21	Monsanto Company	Large scale production of plasminogen activator from normal human colon cells
US5589361A (en) *	1986-03-18	1996-12-31	Genentech, Inc.	Human tissue-type plasminogen activator variant
JPH01500322A (ja) *	1986-03-28	1989-02-09	クリエイティブ　バイオモレクレス，インコーポレーテッド	組織プラスミノーゲンアクテイベーター類縁蛋白質
AU7282687A (en) *	1986-03-28	1987-10-20	Gabriel Alvarado-Urbina	Protein analogues of tissue plasminogen activator
PT84589B (en) *	1986-04-02	1989-05-09	Beecham Group Plc	Process for preparing a fibrinolytic enzyme
GB8608020D0 (en) *	1986-04-02	1986-05-08	Beecham Group Plc	Compounds
AU7090887A (en) *	1986-04-02	1987-10-08	Beecham Group Plc	Plasminogen activators modified in first kringle domain
AU7090687A (en) *	1986-04-02	1987-10-08	Beecham Group Plc	Modified tissue-type plasminogen activators
DE3643158A1 (de) *	1986-04-21	1987-11-19	Boehringer Mannheim Gmbh	Gewebs-plasminogen-aktivator(tpa) - derivat und seine herstellung
DE3613401A1 (de) *	1986-04-21	1987-12-17	Boehringer Mannheim Gmbh	Verfahren zur herstellung von plasminogen-aktivatoren in prokaryonten
JP2585532B2 (ja) *	1986-05-10	1997-02-26	三井東圧化学株式会社	動物細胞の形質転換体及びそれを得る方法並びにその形質転換体を用いてｔ−ＰＡを生産する方法
PT84991B (pt) *	1986-06-06	1990-03-08	Genentech Inc	Processo para a producao de actividador do plasminogenio biologicamente activo
DK345087A (da) *	1986-07-11	1988-01-12	Novo Industri As	Modificeret vaevsplasminogenaktivator
PT86162B (pt) *	1986-11-21	1990-11-20	Smithkline Beecham Corp	Expressao de proteina recombinante
GB8628398D0 (en) *	1986-11-27	1986-12-31	Central Blood Lab Authority	Pharmaceutically-active conjugates
ZA879286B (en)	1986-12-16	1988-10-26	Smith Kline Rit	New plasminogen activators
NL8700013A (nl) *	1987-01-06	1988-08-01	Leuven Res & Dev Vzw	Hybride plasminogeenactivatoren met verbeterde trombolytische eigenschappen en geneesmiddelen die deze plasminogeenactivatoren bevatten.
MY103358A (en) *	1987-04-15	1993-06-30	Novartis Ag	Process for the production of protiens.
US5681713A (en) *	1987-05-08	1997-10-28	Smithkline Beecham Corporation	Expression of heterologous proteins in Drosophila cells
CA1341345C (en) *	1987-05-08	2002-03-05	Hanne Ranch Johansen	Expression of foreign genes in drosophila cells
ATE118815T1 (de) *	1987-06-04	1995-03-15	Zymogenetics Inc	Gewebs-plasminogen-aktivator-analoge mit modifizierten wachstumsfaktordomänen.
WO1988010307A1 (en) *	1987-06-24	1988-12-29	Genentech, Inc.	Balanced constitutive inducible transcription system
US5266474A (en) *	1987-06-24	1993-11-30	Genentech, Inc.	Balanced inducible transcription system
US4935237A (en) *	1988-03-21	1990-06-19	Genentech, Inc.	Processes for the preparation of t-PA mutants
AU2081788A (en) *	1987-07-06	1989-01-30	Genetics Institute Inc.	Novel thrombolytic proteins
IL87276A (en) *	1987-08-03	1995-07-31	Fujisawa Pharmaceutical Co	Analog of tissue plasminogen activator comprising only the kringle 2 and protease domain dna encoding the same processes for the preparation thereof and pharmaceutical compositions containing the same
US5504001A (en) *	1987-11-25	1996-04-02	Zymogenetics, Inc.	Hybrid plasminogen activator
DE3741149A1 (de) *	1987-12-04	1989-06-15	Thomae Gmbh Dr K	Zubereitungsformen zur verhinderung von adhaesionen von organen und organteilen
US5364622A (en) *	1987-12-04	1994-11-15	Dr. Karl Thomae Gmbh	Methods for preventing adhesions to organs and parts of organs by application of tissue plasminogen activator and hydroxyethylcellulose hydrogel
US4929560A (en) *	1988-02-03	1990-05-29	Damon Biotech, Inc.	Recovery of tissue plasminogen activator
JP2576200B2 (ja) *	1988-03-09	1997-01-29	味の素株式会社	生理活性タンパク質の製造法
AU3364589A (en) *	1988-03-22	1989-10-16	Invitron Corporation	Method to enhance tpa production
US5210037A (en) *	1988-03-22	1993-05-11	Centocor Incorporated	Method to enhance TPA production
US5037646A (en) *	1988-04-29	1991-08-06	Genentech, Inc.	Processes for the treatment of vascular disease
US5270198A (en) *	1988-05-20	1993-12-14	Genentech, Inc.	DNA molecules encoding variants of tissue plasminogen activators, vectors, and host cells
US5346824A (en) *	1988-05-20	1994-09-13	Genentech, Inc.	DNA encoding variants of tissue plasminogen activators and expression vectors and hosts thereof
US5023078A (en) *	1988-08-10	1991-06-11	Albert P. Halluin	Plasminogen activator-heparin conjugates
US5262170A (en) *	1988-09-02	1993-11-16	Genentech, Inc.	Tissue plasminogen activator having zymogenic or fibrin specific properties and substituted at amino acid positions 296-299, DNA molecules encoding them, vectors, and host cells
US5714145A (en) *	1988-09-02	1998-02-03	Genentech, Inc.	Tissue plasminogen activator having zymogenic or fibrin specific properties
DE3831714A1 (de) *	1988-09-17	1990-03-22	Basf Ag	Tpa-aehnliche polypeptide, ihre herstellung und verwendung
US5252713A (en) *	1988-09-23	1993-10-12	Neorx Corporation	Polymeric carriers for non-covalent drug conjugation
US5866413A (en) *	1989-03-06	1999-02-02	Board Of Regents Of The University Of Texas System	Pai-1 mutants
EP1029921B1 (en) *	1989-03-06	2002-10-23	The Board of Regents of The University of Texas System	Serpin resistant t-PA; mutants; genes
US5550042A (en) *	1989-03-06	1996-08-27	The Board Of Regents Of The University Of Texas System	Serine protease mutants of the chymotrypsin superfamily resistant to inhibition by their cognate inhibitors and genes encoding the same and serine protease inhibitor mutants and genes encoding the same
ATE132269T1 (de) *	1990-11-30	1996-01-15	Monoclonetics Int	Verfahren zur diagnose chronischer niedriger rückenschmerzen und nackenschmerzen
DK0643772T3 (da) *	1992-06-03	1998-02-02	Genentech Inc	Glycosyleringsvarianter af vævsplasminogenaktivator med forbedrede terapeutiske egenskaber
DE10153601A1 (de)	2001-11-02	2003-05-22	Paion Gmbh	DSPA zur Behandlung von Schlaganfall
JP4595968B2 (ja) *	2007-07-12	2010-12-08	パナソニック電工株式会社	往復式電気かみそりの内刃

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
IL43343A (en) *	1972-10-24	1976-03-31	Lilly Co Eli	Production of plasminogen activator
FR2252842B1 (pl) *	1973-12-03	1977-11-04	Fabre Sa Pierre
FR2310133A2 (fr) *	1975-05-05	1976-12-03	Fabre Sa Pierre	Nouveau medicament comportant un activateur du plasminogene perfectionne
US4245051A (en) *	1978-03-30	1981-01-13	Rockefeller University	Human serum plasminogen activator
DE3015699C2 (de) *	1979-04-26	1982-07-15	Asahi Kasei Kogyo K.K., Osaka	Herstellung eines Plasminogen-Aktivators
ES494343A0 (es) *	1979-07-27	1981-10-01		Procedimiento de obtencion de un activador del plasminogeno
US4370417A (en) *	1980-04-03	1983-01-25	Abbott Laboratories	Recombinant deoxyribonucleic acid which codes for plasminogen activator
NL8003402A (nl) *	1980-06-11	1982-01-04	Leuven Res & Dev Vzw	Nieuwe plasminogeen-activator en farmaceutisch preparaat met trombolytische werking.
JPS5852634B2 (ja) *	1980-12-05	1983-11-24	株式会社林原生物化学研究所	ウロキナ−ゼの製造法
GR79202B (pl) *	1982-05-05	1984-10-22	Genentech Inc
GT198302091A (es) *	1982-05-05	1984-10-25		Activador de plasminogeno de tejido humano a),b),c).

1983
- 1983-05-03 GR GR71273A patent/GR79202B/el unknown
- 1983-05-03 IL IL68561A patent/IL68561A/xx not_active IP Right Cessation
- 1983-05-03 AR AR83292915A patent/AR241654A1/es active
- 1983-05-04 IE IE1024/83A patent/IE54975B1/en not_active IP Right Cessation
- 1983-05-04 PT PT76636A patent/PT76636B/pt unknown
- 1983-05-04 CA CA000427389A patent/CA1293211C/en not_active Expired - Fee Related
- 1983-05-04 GB GB08312221A patent/GB2119804B/en not_active Expired
- 1983-05-04 DE DE198383302501T patent/DE93619T1/de active Pending
- 1983-05-04 EP EP83302501A patent/EP0093619B2/en not_active Expired - Lifetime
- 1983-05-04 RO RO110858A patent/RO90639B/ro unknown
- 1983-05-04 NO NO831575A patent/NO831575L/no unknown
- 1983-05-04 CY CY1341A patent/CY1341A/en unknown
- 1983-05-04 BR BR8302318A patent/BR8302318A/pt unknown
- 1983-05-04 DE DE19833316297 patent/DE3316297A1/de active Granted
- 1983-05-04 DD DD83250611A patent/DD210303A5/de unknown
- 1983-05-04 FR FR8307449A patent/FR2526443B1/fr not_active Expired
- 1983-05-04 ZW ZW104/83A patent/ZW10483A1/xx unknown
- 1983-05-04 ES ES522085A patent/ES522085A0/es active Granted
- 1983-05-04 DK DK198301988A patent/DK174236B1/da not_active IP Right Cessation
- 1983-05-04 DE DE8383302501T patent/DE3380567D1/de not_active Expired
- 1983-05-05 PL PL1983241805A patent/PL152438B1/pl unknown
- 1983-05-05 CS CS833187A patent/CS248705B2/cs unknown
- 1983-05-05 BG BG60834A patent/BG60253B2/bg unknown
- 1983-05-05 OA OA57990A patent/OA07419A/xx unknown
1985
- 1985-05-23 NO NO852065A patent/NO852065L/no unknown
1986
- 1986-06-24 KE KE3647A patent/KE3647A/xx unknown
- 1986-11-20 HK HK885/86A patent/HK88586A/xx unknown
1987
- 1987-12-30 MY MY119/87A patent/MY8700119A/xx unknown
1989
- 1989-02-01 JP JP1023654A patent/JPH0216981A/ja active Granted
1992
- 1992-04-16 JP JP4096448A patent/JP2564444B2/ja not_active Expired - Lifetime

Also Published As

Publication number	Publication date
BR8302318A (pt)	1984-01-10
EP0093619B2 (en)	1996-09-18
FR2526443A1 (fr)	1983-11-10
DE93619T1 (de)	1987-08-13
IE831024L (en)	1983-11-05
GB8312221D0 (en)	1983-06-08
PT76636B (en)	1986-01-10
KE3647A (en)	1986-07-25
PT76636A (en)	1983-06-01
CS248705B2 (en)	1987-02-12
PL152438B1 (en)	1990-12-31
RO90639A (ro)	1987-01-30
GR79202B (pl)	1984-10-22
JPH0216981A (ja)	1990-01-19
BG60253B1 (bg)	1994-03-24
AR241654A1 (es)	1992-10-30
DE3316297C2 (pl)	1989-09-21
ES8501629A1 (es)	1984-12-16
OA07419A (fr)	1984-11-30
DE3316297A1 (de)	1983-11-17
ZW10483A1 (en)	1983-09-21
AU563031B2 (en)	1987-06-25
ES522085A0 (es)	1984-12-16
DK198883A (da)	1983-12-20
DK198883D0 (da)	1983-05-04
DD210303A5 (de)	1984-06-06
GB2119804A (en)	1983-11-23
AU1414283A (en)	1983-11-10
JP2564444B2 (ja)	1996-12-18
HK88586A (en)	1986-11-28
IL68561A0 (en)	1983-09-30
BG60253B2 (bg)	1994-03-24
FR2526443B1 (fr)	1985-12-06
EP0093619B1 (en)	1989-09-13
RO90639B (ro)	1987-01-31
PL241805A1 (en)	1984-07-30
EP0093619A1 (en)	1983-11-09
IL68561A (en)	1991-01-31
DE3380567D1 (en)	1989-10-19
JPH05137583A (ja)	1993-06-01
NO831575L (no)	1983-11-07
GB2119804B (en)	1986-02-26
IE54975B1 (en)	1990-04-11
CA1293211C (en)	1991-12-17
JPH0448440B2 (pl)	1992-08-06
MY8700119A (en)	1987-12-31
DK174236B1 (da)	2002-10-07
NO852065L (no)	1983-11-07

Publication	Publication Date	Title
EP0093619B1 (en)	1989-09-13	Human tissue plasminogen activator, pharmaceutical compositions containing it, processes for making it, and dna and transformed cell intermediates therefor
US4766075A (en)	1988-08-23	Human tissue plasminogen activator
US5763253A (en)	1998-06-09	Methods of preparing tissue plasiminogen activator derivative composition
US4853330A (en)	1989-08-01	Human tissue plasminogen activator
EP0117059B1 (en)	1990-12-27	Methods for producing human tpa and expression vectors therefor
US5702938A (en)	1997-12-30	Human tissue plasminogen activator
US5385732A (en)	1995-01-31	Variants of tissue plasminogen activator, compositions and methods of use for same
US5185259A (en)	1993-02-09	Truncated human tissue plasminogen activator
AU624158B2 (en)	1992-06-04	Variants of plasminogen activators and processes for their production
US5010002A (en)	1991-04-23	Human t-PA production using vectors coding DHFR protein
USH2181H1 (en)	2007-02-06	Human tPA production using vectors coding for DHFR protein
US5268291A (en)	1993-12-07	Human t-PA production using vectors coding for DHFR protein
US5346824A (en)	1994-09-13	DNA encoding variants of tissue plasminogen activators and expression vectors and hosts thereof
BG60507B2 (bg)	1995-06-30	Човешки тъканен плазминогенен активатор
HRP950158A2 (en)	1997-08-31	Human tissue plasminogen activator
SI8310997A (sl)	1997-02-28	Aktivator plazminogena človeškega tkiva