RU2015116745A - Recombinant plasmid DNA pER-TA1 GyrA-AcSer, encoding the serine acetyltransferase capable IN VIVO acetylated N-terminal serine Desacetylthymosin ALPHA 1 and the hybrid protein capable of autocatalytic cleavage with formation Thymosin Alpha 1 HUMAN STRAIN Eschrichia coli C3030 / pER-TA1GyRA-AcSer PRODUCER OF THE SPECIFIED PROTEINS AND METHOD FOR PRODUCING GENE-ENGINEERED THYMOSINE ALPHA 1 HUMAN - Google Patents
Recombinant plasmid DNA pER-TA1 GyrA-AcSer, encoding the serine acetyltransferase capable IN VIVO acetylated N-terminal serine Desacetylthymosin ALPHA 1 and the hybrid protein capable of autocatalytic cleavage with formation Thymosin Alpha 1 HUMAN STRAIN Eschrichia coli C3030 / pER-TA1GyRA-AcSer PRODUCER OF THE SPECIFIED PROTEINS AND METHOD FOR PRODUCING GENE-ENGINEERED THYMOSINE ALPHA 1 HUMAN Download PDFInfo
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- RU2015116745A RU2015116745A RU2015116745/10A RU2015116745A RU2015116745A RU 2015116745 A RU2015116745 A RU 2015116745A RU 2015116745/10 A RU2015116745/10 A RU 2015116745/10A RU 2015116745 A RU2015116745 A RU 2015116745A RU 2015116745 A RU2015116745 A RU 2015116745A
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- acser
- per
- ta1gyra
- alpha
- plasmid dna
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- 239000013612 plasmid Substances 0.000 title claims abstract 11
- 108090000623 proteins and genes Proteins 0.000 title claims abstract 8
- 102000004169 proteins and genes Human genes 0.000 title claims abstract 6
- 108091022908 Serine O-acetyltransferase Proteins 0.000 title claims abstract 5
- 230000015572 biosynthetic process Effects 0.000 title claims abstract 3
- 238000003776 cleavage reaction Methods 0.000 title claims abstract 3
- 230000007017 scission Effects 0.000 title claims abstract 3
- 238000004519 manufacturing process Methods 0.000 title abstract 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 title 1
- 108010078233 Thymalfasin Proteins 0.000 title 1
- 102400000800 Thymosin alpha-1 Human genes 0.000 title 1
- 238000001727 in vivo Methods 0.000 title 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 title 1
- NZVYCXVTEHPMHE-ZSUJOUNUSA-N thymalfasin Chemical compound CC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NZVYCXVTEHPMHE-ZSUJOUNUSA-N 0.000 title 1
- 229960004231 thymalfasin Drugs 0.000 title 1
- 241000588724 Escherichia coli Species 0.000 claims abstract 14
- 101500028961 Homo sapiens Thymosin alpha-1 Proteins 0.000 claims abstract 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract 5
- 239000002594 sorbent Substances 0.000 claims abstract 5
- 229920002101 Chitin Polymers 0.000 claims abstract 4
- 239000000872 buffer Substances 0.000 claims abstract 4
- 239000007853 buffer solution Substances 0.000 claims abstract 4
- 239000012634 fragment Substances 0.000 claims abstract 4
- 230000017730 intein-mediated protein splicing Effects 0.000 claims abstract 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims abstract 4
- 229920001184 polypeptide Polymers 0.000 claims abstract 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract 4
- 239000007983 Tris buffer Substances 0.000 claims abstract 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract 3
- 108090000204 Dipeptidase 1 Proteins 0.000 claims abstract 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract 2
- 108090000790 Enzymes Proteins 0.000 claims abstract 2
- 102000004190 Enzymes Human genes 0.000 claims abstract 2
- 229930182555 Penicillin Natural products 0.000 claims abstract 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract 2
- 239000003242 anti bacterial agent Substances 0.000 claims abstract 2
- 229940088710 antibiotic agent Drugs 0.000 claims abstract 2
- 230000006378 damage Effects 0.000 claims abstract 2
- 230000002068 genetic effect Effects 0.000 claims abstract 2
- 230000001939 inductive effect Effects 0.000 claims abstract 2
- 239000003550 marker Substances 0.000 claims abstract 2
- 229940049954 penicillin Drugs 0.000 claims abstract 2
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract 2
- 238000001179 sorption measurement Methods 0.000 claims abstract 2
- 230000009466 transformation Effects 0.000 claims abstract 2
- 230000001131 transforming effect Effects 0.000 claims abstract 2
- 238000005406 washing Methods 0.000 claims abstract 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 101000605024 Rattus norvegicus Large neutral amino acids transporter small subunit 1 Proteins 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 238000000034 method Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 239000011780 sodium chloride Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract 4
- 108020001507 fusion proteins Proteins 0.000 abstract 2
- 102000037865 fusion proteins Human genes 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 1
- 210000002381 plasma Anatomy 0.000 abstract 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
1. Рекомбинантная плазмидная ДНК pER - TA1GyrA - AcSer, содержащая полицистронную конструкцию для одновременного биосинтеза двух белков - гибридного полипептида, содержащего тимозин α1 человека и интеин, и фермента E.coli - сериновой ацетилтрансферазы в клетках Escherichia coli;NdeI/SapI - фрагмента ДНК плазмиды pTWIN-1;NdeI/SapI - фрагмента ДНК, содержащего адаптированную к этим сайтам последовательность гена рекомбинантного человеческого тимозина α1 и содержащая в качестве генетического маркера ген β-лактамазы, детерминирующий устойчивость трансформированных плазмидой pER - TA1GyrA - AcSer клеток E.coli к пенициллиновым антибиотикам; уникальные сайты узнавания рестрикционных эндонуклеаз, расположенные на следующем расстоянии влево от сайта BamHI - Pstl - 575 п.о., NdeI -: 1448 п.о., XbaI - 1487 п.о., EcoRV - 3520 п.о., HpaI - 3576 п.о.2. Штамм Escherichia coli С3030/ pER - TA1GyrA - AcSer, продуцирующий гибридный полипептид, содержащий дезацетилтимозина α1 человека и интеин, а также сериновую ацетилтрансферазу, полученный путем трансформации клеток штамма Escherichia coli С3030 рекомбинантной плазмидной ДНК pER - TA1GyrA - AcSer по п. 1.3. Способ получения рекомбинантного тимозина α1 человека, включающий трансформацию штамма Escherichia coli С3030 плазмидной ДНК pER - TA1GyrA - AcSer, по п. 1, культивирование штамма-продуцента Escherichia coli С3030/ pER - TA1GyrA - AcSer по п. 2, отделение гибридного белка TA1GyrA в растворимой форме после разрушения клеток с помощью ультразвукового дезинтегратора в буферном растворе (50 мМ Tris/HCl, 10 мM EDTA, 1 мM PMSF), адсорбирование гибридного белка на колонке с хитиновым сорбентом (NEB, England), индуцирование его автокаталитического расщепления промыванием хитинового сорбента буферным раствором, содержащим уравновешенным в буфере, содерж�1. Recombinant plasmid DNA pER - TA1GyrA - AcSer containing a polycistronic construct for the simultaneous biosynthesis of two proteins - a hybrid polypeptide containing human thymosin α1 and intein, and E. coli enzyme - serine acetyltransferase in Escherichia coli cells; NdeI / SapI fragment of plasmid DNA pTWIN-1; NdeI / SapI - DNA fragment containing the recombinant human thymosin α1 gene sequence adapted to these sites and containing the β-lactamase gene determining the stability of transformed plasmas as a genetic marker Doi pER - TA1GyrA - AcSer E.coli cells to penicillin antibiotics; unique recognition sites of restriction endonucleases located at the following distance to the left of the BamHI site - Pstl - 575 bp, NdeI -: 1448 bp, XbaI - 1487 bp, EcoRV - 3520 bp, HpaI - 3576 bp 2. The Escherichia coli strain C3030 / pER - TA1GyrA - AcSer producing a hybrid polypeptide containing human deacetylthymosin α1 and intein, as well as the serine acetyltransferase obtained by transformation of Escherichia coli C3030 strain cells with recombinant plasmid pER - AcSerg IgG plasmid DNA. A method for producing recombinant human thymosin α1, comprising transforming the Escherichia coli strain C3030 plasmid DNA pER - TA1GyrA - AcSer, according to claim 1, culturing the producer strain Escherichia coli C3030 / pER - TA1GyrA - AcSer according to claim 2, separating the TAA fusion protein into TA1 fusion protein after destruction of the cells using an ultrasonic disintegrator in a buffer solution (50 mM Tris / HCl, 10 mM EDTA, 1 mM PMSF), adsorption of the hybrid protein on a column with chitin sorbent (NEB, England), inducing its autocatalytic cleavage by washing the chitin sorbent with a buffer solution ohm containing equilibrated in buffer, containing
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2015116745/10A RU2593172C2 (en) | 2015-05-05 | 2015-05-05 | RECOMBINANT PLASMID DNA pER-TA1 GyrA-AcSer CODING SERINE ACETYLTRANSFERASE CAPABLE OF in vivo ACETYLATION OF N-TERMINAL SERINE DEACETYL-THYMOSIN α1 AND HYBRID PROTEIN CAPABLE OF AUTOCATALYTIC BREAKDOWN TO FORM HUMAN THYMOSIN α1, STRAIN OF Eschrichia coli C3030/pER-TA1GyrA-AcSer PRODUCER OF SAID PROTEINS AND METHOD OF PRODUCING GENETICALLY ENGINEERED HUMAN THYMOSIN |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2015116745/10A RU2593172C2 (en) | 2015-05-05 | 2015-05-05 | RECOMBINANT PLASMID DNA pER-TA1 GyrA-AcSer CODING SERINE ACETYLTRANSFERASE CAPABLE OF in vivo ACETYLATION OF N-TERMINAL SERINE DEACETYL-THYMOSIN α1 AND HYBRID PROTEIN CAPABLE OF AUTOCATALYTIC BREAKDOWN TO FORM HUMAN THYMOSIN α1, STRAIN OF Eschrichia coli C3030/pER-TA1GyrA-AcSer PRODUCER OF SAID PROTEINS AND METHOD OF PRODUCING GENETICALLY ENGINEERED HUMAN THYMOSIN |
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| Publication Number | Publication Date |
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| RU2015116745A true RU2015116745A (en) | 2015-10-10 |
| RU2593172C2 RU2593172C2 (en) | 2016-07-27 |
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| RU2015116745/10A RU2593172C2 (en) | 2015-05-05 | 2015-05-05 | RECOMBINANT PLASMID DNA pER-TA1 GyrA-AcSer CODING SERINE ACETYLTRANSFERASE CAPABLE OF in vivo ACETYLATION OF N-TERMINAL SERINE DEACETYL-THYMOSIN α1 AND HYBRID PROTEIN CAPABLE OF AUTOCATALYTIC BREAKDOWN TO FORM HUMAN THYMOSIN α1, STRAIN OF Eschrichia coli C3030/pER-TA1GyrA-AcSer PRODUCER OF SAID PROTEINS AND METHOD OF PRODUCING GENETICALLY ENGINEERED HUMAN THYMOSIN |
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| CN107828811A (en) * | 2017-10-24 | 2018-03-23 | 湖南方盛制药股份有限公司 | Plasmid standard, its preparation method and its application of the target gene of thymalfasin containing people |
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| RU2055895C1 (en) * | 1992-07-29 | 1996-03-10 | Всесоюзный научно-исследовательский институт прикладной микробиологии | METHOD OF PREPARING RECOMBINANT HUMAN α1 THYMOSINE |
| RU2225443C1 (en) * | 2002-07-25 | 2004-03-10 | Общество с ограниченной ответственностью "Научно-производственное предприятие "Фармаклон" | RECOMBINANT PLASMID DNA ENCODING SYNTHESIS, METHOD FOR PREPARING AND PREPARATION OF RECOMBINANT HYBRID PROTEIN α-TUMOR NECROSIS FACTOR THYMOSIN-α1. |
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