RU2662888C1 - PLASMID EXPRESSING ALKALINE SERINE PROTEINASE COMPRISING A GENE EXPRESSING S1 FAMILY ALKALINE SERINE PROTEINASE FROM STREPTOMYCES AVERMITILIS VKM AC-1301, STRAIN ESCHERICHIA COLI M15 (pRep4, pQE30-A2.1) - PRODUCER OF THE PROTEINASE - Google Patents
PLASMID EXPRESSING ALKALINE SERINE PROTEINASE COMPRISING A GENE EXPRESSING S1 FAMILY ALKALINE SERINE PROTEINASE FROM STREPTOMYCES AVERMITILIS VKM AC-1301, STRAIN ESCHERICHIA COLI M15 (pRep4, pQE30-A2.1) - PRODUCER OF THE PROTEINASE Download PDFInfo
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- RU2662888C1 RU2662888C1 RU2017111639A RU2017111639A RU2662888C1 RU 2662888 C1 RU2662888 C1 RU 2662888C1 RU 2017111639 A RU2017111639 A RU 2017111639A RU 2017111639 A RU2017111639 A RU 2017111639A RU 2662888 C1 RU2662888 C1 RU 2662888C1
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- 239000013612 plasmid Substances 0.000 title claims abstract description 8
- 241001596967 Escherichia coli M15 Species 0.000 title claims abstract description 7
- 241001468227 Streptomyces avermitilis Species 0.000 title claims abstract description 6
- 102000012479 Serine Proteases Human genes 0.000 title claims abstract 8
- 108010022999 Serine Proteases Proteins 0.000 title claims abstract 8
- 102000035195 Peptidases Human genes 0.000 title abstract description 10
- 108091005804 Peptidases Proteins 0.000 title abstract description 10
- 235000019833 protease Nutrition 0.000 title abstract description 10
- 108090000623 proteins and genes Proteins 0.000 title description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 230000001131 transforming effect Effects 0.000 claims abstract 2
- 239000003599 detergent Substances 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 241000203809 Actinomycetales Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
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- 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)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- 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)
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Abstract
Description
Бактериальные протеиназы – это ферменты, катализирующие гидролиз пептидных связей в белках и пептидах. Микробные протеиназы составляют одну из важных групп промышленно и коммерчески получаемых ферментов, объемы продаж которых составляют примерно 2/3 всех продаж ферментов. Динамика роста количества патентов на получение протеиназ является наиболее высокой среди промышленно значимых ферментов.Bacterial proteinases are enzymes that catalyze the hydrolysis of peptide bonds in proteins and peptides. Microbial proteinases are one of the important groups of industrially and commercially obtained enzymes, with sales of approximately 2/3 of all enzyme sales. The growth dynamics of the number of patents for proteinases is the highest among industrially significant enzymes.
В качестве источника генетического материала, кодирующего ранее неохарактеризованную протеиназу семейства S1 (далее по тексту белок A2.1), был выбран штамм Streptomyces avermitilis - VKM Ас-1301, относящийся к порядку Actinomycetales.As a source of genetic material encoding a previously uncharacterized proteinase of the S1 family (hereinafter referred to as protein A2.1), the strain Streptomyces avermitilis - VKM Ac-1301, which belongs to the Actinomycetales order, was selected.
Для получения искомого гена была проведена амплификация с использованием ДНК-зависимой ДНК-полимеразы Q5 и праймерами:To obtain the desired gene, amplification was performed using DNA-dependent DNA polymerase Q5 and primers:
А2.1_F_wSP_BamHI: TATATGGATCCGCCATCGAGCCGCCCA2.1_F_wSP_BamHI: TATATGGATCCGCCATCGAGCCGCCC
А2.1_R_HindIII: TATATAAGCTTTCAGAGACGCTGCCACAA2.1_R_HindIII: TATATAAGCTTTCAGAGACGCTGCCACA
При амплификации искомого гена последовательность, кодирующая сигнальный пептид, была исключена. Полученный ПЦР-продукт был обработан эндонуклеазами рестрикции BamHI и HindIII и клонирован в экспрессионный вектор pQE30 (pREP4). Лигазную смесь трансформировали в компетентные клетки Е. coli M15 (pREP4), отдельные клоны трансформантов E. coli анализировались на наличие вставки с помощью ПЦР с ДНК–зависимой ДНК–полимеразой Q5, с праймерами к плазмиде. Клоны со вставкой пересевали на ночную культуру для последующего выделения плазмидной ДНК pQE30 (pREP4, pQE30-A2.1). Анализ клонируемой последовательности ДНК проводился методом секвенирования. Нуклеотидная последовательность гена, кодирующего рекомбинантный белок, представлена в перечне последовательностей SEQ ID NO: 1, а карта полученной плазмидной ДНК представлена на фиг. 1.When amplifying the desired gene, the sequence encoding the signal peptide was excluded. The resulting PCR product was processed with restriction endonucleases BamHI and HindIII and cloned into the expression vector pQE30 (pREP4). The ligase mixture was transformed into competent E. coli M15 cells ( pREP4 ) , individual clones of E. coli transformants were analyzed for insertion by PCR with Q5 DNA-dependent DNA polymerase, with plasmid primers. Clones with the insert were subcultured onto overnight culture for subsequent isolation of pQE30 plasmid DNA (pREP4, pQE30-A2.1). Analysis of the cloned DNA sequence was carried out by sequencing. The nucleotide sequence of the gene encoding the recombinant protein is shown in the sequence listing of SEQ ID NO: 1, and a map of the obtained plasmid DNA is shown in FIG. one.
Полученный экспрессионный штамм E. coli M15 (pRep4, pQE30-A2.1) позволяет получить протеиназу А2.1 молекулярной массой 44,92 кДа, pH оптимум 8 (при гидролизе 2% денатурированного гемоглобина) и 10–11 (при гидролизе 2% казеина), температурный оптимум 60°С. Показано, что активность протеиназы А2.1 не ингибируется полностью в присутствии SDS и незначительно изменяется в присутствии Triton X–100, Twin 20. Полученные результаты являются предпосылкой для возможного применения данной протеиназы в составе моющих средств.The resulting expression strain of E. coli M15 (pRep4, pQE30-A2.1) allows to obtain proteinase A2.1 with a molecular weight of 44.92 kDa, pH optimum of 8 (for hydrolysis of 2% denatured hemoglobin) and 10–11 (for hydrolysis of 2% casein ), temperature optimum 60 ° С. It was shown that the activity of proteinase A2.1 is not completely inhibited in the presence of SDS and slightly changes in the presence of Triton X – 100, Twin 20. The results obtained are a prerequisite for the possible use of this proteinase in detergents.
Claims (2)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2017111639A RU2662888C1 (en) | 2017-04-06 | 2017-04-06 | PLASMID EXPRESSING ALKALINE SERINE PROTEINASE COMPRISING A GENE EXPRESSING S1 FAMILY ALKALINE SERINE PROTEINASE FROM STREPTOMYCES AVERMITILIS VKM AC-1301, STRAIN ESCHERICHIA COLI M15 (pRep4, pQE30-A2.1) - PRODUCER OF THE PROTEINASE |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| RU2017111639A RU2662888C1 (en) | 2017-04-06 | 2017-04-06 | PLASMID EXPRESSING ALKALINE SERINE PROTEINASE COMPRISING A GENE EXPRESSING S1 FAMILY ALKALINE SERINE PROTEINASE FROM STREPTOMYCES AVERMITILIS VKM AC-1301, STRAIN ESCHERICHIA COLI M15 (pRep4, pQE30-A2.1) - PRODUCER OF THE PROTEINASE |
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| RU2662888C1 true RU2662888C1 (en) | 2018-07-31 |
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| RU2017111639A RU2662888C1 (en) | 2017-04-06 | 2017-04-06 | PLASMID EXPRESSING ALKALINE SERINE PROTEINASE COMPRISING A GENE EXPRESSING S1 FAMILY ALKALINE SERINE PROTEINASE FROM STREPTOMYCES AVERMITILIS VKM AC-1301, STRAIN ESCHERICHIA COLI M15 (pRep4, pQE30-A2.1) - PRODUCER OF THE PROTEINASE |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993000925A1 (en) * | 1991-07-01 | 1993-01-21 | Amgen Inc. | Isolation and characterization of a novel protease from streptomyces lividans |
| US20050112579A1 (en) * | 2002-07-02 | 2005-05-26 | Dendreon San Diego Llc, A Delaware Corporation | Nucleic acid molecules encoding serine protease 16, the encoded polypeptides and methods based thereon |
| RU2486243C1 (en) * | 2011-10-26 | 2013-06-27 | Федеральное государственное бюджетное учреждение науки Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А.Овчинникова Российской академии наук (ИБХ РАН) | POLYNUCLEOTIDE ENCODING MUTANT RECOMBINANT IgA1 PROTEASE OF Neisseria meningitidis OF SEROGROUP B, RECOMBINANT PLASMID DNA COMPRISING SAID POLYNUCLEOTIDE, HOST CELL CONTAINING SAID PLASMID DNA, RECOMBINANT IgA1 PROTEASE OF Neisseria memingitidis OF SEROGROUP B, METHOD OF PRODUCING MATURE FORM OF IgA1 PROTEASE |
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- 2017-04-06 RU RU2017111639A patent/RU2662888C1/en not_active IP Right Cessation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993000925A1 (en) * | 1991-07-01 | 1993-01-21 | Amgen Inc. | Isolation and characterization of a novel protease from streptomyces lividans |
| US20050112579A1 (en) * | 2002-07-02 | 2005-05-26 | Dendreon San Diego Llc, A Delaware Corporation | Nucleic acid molecules encoding serine protease 16, the encoded polypeptides and methods based thereon |
| RU2486243C1 (en) * | 2011-10-26 | 2013-06-27 | Федеральное государственное бюджетное учреждение науки Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А.Овчинникова Российской академии наук (ИБХ РАН) | POLYNUCLEOTIDE ENCODING MUTANT RECOMBINANT IgA1 PROTEASE OF Neisseria meningitidis OF SEROGROUP B, RECOMBINANT PLASMID DNA COMPRISING SAID POLYNUCLEOTIDE, HOST CELL CONTAINING SAID PLASMID DNA, RECOMBINANT IgA1 PROTEASE OF Neisseria memingitidis OF SEROGROUP B, METHOD OF PRODUCING MATURE FORM OF IgA1 PROTEASE |
Non-Patent Citations (1)
| Title |
|---|
| NCBI Reference Sequence: WP_010982438.1, 15.05.2013; Найдено в Инетрнет 06.09.2017 по адресу: www.ncbi.nlm.nih.gov/protein?cmd=Retrieve&dopt=GenPept&list_uids=499291180#sequence_WP-010982438.1. * |
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