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WO2003018742A2 - L-asparaginase recombinante d'erwinia carotovora - Google Patents

L-asparaginase recombinante d'erwinia carotovora Download PDF

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Publication number
WO2003018742A2
WO2003018742A2 PCT/RU2002/000405 RU0200405W WO03018742A2 WO 2003018742 A2 WO2003018742 A2 WO 2003018742A2 RU 0200405 W RU0200405 W RU 0200405W WO 03018742 A2 WO03018742 A2 WO 03018742A2
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WO
WIPO (PCT)
Prior art keywords
gene
aηά
recombinant
asparaginase
investigation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/RU2002/000405
Other languages
English (en)
Russian (ru)
Other versions
WO2003018742A3 (fr
Inventor
Mikhail Anatolevich Eldarov
Aleksandr Aleksandrovich Zhgun
Yury Viktorovich Gervaziev
Svetlana Serebedzhanovna Aleksandrova
Vladimir Georgievich Bogush
Konstantin Vasilevich Sidoruk
Elena Vasilevna Sveshnikova
Anna Arkadevna Borisova
Natalya Mikhailovna Omelnyuk
Aleksandr Ivanovich Archakov
Konstantin Georgievich Skryabin
Nikolai Nikolaevich Sokolov
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GOSUDARSTVENNOE UCHREZHDENIE NAUCHNO-ISSLEDOVATELSKY INSTITUT BIOMEDITSINSKOY KHIMII IM OREKHOVICHA ROSSIISKOY AKADEMII MEDITSINSKIKH NAUK
Gosudarstvennoe Uchrezhdenie Tsentr 'bioinzheneriya' Rossiiskoy Akademii Nauk
Original Assignee
GOSUDARSTVENNOE UCHREZHDENIE NAUCHNO-ISSLEDOVATELSKY INSTITUT BIOMEDITSINSKOY KHIMII IM OREKHOVICHA ROSSIISKOY AKADEMII MEDITSINSKIKH NAUK
Gosudarstvennoe Uchrezhdenie Tsentr 'bioinzheneriya' Rossiiskoy Akademii Nauk
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.)
Filing date
Publication date
Priority claimed from RU2001123442/13A external-priority patent/RU2221868C2/ru
Priority claimed from RU2002108505/13A external-priority patent/RU2224797C2/ru
Application filed by GOSUDARSTVENNOE UCHREZHDENIE NAUCHNO-ISSLEDOVATELSKY INSTITUT BIOMEDITSINSKOY KHIMII IM OREKHOVICHA ROSSIISKOY AKADEMII MEDITSINSKIKH NAUK, Gosudarstvennoe Uchrezhdenie Tsentr 'bioinzheneriya' Rossiiskoy Akademii Nauk filed Critical GOSUDARSTVENNOE UCHREZHDENIE NAUCHNO-ISSLEDOVATELSKY INSTITUT BIOMEDITSINSKOY KHIMII IM OREKHOVICHA ROSSIISKOY AKADEMII MEDITSINSKIKH NAUK
Priority to AU2002332371A priority Critical patent/AU2002332371A1/en
Publication of WO2003018742A2 publication Critical patent/WO2003018742A2/fr
Publication of WO2003018742A3 publication Critical patent/WO2003018742A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Le ⁇ a ⁇ s ⁇ vennye ⁇ my ba ⁇ e ⁇ ialny ⁇ L-as ⁇ a ⁇ aginaz on ⁇ yazhenie desya ⁇ ile ⁇ y shi ⁇ is ⁇ lzuyu ⁇ sya in ⁇ n ⁇ l ⁇ giches ⁇ y ⁇ a ⁇ i ⁇ e for treating ⁇ s ⁇ y ⁇ lim ⁇ blas ⁇ ny ⁇ ley ⁇ z ⁇ v, and lim ⁇ - ⁇ e ⁇ i ⁇ ul ⁇ sa ⁇ m chel ⁇ ve ⁇ a ( ⁇ g ⁇ zyu ⁇ D.S,] ⁇ . [1985].
  • a solvable invention is the production of microscopic activity with high specific activity and lowered glutalin relevance. The problem was solved by
  • Example 1 Identification of the Strain ⁇ mm ⁇ g ⁇ . industrial L-asparaginase with high asparticase and low glutaminase activity
  • P ⁇ ayme ⁇ y is ⁇ lz ⁇ vali in PTS ⁇ with ⁇ b ⁇ azts ⁇ m gen ⁇ mn ⁇ y D ⁇ isolated from sh ⁇ amma ⁇ g ⁇ g ⁇ g ⁇ s ⁇ g ⁇ g ⁇ ⁇ a 204.
  • the volume of the reaction mixture was 50 ⁇ l of a single PCB buffer for--polimerase ( ⁇ vit iser sait »» »)»)), the percentage of the concentration was 0.5 m, the percentage was 0.5 ⁇ After amplification in 5 ⁇ l of the sample by the elec- tric elec- trophoresis in 1% agar gel, the homogeneous homogenous reaction of the particle size 300 was identified.
  • the volume of the reactive mixture was. 50 ⁇ l of a single PCB buffer for a mixture of ⁇ ⁇ and ⁇ polymer, a concentration of ⁇ - 0.4 m ⁇ , a concentration of ⁇ - - - 1 ⁇ m, a percentage of D ⁇ - 20 ng / PCRs were operated under the following conditions - heating - 2 min, 94 °, then 25 cycles of amplification - 94 °, 40 "; 50 °, 40"; 68 °, 5 '.
  • the samples obtained on the basis of the sample and the ⁇ method of electrolysis in 1% agar gel were used to identify fragments of sizes 1200 and 5000 bp, respectively. Samples were isolated from the gel (Example 2) and partitioned by nucleotide sequencing by automatically sequencing for a total of 207 seconds. The installed complete investigation has been carried out in the liver before issue 8 ⁇ , GO ⁇ 07.
  • Is ⁇ lz ⁇ vannye ⁇ ayme ⁇ y ⁇ g ⁇ anichivayu ⁇ ⁇ agmen ⁇ gene ⁇ S ⁇ - ⁇ . ⁇ 8, v ⁇ lyuchayuschy initsia ⁇ ny ⁇ d ⁇ n ⁇ anslyatsii ⁇ , ⁇ as ⁇ l ⁇ zhenny in ⁇ l ⁇ zhenii 48 ⁇ sled ⁇ va ⁇ eln ⁇ s ⁇ i 8 ⁇ , GO ⁇ 07 and ⁇ e ⁇ mina ⁇ ny ⁇ d ⁇ n ⁇ , ⁇ as ⁇ l ⁇ zhenny in ⁇ l ⁇ zhenii 1093 ⁇ y ⁇ sled ⁇ va ⁇ eln ⁇ s ⁇ i same.
  • a specific full-sized amplification product includes a complete transducer investigation of the SCC gene.
  • the following amplification conditions were used: initial denaturation - 94 °, 60 ", then 30 cycles of amplification - 94 °, 40"; 50 °, 40 "; 72 °, 90".
  • the reaction mixture was separated by 1% agar gel and the reaction product was detected - a single reaction element of 1100 p.p.
  • the substance was isolated from the gel and removed in vectrum ⁇ - ⁇ (" ⁇ beau ⁇ ", ⁇ ), as described in Example 2. From the obtained individual tips 11 plants isolated the standard method and analyzed it by means of hydrolysis of the organic process and separation of the products of the business unit in 1%.
  • EXAMPLE 4 Expression of the ⁇ - ⁇ 8 gene in the cells of the Russian Academy of Sciences through the control panel.
  • the plasmid was disassembled together with W + W, removed from the agar gel with a fragment of 2560 ⁇ .n. Two fragments of the league were removed, they took away the risks of strain ⁇ 9, stable at the same time, ampicillin and canamycin.
  • plasmid D was isolated, hydrolyzed by its natural product and detected the presence of hydrolysis products of size 3 T.p.n. and 2.5 ⁇ . ⁇ .n.
  • the physical and genetic pathway of the obtained such plasmid species is shown in Fig. 1.
  • a sulphate-ammonia fraction (22.5 ml) was applied to a ring with a safe ⁇ -25 (5 x 45 cm);
  • the speed of application of the buffer when applying and gel filtration is 400 ml / hour.
  • L-paraphaginase is eluted from the S-phase in the range of ⁇ C ⁇ concentration of 0.45 to 0.5 ⁇ . They received about 225 000 100 activity of the enzyme in 100-120 ml of buffer with an output of .75.6% (Table 3).
  • the combined active fractions suppressed the ultrafiltration by pressure on the system “Millipore” (US) with the use of an ultrafiltration membrane for 30 000 days.
  • composition of a recombinant L-aspartase ⁇ g. with ⁇ in the aforementioned above makes up about 95%, and a small.
  • the mass of the subunit of the fragment was about 36 kDa, which follows from the analysis of the preparation by the gel method of the elec- troelectomy in DDS-PG (Fig. 2).
  • the ⁇ C ⁇ - ⁇ 8 gene cures a polypeptide with an amino acid test indicated in the liver of the investigations on July 8 8., which is a result of an accident.
  • Protein samples were dissolved in 30% acetyl, containing 0.1% acid and applied to the membranes of ⁇ ⁇ ⁇ ⁇ ⁇ (USA).
  • the sequencing was performed on the site of the United Kingdom (Germany, model 816), equipped with analysis of the environment (United States, United States)
  • ⁇ - ⁇ has a wide range of ⁇ in the range of ⁇ from 7.5 to 9.0; the mass of the subunit is about 36 kDa.
  • a temperature of optimum (55 ° C) and a high specific activity of the SSC-3 see table 3.
  • the recombinant b-paraphase of ⁇ .cie ⁇ 1969 ⁇ has a very low content of glutamine, compared with the corresponding ⁇ ⁇ ⁇ ............ Skuzh ⁇ ket ierik, the hydropathy of parasitic paralysis is responsible for the many toxic effects of the drug and the pharmaceutical use.
  • the negligible glutaminase activity of the USA can cause the low toxicity of medical preparations of this medicine and their improved pharmaceuticals. 17

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé pour isoler la séquence d'ADN codant pour la L-asparaginase recombinante de la bactérie Erwinia carotovora. On fabrique une préparation de peptide homogène qui correspond à la L-asparaginase recombinante de la bactérie Erwinia carotovora et qui est isolée en utilisant une souche bactérienne productrice.
PCT/RU2002/000405 2001-08-22 2002-08-21 L-asparaginase recombinante d'erwinia carotovora Ceased WO2003018742A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002332371A AU2002332371A1 (en) 2001-08-22 2002-08-21 Recombinant l-asparaginase less thanigreater thanerwinia caratovoraless than/igreater than

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
RU2001123442/13A RU2221868C2 (ru) 2001-08-22 2001-08-22 Ген l-аспарагиназы erwinia carotovora и штамм escherichia coli вкпм № в-8174 - продуцент l-аспарагиназы erwinia carotovora
RU2001123442 2001-08-22
RU2002108505/13A RU2224797C2 (ru) 2002-04-04 2002-04-04 Рекомбинантная плазмидная днк pacyclans для переноса и экспрессии в клетках escherichia coli гена l-аспарагиназы erwinia carotovora (ecar-lans) и способ получения рекомбинантной ecar-lans из биомассы штамма e.coli-продуцента
RU2002108505 2002-04-04

Publications (2)

Publication Number Publication Date
WO2003018742A2 true WO2003018742A2 (fr) 2003-03-06
WO2003018742A3 WO2003018742A3 (fr) 2003-08-07

Family

ID=26654092

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2002/000405 Ceased WO2003018742A2 (fr) 2001-08-22 2002-08-21 L-asparaginase recombinante d'erwinia carotovora

Country Status (2)

Country Link
AU (1) AU2002332371A1 (fr)
WO (1) WO2003018742A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011003886A1 (fr) * 2009-07-06 2011-01-13 Alize Pharma Ii L-asparaginase pegylée
US10174302B1 (en) 2017-06-21 2019-01-08 Xl-Protein Gmbh Modified L-asparaginase
CN110256535A (zh) * 2019-06-14 2019-09-20 中国石油大学(华东) L-天门冬酰胺酶XiDL及其编码基因和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8519753D0 (en) * 1985-08-06 1985-09-11 Health Lab Service Board Production of 1-asparaginase
AU6773487A (en) * 1985-12-13 1987-06-30 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Process for manufacture of l-asparaginase from erwinia carotovora
WO1998056410A1 (fr) * 1997-06-09 1998-12-17 Childrens Hospital Los Angeles Utilisation de l'asparaginase wolinella succinogenes pour traiter des maladies associees a la dependance de l'asparagine

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011003886A1 (fr) * 2009-07-06 2011-01-13 Alize Pharma Ii L-asparaginase pegylée
WO2011003633A1 (fr) * 2009-07-06 2011-01-13 Alize Pharma Ii L-asparaginase pegylée
CN102573917A (zh) * 2009-07-06 2012-07-11 阿利兹第二药物公司 聚乙二醇化的l-天冬酰胺酶
EA021168B1 (ru) * 2009-07-06 2015-04-30 Ализе Фарма Ii Пегилированная l-аспарагиназа
US9920311B2 (en) 2009-07-06 2018-03-20 Jazz Pharmaceuticals Ii Sas PEGylated L-asparaginase
US11046946B2 (en) 2009-07-06 2021-06-29 Jazz Pharmaceuticals Ii Sas PEGylated L-asparaginase
USRE49736E1 (en) 2009-07-06 2023-11-28 Jazz Pharmaceuticals Ii Sas Pegylated L-asparaginase
US12441992B2 (en) 2009-07-06 2025-10-14 Jazz Pharmaceuticals Ii Sas Method of treating AML with PEGylated l-asparaginase
US10174302B1 (en) 2017-06-21 2019-01-08 Xl-Protein Gmbh Modified L-asparaginase
US11802279B2 (en) 2017-06-21 2023-10-31 Jazz Pharmaceuticals Ireland Ltd. Modified L-asparaginase
CN110256535A (zh) * 2019-06-14 2019-09-20 中国石油大学(华东) L-天门冬酰胺酶XiDL及其编码基因和应用
CN110256535B (zh) * 2019-06-14 2021-01-01 中国石油大学(华东) L-天门冬酰胺酶XiDL及其编码基因和应用

Also Published As

Publication number Publication date
AU2002332371A1 (en) 2003-03-10
WO2003018742A3 (fr) 2003-08-07

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