RU2007139262A - Recombinant plasmid pHINS21, encoding a hybrid protein with human pro-insulin, a strain of Escherichia coli bacteria JM109 / pHINS21 - producer of pro-granulose-white hyalurum - Google Patents

Recombinant plasmid pHINS21, encoding a hybrid protein with human pro-insulin, a strain of Escherichia coli bacteria JM109 / pHINS21 - producer of pro-granulose-white hyalurum Download PDF

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Publication number
RU2007139262A
RU2007139262A RU2007139262/13A RU2007139262A RU2007139262A RU 2007139262 A RU2007139262 A RU 2007139262A RU 2007139262/13 A RU2007139262/13 A RU 2007139262/13A RU 2007139262 A RU2007139262 A RU 2007139262A RU 2007139262 A RU2007139262 A RU 2007139262A
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Russia
Prior art keywords
phins21
hybrid protein
producer
human proinsulin
strain
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RU2007139262/13A
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Russian (ru)
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RU2376368C2 (en
Inventor
Петр Иванович Родионов (RU)
Петр Иванович Родионов
Петр Петрович Родионов (RU)
Петр Петрович Родионов
Вадим Васильевич Шматченко (RU)
Вадим Васильевич Шматченко
Алексей Вячеславович Степанов (RU)
Алексей Вячеславович Степанов
Александр Николаевич Байдусь (RU)
Александр Николаевич Байдусь
Наталья Анатольевна Шматченко (RU)
Наталья Анатольевна Шматченко
Тарас Алексеевич Горкун (RU)
Тарас Алексеевич Горкун
Original Assignee
Общество с ограниченной ответственностью "ГЕРОФАРМ" (RU)
Общество С Ограниченной Ответственностью "Герофарм"
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Priority to RU2007139262/13A priority Critical patent/RU2376368C2/en
Priority to EA201070461A priority patent/EA201070461A1/en
Priority to PCT/RU2008/000658 priority patent/WO2009054754A1/en
Publication of RU2007139262A publication Critical patent/RU2007139262A/en
Application granted granted Critical
Publication of RU2376368C2 publication Critical patent/RU2376368C2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Abstract

1. Рекомбинантная плазмида pHINS21 (фиг.1), содержащая ДНК, кодирующая гибридный белок размером 143 аминокислоты (фиг.2), в котором аминокислотные последовательности лидерного пептида, представляющего собой N-концевой фрагмент гамма-интерферона человека, и проинсулина человека соединены пептидным линкером, содержащим сайт расщепления энтерокиназы, при этом указанная плазмида имеет молекулярную массу 2,36 МДа и включает уникальные сайты узнавания эндонуклеазами рестрикции, расположенные на следующем расстоянии вправо от сайта EcoRI: BamHI - 159 п.о., HpaI - 185 п.о., HindIII - 442 п.о., PvuI - 1382 п.о. ! 2. Трансформированная клетка Escherichia coli, содержащая рекомбинантную плазмидную ДНК pHINS21 по п.1, - продуцент гибридного белка, содержащего проинсулин человека. ! 3. Штамм бактерий Escherichia coli JM109/pHINS21 - продуцент гибридного белка, содержащего проинсулин человека. ! 4. Способ получения проинсулина человека, включающий ! культивирование штамма-продуцента Escherichia coli, ! разрушение бактериальных клеток, ! выделение телец включения, их растворение в буфере, содержащем мочевину и дитиотреитол, ! ренатурацию и очистку ренатурированного гибридного белка с проинсулином человека, ! расщепление гибридного белка высокоспецифичной протеиназой, ! очистку и получение целевого продукта. ! 5. Способ по п.4, отличающийся тем, что ! в качестве штамма-продуцента используется штамм E.coli JM109/pHINS21, ! расщепление гибридного белка с проинсулином человека проводят энтерокиназой (или ее каталитической субъединицей), ! полученный после ферментативного расщепления проинсулин очищают ионообменной хроматографией на сорбентах с сульфопропильными группами. ! 6. Способ по п.5, отличающийся тем, ч�1. Recombinant plasmid pHINS21 (FIG. 1), containing DNA encoding a 143 amino acid fusion protein (FIG. 2), in which the amino acid sequences of the leader peptide, which is the N-terminal fragment of human gamma interferon, and human proinsulin are connected by a peptide linker containing the enterokinase cleavage site, wherein said plasmid has a molecular weight of 2.36 MDa and includes unique recognition sites of restriction endonucleases located at the following distance to the right of the EcoRI site: BamHI - 159 bp, HpaI - 185 bp, HindIII - 442 bp, PvuI - 1382 bp ! 2. A transformed Escherichia coli cell containing the recombinant plasmid DNA pHINS21 according to claim 1, is a producer of a hybrid protein containing human proinsulin. ! 3. The bacterial strain Escherichia coli JM109 / pHINS21 is a producer of a hybrid protein containing human proinsulin. ! 4. A method of obtaining human proinsulin, including! cultivation of the producer strain Escherichia coli,! destruction of bacterial cells! isolation of inclusion bodies, their dissolution in a buffer containing urea and dithiothreitol,! renaturation and purification of the renatured hybrid protein with human proinsulin,! fusion protein cleavage with highly specific proteinase! purification and obtaining the target product. ! 5. The method according to claim 4, characterized in that! E.coli strain JM109 / pHINS21,! is used as the producer strain. cleavage of the hybrid protein with human proinsulin is performed by enterokinase (or its catalytic subunit),! The proinsulin obtained after enzymatic cleavage is purified by ion exchange chromatography on sorbents with sulfopropyl groups. ! 6. The method according to claim 5, characterized in that

Claims (6)

1. Рекомбинантная плазмида pHINS21 (фиг.1), содержащая ДНК, кодирующая гибридный белок размером 143 аминокислоты (фиг.2), в котором аминокислотные последовательности лидерного пептида, представляющего собой N-концевой фрагмент гамма-интерферона человека, и проинсулина человека соединены пептидным линкером, содержащим сайт расщепления энтерокиназы, при этом указанная плазмида имеет молекулярную массу 2,36 МДа и включает уникальные сайты узнавания эндонуклеазами рестрикции, расположенные на следующем расстоянии вправо от сайта EcoRI: BamHI - 159 п.о., HpaI - 185 п.о., HindIII - 442 п.о., PvuI - 1382 п.о.1. Recombinant plasmid pHINS21 (FIG. 1), containing DNA encoding a 143 amino acid fusion protein (FIG. 2), in which the amino acid sequences of the leader peptide, which is the N-terminal fragment of human gamma interferon, and human proinsulin are connected by a peptide linker containing the enterokinase cleavage site, wherein said plasmid has a molecular weight of 2.36 MDa and includes unique recognition sites of restriction endonucleases located at the following distance to the right of the EcoRI site: BamHI - 159 bp, HpaI - 185 bp, HindIII - 442 bp, PvuI - 1382 bp 2. Трансформированная клетка Escherichia coli, содержащая рекомбинантную плазмидную ДНК pHINS21 по п.1, - продуцент гибридного белка, содержащего проинсулин человека.2. A transformed Escherichia coli cell containing the recombinant plasmid DNA pHINS21 according to claim 1, is a producer of a hybrid protein containing human proinsulin. 3. Штамм бактерий Escherichia coli JM109/pHINS21 - продуцент гибридного белка, содержащего проинсулин человека.3. The bacterial strain Escherichia coli JM109 / pHINS21 is a producer of a hybrid protein containing human proinsulin. 4. Способ получения проинсулина человека, включающий4. A method of obtaining human proinsulin, including культивирование штамма-продуцента Escherichia coli,cultivation of the producer strain of Escherichia coli, разрушение бактериальных клеток,destruction of bacterial cells, выделение телец включения, их растворение в буфере, содержащем мочевину и дитиотреитол,isolation of inclusion bodies, their dissolution in a buffer containing urea and dithiothreitol, ренатурацию и очистку ренатурированного гибридного белка с проинсулином человека,renaturation and purification of the renatured hybrid protein with human proinsulin, расщепление гибридного белка высокоспецифичной протеиназой,cleavage of the hybrid protein with a highly specific proteinase, очистку и получение целевого продукта.purification and obtaining the target product. 5. Способ по п.4, отличающийся тем, что5. The method according to claim 4, characterized in that в качестве штамма-продуцента используется штамм E.coli JM109/pHINS21,as a producer strain, the strain E. coli JM109 / pHINS21 is used, расщепление гибридного белка с проинсулином человека проводят энтерокиназой (или ее каталитической субъединицей),cleavage of the hybrid protein with human proinsulin is carried out by enterokinase (or its catalytic subunit), полученный после ферментативного расщепления проинсулин очищают ионообменной хроматографией на сорбентах с сульфопропильными группами.The proinsulin obtained after enzymatic cleavage is purified by ion exchange chromatography on sorbents with sulfopropyl groups. 6. Способ по п.5, отличающийся тем, что6. The method according to claim 5, characterized in that после ферментативного расщепления гибридного белка очистку проинсулина проводят хроматографией на SP-сефарозе FF в 0,05-0,2M аммоний-ацетатном буфере, рН 3,0-6,0, содержащем 1-2М мочевину, а сорбированный белок элюируют линейным градиентом хлористого калия от 0 до 0,5М в уравновешивающем буфере.after enzymatic cleavage of the hybrid protein, proinsulin is purified by chromatography on SP-Sepharose FF in 0.05-0.2M ammonium acetate buffer, pH 3.0-6.0, containing 1-2M urea, and the sorbed protein is eluted with a linear gradient of potassium chloride from 0 to 0.5 M in a balancing buffer.
RU2007139262/13A 2007-10-22 2007-10-22 STRAIN OF BACTERIA Escherichia coli JM109/pHINS21 - PRODUCER OF HYBRID PROTEIN WITH HUMAN PROINSULIN AND METHOD FOR PRODUCTION OF HUMAN PROINSULIN RU2376368C2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
RU2007139262/13A RU2376368C2 (en) 2007-10-22 2007-10-22 STRAIN OF BACTERIA Escherichia coli JM109/pHINS21 - PRODUCER OF HYBRID PROTEIN WITH HUMAN PROINSULIN AND METHOD FOR PRODUCTION OF HUMAN PROINSULIN
EA201070461A EA201070461A1 (en) 2007-10-22 2008-10-21 RECOMMENDED COOPERATION COOPERATIVE
PCT/RU2008/000658 WO2009054754A1 (en) 2007-10-22 2008-10-21 PHINS21 RECOMBINANT PLASMID FOR ENCODING HYBRID PROTEIN WITH HUMAN PROINSULIN, AN Escherichia coli JM109/pHINS21 BACTERIA STRAIN AS A PRODUCER OF HYBRID PROTEIN WITH HUMAN PROINSULIN AND A HUMAN PROINSULIN PRODUCING METHOD

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RU2007139262/13A RU2376368C2 (en) 2007-10-22 2007-10-22 STRAIN OF BACTERIA Escherichia coli JM109/pHINS21 - PRODUCER OF HYBRID PROTEIN WITH HUMAN PROINSULIN AND METHOD FOR PRODUCTION OF HUMAN PROINSULIN

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RU2376368C2 RU2376368C2 (en) 2009-12-20

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WO2013015697A1 (en) * 2011-07-28 2013-01-31 Mabion S.A. A recombinant protein, a polynucleotide encoding it as well as a method of obtaining insulin or its an analogue
RU2728611C1 (en) * 2019-05-24 2020-07-30 Закрытое акционерное общество "ФАРМ-ХОЛДИНГ" Recombinant plasmid dna pf265 coding hybrid polypeptide containing human proinsulin, and bacterial strain escherichia coli - producer of hybrid polypeptide containing human proinsulin

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US4751180A (en) * 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
RU2208637C1 (en) * 2002-04-16 2003-07-20 Институт биоорганической химии им. М.М. Шемякина и Ю.А. Овчинникова РАН Method for preparing gene-engineering human insulin
RU2263147C1 (en) * 2004-02-16 2005-10-27 Открытое акционерное общество "Национальные биотехнологии" RECOMBINANT PLASMID DNA pHINSO5 ENCODING HYBRID POLYPEPTIDE COMPRISING HUMAN PROINSULIN, ESHERICHIA COLI CELL TRANSFORMED WITH RECOMBINANT PLASMID DNA pHINS05 AND STRAIN OF BACTERIUM ESCHERICHIA COLI JM109/pHINS05 AS PRODUCER OF HYBRID POLYPEPTIDE COMPRISING HUMAN PROINSULIN

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