RU2000102364A - VECTOR FOR EXPRESSION OF A HETEROLOGICAL PROTEIN AND METHODS OF EXTRASION OF A RECOMBINANT PROTEIN AND CLEANING OF THE IDENTIFIED RECOMBINANT INSULIN - Google Patents

VECTOR FOR EXPRESSION OF A HETEROLOGICAL PROTEIN AND METHODS OF EXTRASION OF A RECOMBINANT PROTEIN AND CLEANING OF THE IDENTIFIED RECOMBINANT INSULIN

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Publication number
RU2000102364A
RU2000102364A RU2000102364/13A RU2000102364A RU2000102364A RU 2000102364 A RU2000102364 A RU 2000102364A RU 2000102364/13 A RU2000102364/13 A RU 2000102364/13A RU 2000102364 A RU2000102364 A RU 2000102364A RU 2000102364 A RU2000102364 A RU 2000102364A
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RU
Russia
Prior art keywords
cell membrane
sequence encoding
vector
protein
recombinant
Prior art date
Application number
RU2000102364/13A
Other languages
Russian (ru)
Other versions
RU2222598C2 (en
Inventor
Спартаку АСТОЛФИ-ФИЛЬЮ
ЛИМА Биатрис Долабела ДИ
Джозеф Эрнст ТИМАНН
СОЗА Элоиза Рибейру Тунес ДИ
Люсиано ВИЛЕЛА
Original Assignee
Универсидади Ди Бразилиа
Биобрас С.А.
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.)
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Priority claimed from US08/886,967 external-priority patent/US6068993A/en
Application filed by Универсидади Ди Бразилиа, Биобрас С.А. filed Critical Универсидади Ди Бразилиа
Publication of RU2000102364A publication Critical patent/RU2000102364A/en
Application granted granted Critical
Publication of RU2222598C2 publication Critical patent/RU2222598C2/en

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Claims (15)

1. Вектор, включающий молекулу нуклеиновой кислоты, для экспрессии, по крайней мере, одного гетерологичного белка, при том, что упомянутый вектор включает следующие функционально соединенные участки в направлении 5' --> 3':
(i) последовательность, кодирующая сайт начала репликации;
(ii) последовательность, кодирующая промотор;
(iii) последовательность, кодирующая инициирующий сегмент;
(iv) последовательность, кодирующая, по крайней мере, один рестрикционный сайт, предназначенный для встраивания гетерологичной ДНК, кодирующей, по крайней мере, один гетерологичный белок и
(v) последовательность, кодирующая сайт терминации транскрипции.1. A vector, including a nucleic acid molecule, for the expression of at least one heterologous protein, while the said vector includes the following functionally connected sections in the direction 5 '->3':
(i) a sequence encoding a replication start site;
(ii) a sequence encoding a promoter;
(iii) a sequence encoding a trigger segment;
(iv) a sequence encoding at least one restriction site for embedding a heterologous DNA encoding at least one heterologous protein; and
(v) a sequence encoding a transcription termination site. 2. Вектор по п. 1, отличающийся тем, что молекулой нуклеиновой кислоты является ДНК. 2. The vector according to claim 1, characterized in that the nucleic acid molecule is DNA. 3. Вектор по п. 1, отличающийся тем, что инициирующий сегмент включает сайт инициации трансляции. 3. The vector according to claim 1, characterized in that the initiating segment includes a translation initiation site. 4. Вектор по п. 1, отличающийся тем, что после (i) и перед (ii) находится последовательность, кодирующая селективный маркер. 4. The vector according to claim 1, characterized in that after (i) and before (ii) there is a sequence encoding a selective marker. 5. Вектор по п. 1, отличающийся тем, что он является плазмидой для экспрессирования в грамм-отрицательной бактерии. 5. The vector according to claim 1, characterized in that it is a plasmid for expression in a gram-negative bacterium. 6. Вектор, определенный в п. 5, отличающийся тем, что грамм-отрицательной бактерией является кишечная палочка Е. coli, сайт начала репликации происходит из плазмиды pUC8, инициирующий сегмент является сайтом инициации трансляции, включающим синтетический участок Шайна-Дальгарно, происходящий из гена 10 фага Т7, имеется последовательность, кодирующая селективный маркер, расположенная между (i) и (ii), включающая последовательность, кодирующую резистентность к тетрациклину, промотором является промотор РL и сайтом терминации транскрипции является Rho-независимый.6. The vector as defined in claim 5, characterized in that the E. coli E. coli is a gram-negative bacterium, the origin of replication originates from the plasmid pUC8, the initiating segment is the translation initiation site, including the Shine-Dalgarno synthetic region, originating from the gene 10 of phage T7, there is a sequence encoding a selectable marker located between (i) and (ii), including a sequence encoding a tetracycline resistance, the promoter is the R L promoter and the transcription termination site is Rho-independent. 7. Вектор по п. 1, отличающийся тем, что по крайней мере, один рестрикционный сайт кодирует множественные рестрикционные сайты. 7. The vector according to claim 1, characterized in that at least one restriction site encodes multiple restriction sites. 8. Вектор по п. 7, отличающийся тем, что множественные рестрикционные сайты представляют собой NcoI, EcoRI, StuI, PstI, BamHI и BspEI. 8. The vector according to claim 7, characterized in that the multiple restriction sites are NcoI, EcoRI, StuI, PstI, BamHI and BspEI. 9. Вектор по п. 1, отличающийся тем, что гетерологичным белком является проинсулин. 9. The vector according to claim 1, characterized in that the heterologous protein is proinsulin. 10. Способ экстрагирования рекомбинантного белка изнутри клеток рекомбинантных грамм-отрицательных бактерий, имеющих клеточную мембрану, без лизирования бактерий, включающий следующие этапы:
(a) пермеабилизация клеточной мембраны путем обработки бактерий детергентом при соблюдении условий, достаточных для разделения нативных клеточных белков от клеточной мембраны без разделения рекомбинантного белка от клеточной мембраны;
(b) солюбилизация рекомбинантного белка и клеточной мембраны;
(c) отделение рекомбинантного белка от клеточной мембраны.10. A method of extracting a recombinant protein from within cells of recombinant gram-negative bacteria having a cell membrane without lysing bacteria, comprising the following steps:
(a) permeabilization of the cell membrane by treating the bacteria with a detergent under sufficient conditions to separate native cell proteins from the cell membrane without separating the recombinant protein from the cell membrane;
(b) solubilization of the recombinant protein and cell membrane;
(c) separating the recombinant protein from the cell membrane. 11. Способ по п. 10, отличающийся тем, что детергентом, используемым на этапе (а), является Тритон Х-100. 11. The method according to p. 10, characterized in that the detergent used in step (a) is Triton X-100. 12. Способ по п. 10, отличающийся тем, что на этапе (а) условия, являющиеся достаточными для отделения нативных клеточных белков от клеточной мембраны, представлены центрифугированием, и полученный в результате осадок содержит рекомбинантный белок с клеточной мембраной. 12. The method according to p. 10, characterized in that at step (a) the conditions that are sufficient to separate the native cell proteins from the cell membrane are represented by centrifugation, and the resulting pellet contains a recombinant protein with a cell membrane. 13. Способ по п. 10, отличающийся тем, что на этапе (b) солюбилизация осуществляется путем обработки рекомбинантного белка с клеточной мембраной этапа (b) мочевиной. 13. The method according to p. 10, characterized in that in step (b), the solubilization is carried out by processing a recombinant protein with a cell membrane of the step (b) urea. 14. Способ по п. 10, отличающийся тем, что на этапе (c) отделение осуществляется центрифугированием, а полученная в результате надсадочная фракция содержит рекомбинантный белок. 14. The method according to p. 10, characterized in that at step (c) the separation is carried out by centrifugation, and the resulting supernatant fraction contains a recombinant protein. 15. Способ очистки выделенного рекомбинантного инсулина человека, включающий:
(a) подвергание выделенного рекомбинантного проинсулина человека сульфитолизу и отделение получаемого жидкого продукта;
(b) обработка жидкого продукта, полученного на этапе (а), на хроматографической никель-хелатирующей колонке с получением элюата;
(c) ренатурация элюата, полученного на этапе (b);
(d) превращение продукта, полученного на этапе (с), с использованием трипсина и карбоксипептидазы-В;
(e) хроматографическая обработка продукта, полученного на этапе (d), с получением очищенного выделенного рекомбинантного инсулина человека.15. The method of purification of the selected recombinant human insulin, including:
(a) subjecting the isolated recombinant human proinsulin to sulfitolysis and separating the resulting liquid product;
(b) processing the liquid product obtained in step (a) on a chromatographic nickel-chelating column to obtain an eluate;
(c) renaturation of the eluate obtained in step (b);
(d) converting the product obtained in step (c) using trypsin and carboxypeptidase-B;
(e) chromatographic treatment of the product obtained in step (d) to obtain purified isolated recombinant human insulin.
RU2000102364/13A 1997-07-02 1998-07-02 Vector for expression of heterologous protein, plasmid (variants) RU2222598C2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/886,967 US6068993A (en) 1997-07-02 1997-07-02 Vector for expression of heterologous protein and methods for extracting recombinant protein and for purifying isolated recombinant insulin
US08/886,967 1997-07-02

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RU2000102364A true RU2000102364A (en) 2002-03-27
RU2222598C2 RU2222598C2 (en) 2004-01-27

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US (4) US6068993A (en)
EP (1) EP0996713B1 (en)
AT (1) ATE278776T1 (en)
BR (1) BR9810650B1 (en)
CA (2) CA2687542C (en)
DE (1) DE69826864T2 (en)
IL (2) IL133832A0 (en)
IN (1) IN187721B (en)
PL (1) PL194141B1 (en)
RU (1) RU2222598C2 (en)
WO (1) WO1999001548A2 (en)

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