vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2019-64-1-7-17vestib-403Research ArticleСтатьиУсловия выделения рекомбинантного бычьего альфа-интерферона из телец включения E. coliSelection conditions of the recombinant bovine α-interferon from E. coli inclusion bodiesЖидецкийА. В.ZhydzetskiA. V.

Жидецкий Александр Вячеславович – м л. науч. c отрудник.

ул. Курчатова, 10, 220030, г. Минск.

Alexander V. Zhydzetski – Junior researcher.

10, Kurchatov Str., 220030, Minsk.

Zhydzetski@gmail.comПотаповичМ. И.PatapovichM. I.

Потапович Максим Иосифович – заведующий лабораторией.

ул. Курчатова, 10, 220030, г. Минск.

Maksim I. Patapovich – Head of the Laboratory.

10, Kurchatov Str., 220030, Minsk.

mipatapovich@gmail.comКудинаИ. В.KudinaI. V.

Кудина Ирина Владимировна – мл. науч. сотрудник.

ул. Курчатова, 10, 220030, г. Минск.

Irina V. Kudina – Junior researcher.

10, Kurchatov Str., 220030, Minsk.

Kuddya@gmail.comПрокулевичВ. А.PrakulevichU. A.

Прокулевич Владимир Антонович – д-р биол. наук, профессор, заведующий кафедрой.

ул. Курчатова, 10, 220030, г. Минск.

Uladzimir А. Prakulevich – D . S c. ( Biol.), P rofessor, Head of the Department.

10, Kurchatov Str., 220030, Minsk.

prokulevich@mail.ruШолухМ. В.SholukhM. V.

Шолух Михаил Васильевич – канд. биол. наук, доцент, заведующий лабораторией.

ул. Курчатова, 10, 220030, г. Минск.

Mikhail М. Sholukh – Ph. D. (Biol.), Assistant professor, Head of the Laboratory.

10, Kurchatov Str., 220030, Minsk.

msholukh@mail.ruБелорусский государственный университет.Belarusian State University.201913022019641717Copyright © Жидецкий А.В., Потапович М.И., Кудина И.В., Прокулевич В.А., Шолух М.В., 20192019Жидецкий А.В., Потапович М.И., Кудина И.В., Прокулевич В.А., Шолух М.В.Zhydzetski A.V., Patapovich M.I., Kudina I.V., Prakulevich U.A., Sholukh M.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/403

Эффективность рекомбинантного бычьего альфа-интерферона (rbIFN-α), как и других белков данной группы, обусловлена тем, что при проведении антивирусной терапии он стимулирует и модулирует противовирусную активность ряда клеток-мишеней в ответ на вирусную атаку.Данное исследование посвящено определению оптимальных условий выделения, очистки и рефолдинга методом разведения rbIFN-α из телец включения (ТВ) E. coli. Основными методами являлись спектрофотометрические, электрофоретические, хроматографические и рефолдинг методом разведения.Двухстадийная отмывка ТВ растворами на основе 50 ммоль/л Трис, 50 ммоль/л NaCl и 3,5 моль/л мочевины и их последующая солюбилизация в растворе с 50 ммоль/л Трис-HCl, pH 9,0, 8 моль/л мочевины и 20 ммоль/л β-меркаптоэтанола позволили получить целевой белок в мономерной форме с чистотой 53,18 ± 9,3 %. Применение тандемной ионообенной хроматографии на последовательно соединенных колонках с DE 52 целлюлозой и toyopearl DEAE-650 M дало возможность получить целевой rbIFN-α с чистотой 80,7 ± 8,6 %. В результате скрининга основных характеристик определен следующий состав рефолдинга буфера для проведения ренатурации рекомбинантного rbIFN-α: 10 ммоль/л NaPB, pH 7,4, 0,4 моль/л сахароза, 1 ммоль/л ЭДТА, 1 ммоль/л L-Цис, 0,1 ммоль/л L-цистина, 0,05 % Kolliphor EL. На конечном этапе сбора целевого белка с помощью данной системы выход мономерной формы rbIFN-α составил 20,44 ± 1,1 %, чистота – 98,43 %, активность – (5 ± 3,6)•106 МЕ/мг.

Like other proteins of the cytokine family, bovine α-interferon activates and modulates antiviral state of the target cells and inhibits division and growth of the infected cells which makes it an excellent candidate as a new antiviral therapeutic agent.This study is concerned with the determination of the optimal isolation, purification and refolding conditions of the recombinant bovine interferon-α (rbIFN-α) from inclusion bodies (IBs). Main methods used were UV/Visible spectroscopy, electrophoresis, liquid chromatography and refolding by dilution.It was found that two step IBs washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity. Further application of the anion-exchange tandem chromatography on DE 52 cellulose and toyopearl DEAE-650 M gives a possibility to remove the major impurities and obtain rbIFN-α with 80.7 ± 8.6 % purity. Refolding by dilution in the buffer containing 20 mmol/l NaPB, рН 7.4, 0.4 mol/l sucrose, 1 mmol/l L-Cys, 0.1 mmol/l L-Cystine, 1 mmol/l EDTA, 0.05 % Kolliphor EL at 10 °C followed by the protein collection allows to get the recombinant rbIFN-α in homogeneous state, with 98.43 % purity and antiviral activity about (5 ± 3.6)•106 U/mg.

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The authors declare that there are no conflicts of interest present.