Новый эффективный подход для получения рекомбинантных белков основного фактора роста фибробластов (FGF-2) и лиганд-связывающего внеклеточного домена рецептора II типа TGF-β (TβRII-ED) в E. coli тема диссертации и автореферата по ВАК РФ 03.01.04, кандидат биологических наук Елистратов, Павел Алексеевич

• Елистратов, Павел Алексеевич
• кандидат биологических наук
• 2011

Оглавление диссертации кандидат биологических наук Елистратов, Павел Алексеевич

Содержание

Список используемых сокращений

1. Введение

2. Цели и задачи работы

3. Литературный обзор

3.1. Общая характеристика факторов роста

3.2. Основной фактор роста фибробластов РОБ

3.2.1. Общая характеристика РОР

3.2.2. Взаимодействие РОР-2 с рецепторами

3.2.3. Изоформы РОР

3.2.4. Структура ТО¥~

3.2.5. Биологическая активность и функции РОР

3.3. Трансформирующий фактор роста (31 (ГСР-р1) и его рецептор типа II (ТРЯП)

3.3.1. Суперсемейство белков ТОР-Р

3.3.2. Структура ГСР-р

3.3.3. Рецепторы и механизмы внутриклеточных сигнальных путей

ТвР-Р

3.3.4. Биологическая активность и функции ТОР-р

3.4. Экспрессия и ренатурация рекомбинантных белков

3.4.1. Слитные рекомбинантные белки

3.4.2. Экспрессия рекомбинантных белков

3.4.3. Расщепление слитных белков

3.4.4. Ренатурация рекомбинантных белков

4. Материалы и методы

4.1. Материалы

4.2. Методы

4.2.1. Конструирование плазмидной ДНК

4.2.2. Сайт-направленный мутагенез

4.2.3. PIPES-трансформация, приготовление компетентных клеток E.coli штамма XL-1 Blue

4.2.4. Получение компетентных клеток E.coli BL21(DE3)

4.2.5. Трансформация клеток E.coli плазмидной ДНК

4.2.6. Выделение плазмидной ДНК

4.2.7. Электрофоретический анализ ДНК в агарозном геле

4.2.8. Определение концентрации белка в растворе

4.2.9. Электрофоретический анализ белков в полиакриламидном

4.2.10. Экспрессия белка FGF-2 и его мутантного варианта и белка

TßRII

4.2.11. Очистка и расщепление белка FGF

4.2.12. Идентификация белков по N-концевой последовательности

4.2.13. Метод оценки жизнеспособности клеток с метилтиазолтетразолием (МТТ-тест)

4.2.14. Очистка, ренатурация и расщепление белка TßRII-ED

4.2.15. Очистка TßRII-ED с помощью метода обратнофазной HPLC хроматографии

4.2.16. MALDI-TOF масс-спектрометрия

4.2.17. Метод спектроскопии кругового дихроизма

4.2.18. !НЯМР -спектроскопия

4.2.19. Исследование связывания белка-рецептора TßRII-ED с лигандом TGF-ßl методом ELISA

5. Результаты и обсуждение

5.1. Синтез генов FGF-2 и TßRII-ED и клонирование их в экспрессионные векторы

5.2. Экспрессия слитного белка Trx/FGF2 в E.coli

5.3. Очистка слитного белка Trx/FGF

5.4. Расщепление слитного белка Trx/FGF-2 энтеропептидазой и очистка целевого белка FGF

5.5. Исследование физико-химических свойств полученного рекомбинантного белка FGF

5.6. Введение C78S и C96S мутаций в ген FGF-2 и получение экспрессирующего вектора pET32a/FGF-2/C78S/C96S

5.7. Экспрессия и очистка мутантного варианта FGF-2/C78S/C96S

5.8. Проверка биологической активности очищенного препарата FGF-2 и его мутантного варианта FGF-2/C78S/C96S на культуре клеток

5.9. Преимущества разработанной методики получения и очистки рекомбинантного FGF

5.10. Экспрессия слитного белка Trx/TßRII-ED в E.coli

5.11. Очистка и расщепление слитного белка Trx/TßRII-ED энтеропептидазой и очистка целевого белка TßRII-ED

5.12. Исследование физико-химических свойств полученного рекомбинантного белка TßRII-ED

5.13. Исследование связывания белка-рецептора TßRII-ED с лигандом TGF-ßl методом ELISA

5.14. Преимущества разработанной методики получения и очистки рекомбинантного белка-рецептора TßRII-ED

6. Выводы

Заключение диссертации по теме «Биохимия», Елистратов, Павел Алексеевич

6. Выводы

1. Получен штамм-продуцент Е. coli BL21(DE3)/pET-32a/FGF-2 для получения фактора роста фибробластов FGF-2 и его мутантного варианта FGF-2/C78S/C96S. Целевые белки экспрессировали в составе слитного белка с тиоредоксином.

2. Разработаны методы очистки, позволившие получить примерно 100 мг белка FGF-2 и 120 мг FGF-2/C78S/C96S из 1 литра культуры клеток.

3. Показано, что полученные препараты FGF-2 и FGF-2/C78S/C96S обладают одинаково высокой стабильностью и пролиферативной активностью по отношению к фибробластам мыши.

4. Получен штамм-продуцент Е. coli BL21(DE3)/pET-32a/TßMI-ED для экспресии лиганд-связывающего домена рецептора II типа цитокина TGFD в составе гибридного белка тиоредоксин/TßRII-EDD □

5. Разработаны методы ренатурации и очистки, позволившие получить примерно 140 мг стабильного белка TßRÜ-ED из 1 литра культуры клеток.

6. Показано, что полученный гомогенный препарат TßRII-ED имеет столь же высокую лиганд-связывающую активность по отношению к TGFß, как и аналогичный рецептор, полученный из клеток млекопитающих.

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