Новый эффективный подход для получения рекомбинантных белков основного фактора роста фибробластов (FGF-2) и лиганд-связывающего внеклеточного домена рецептора II типа TGF-? (T?RII-ED) в E. coli

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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. Показано, что полученный гомогенный препарат T? RII-ED имеет столь же высокую лиганд-связывающую активность по отношению к TGF?, как и аналогичный рецептор, полученный из клеток… Читать ещё >

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
T?RII
- 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 T? RII-ED
- 4. 2. 15. Очистка T? RII-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 T? RII-ED с лигандом TGF-?l методом ELISA
5. Результаты и обсуждение
- 5. 1. Синтез генов FGF-2 и T? RII-ED и клонирование их в экспрессионные векторы
- 5. 2. Экспрессия слитного белка Trx/FGF2 в E. col
- 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/T?RII-ED в E. col
- 5. 11. Очистка и расщепление слитного белка Trx/T?RII-ED энтеропептидазой и очистка целевого белка T? RII-ED
- 5. 12. Исследование физико-химических свойств полученного рекомбинантного белка T? RII-ED
- 5. 13. Исследование связывания белка-рецептора T? RII-ED с лигандом TGF-?l методом ELISA
- 5. 14. Преимущества разработанной методики получения и очистки рекомбинантного белка-рецептора T? RII-ED
6. Выводы

Новый эффективный подход для получения рекомбинантных белков основного фактора роста фибробластов (FGF-2) и лиганд-связывающего внеклеточного домена рецептора II типа TGF-? (T?RII-ED) в E. coli (реферат, курсовая, диплом, контрольная)

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/T?MI-ED для экспресии лиганд-связывающего домена рецептора II типа цитокина TGFD в составе гибридного белка тиоредоксин/Tß-RII-EDD ?

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

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

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