Каталитическая субъединица энтеропептидазы человека: получение, характеризация ферментативных свойств и моделирование мутаций, облегчающих ренатурацию и повышающих специфичность фермента
Диссертация
В настоящей работе был получен активный препарат рекомбинантной легкой цепи энтеропептидазы человека высокой степени чистоты. Исходя из сложности сборки подобной протеиназы при использовании прокариотической системы экспрессии был выбран выриант производства целевого белка в виде единой полипептидной цепи с тиоредоксином в нерастворимом виде для последующей его ренатурации. Использование схемы… Читать ещё >
Содержание
- СПИСОК ИСПОЛЬЗОВАННЫХ СОКРАЩЕНИЙ
- 1. ВВЕДЕНИЕ
- 2. ОБЗОР ЛИТЕРАТУРЫ
- 2. 1. Энтеропептидаза — ключевой фермент пищеварения
- 2. 1. 1. История открытия и физиологическая роль
- 2. 1. 2. Структура энтеропептидазы
- 2. 1. 3. Экспрессия гена энтеропептидазы
- 2. 2. Взаимодействия сериновых протеиназ с субстратом при ферментативном катализе
- 2. 2. 1. Специфичность сериновых протеиназ
- 2. 2. 2. Сайты распознавания субстрата
- 2. 2. 3. Распознавание субстратов во время катализа
- 2. 1. Энтеропептидаза — ключевой фермент пищеварения
- 2. 2. 4. Как удаленные взаимодействия влияют на катализ?
- 2. 3. Экспрессия рекомбинантных гибридных белков и их ренатурация
- 2. 3. 1. Экспрессия рекомбинантных белков
- 2. 3. 2. Гибридные (слитные) белки
- 2. 3. 3. Ренатурация рекомбинантных белков
- 3. 1. Материалы
- 3. 1. 1. Реактивы и ферментные препараты
- 3. 1. 2. Штаммы и плазмидные вектора
- 3. 1. 3. Питательные среды для роста бактерий
- 3. 1. 4. Синтетические олигонуклеотиды
- 3. 2. Методы
- 3. 2. 1. Трансформация клеток Е. coli плазмидной ДНК
- 3. 2. 2. Выделение плазмидной ДНК
- 3. 2. 3. Электрофоретический анализ ДНК в агарозном геле
- 3. 2. 4. Количественные определения препаратов ДНК и примесей РНК
- 3. 2. 5. Выделение ДНК из агарозного геля
- 3. 2. 6. Определение нуклеотидной последовательности (секвенирование) ДНК
- 3. 2. 7. Конструирование рекомбинантной плазмидной ДНК pET-32/L-HEP
- 3. 2. 7. Олигонуклеотид-направленный мутагенез
- 3. 2. 8. Экспрессия гена L-HEP и ее мутантных вариантов
- 3. 2. 9. Идентификация белков по N-концевой последовательности
- 3. 2. 10. Ренатурация гибридных белков с тиоредоксином в качестве белка-носителя
- 3. 2. 11. Очистка активной L-HEP и ее мутантных вариантов
- 3. 2. 12. Определение количества белка в растворах и гелях
- 3. 2. 13. Измерение каталитической активности L-HEP и ее мутантных вариантов
- 3. 2. 14. Реакции ингибирования каталитической акитвности L-HEP
- 3. 2. 15. Построение пространственных моделей L-HEP и ее мутантных вариантов
- 3. 2. 16. Молекулярная динамика каталитических субъединиц энтеропептидаз
- 3. 2. 17. Оценка мутантных вариантов L-HEP
- 3. 2. 18. Моделирование взаимодействий L-BEP, L-HEP и ее мутантных вариантов с пептидными субстратами
- 4. 1. Синтез гена L-HEP, его клонирование в плазмидные вектора и подбор экспрессионной системы
- 4. 2. Ренатурация слитного белка Trx/L-HEP и очистка активного фермента L-HEP
- 4. 3. Исследование ферментативных свойств L-HEP
- 4. 4. Построение пространственной модели L-HEP
- 4. 5. Моделирование и получение мутантных вариантов L-HEP с увеличенным выходом ренатурации
- 4. 6. Исследование специфичности L-HEP
- 4. 7. Моделирование взаимодействия активного центра энтеропептидазы с пептидными субстратами
- 4. 8. Получение и исследование каталитических свойств мутантных вариантов L-HEP с заменами R96K, K219Q и R96K/K219Q
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- Выражаю глубокую благодарность своим научным руководителям, д.б.н., профессору Д. А. Долгих и к.б.н. М. Э. Гаспарян, за доброе внимание, поддержку начинаний и своевременную, и потому неоценимую, помощь при выполнении моей диссертационной работы.
- Я очень признателен моему консультанту по молекулярному моделированию к.ф.-м.н. Ю. А. Косинскому за терпеливое обучение методам и ценные обсуждения результатов.
- Также не могу не отметить важную роль моей семьи и друзей в стабилизации фона проводимых исследований: благодарю их и надеюсь, что смогу быть полезен в ответ.