Синтез и скрининг нативных белковых библиотек в бесклеточной системе трансляции на основе экстракта из зародышей пшеницы тема диссертации и автореферата по ВАК РФ 03.00.04, кандидат химических наук Александров, Александр Николаевич

• Александров, Александр Николаевич
• кандидат химических наук
• 2003

Оглавление диссертации кандидат химических наук Александров, Александр Николаевич

1. Введение.

2. Обзор литературы.

2.1 Структура мРНК и регуляция трансляции.

2.1.1 Роль ком в регуляции трансляции.

2.1.2 Роль поли(А)- последовательности в трансляции.

2.1.3 Кооперативное действие кепа и поли(А)-последовательиости на эффективность трансляции.

2.1.4 Стабильность мРНКв бесклеточных системах трансляции.

2.1.5 Общие черты нетранслируемых областей мРНК эукариот.

2.1.6 Окружение стартового кодонамРНК.

2.2 Бесклеточиая система трансляции из зародышей пшеницы.

2.2.1 Ионная сила и концентрация в ЭЗП.

2.2.2 Влияние полиаминов. уровня А'ГФ и тРНКна биосинтез белка в ЭЗП.

2.2.3 Эндогенные активности ЭЗП.

2.3 Терминация трансляции, котрансляционное сворачивание белков и рибосомный дисплей.

2.3.1 Терминация трансляции и её регуляция.

2.3.2 Котрансляционное сворачивание белков.

2.3.3 Рибосомный дисплеи и гибриды мРНК-белок - основные методы отбора белков in vitro.

2.3.4 Достоинства и недостатки систем отбора in vitro.

3. Экспериментальная часть.

4. Результаты и их обсуждение.

4.1. Выделение бесклеточнои системы трансляции из зародышей пшеницы (ЭЗП) и аналитический вариант трансляции в этой системе.

4.1.1 мРНК и генетические конструкции.

4.1.2 Приготовление экстракта из зародышей пшеницы и аналитический вариант трансляции.

4.1.3 Качественный анализ целостности мРНК в условиях трансляции в ЭЗП. . 82 4.2 Синтез и скринннг белковой библиотеки из печени мыши в ЭЗП.

4.2.1 Исследование агрегации продуктов трансляции с компонентами ЭЗП.

4.2.2 Определение чувствительности скрининга в рибосомиом дисплее.

4.2.3 Синтез пативнои белковой библиотеки из печени мыши и ее скрининг на иммобилизованном метотрексате (ингибиторе активности ЦГФР).

5. Выводы.

Введение диссертации (часть автореферата) на тему «Синтез и скрининг нативных белковых библиотек в бесклеточной системе трансляции на основе экстракта из зародышей пшеницы»

В настоящее время одним из ключевых методов изучения межмолекулярных взаимодействий является функциональный отбор биологически активных молекул in vitro. Разработанные ранее методы такого отбора оказались применимы только для РНК и ДНК со специфическими структурными и каталитическими свойствами [lieumiry, 1992; Jiartel, 1993; Osborn, 1997; Famulok, 2000; Hasselberth, 2000].

В отличие от скрининга нуклеиновых кислот функциональный отбор белков и пептидов in vitro требует физической связи между белком и кодирующей его нуклеиновой кислотой. Такая связь может быть реализована описанными ранее методами фагового дисплея [Smith, 1985] и дисплея на плазмидах [Schatz, 1996]. К сожалению, указанные методы отбора ограничены представительностью наборов РЖ (ДНК). Так, библиотеки фагового дисплея лимитированы эффективностью трансфекции, которая составляет величину менее 109 независимых рекомбинантов.

Недавно была описана аналогичная фаговому дисплею стратегия функционального отбора белков, основанная на связи синтезированных полипептидов с прокариотическими (.E.coli [Matteakis, 1994; Hanes, 1997; 1999]) и эукариотическими (ретикулоциты кролика [Hanes, 1999; Не, 1997; Makeyev, 1999] и зародыши пшеницы [Gersuk, 1997]) рибосомами. По аналогии с фаговым дисплеем этот метод скрининга получил название рибосомного дисплея. В этом случае количество последовательностей, которые могут быть представлены на рибосомах, значительно больше (более Ю12), чем это достижимо в фаговом дисплее. Однако оказалось, что метод рибосомного дисплея обладает существенным недостатком - он требует введения в скрининг мРНК без терминаторных кодонов. Это обстоятельство существенно затрудняет скрининг мРНК и кДНК библиотек в рибосомном дисплее.

В связи с вышеизложенным, целью настоящей работы является:

1. Оптимизация бесклеточной системы трансляции in vitro из зародышей пшеницы (экстракт зародышей пшеницы, ЭЗП) для скрининга в рибосомном дисплее мРНК (кДНК) библиотек.

2. Проведение отбора днгпдрофолатредуктазы из полн(А+)РНК клеток печени мыши методом рибосомного дисплея на основе ЭЗП.

2. Обзор литературы

Типичный скрининг в рибосомном дисплее объединяет четыре основные стадии: 1) трансляцию набора мРНК в выбранной системе трансляции in vitro и ко-трансляционное сворачивание продуктов трансляции;

2) отбор на молекуле-мишени синтезированного полипептида (полипептидов) в составе комплекса с рибосомой и кодирующей полипептид мРНК;

3) аплификацию отобранных мРНК (ОТ-ПЦР);

4) секвенирование кДНК (или клонирование последней и определение функциональной активности продукта экспрессии отобранного гена).

Ключевой стадией рибосомного дисплея является трансляция in vitro и ко-трансляционное сворачивание продуктов трансляции. Этим двум процессам и уделено основное внимание в настоящем обзоре. Поскольку невозможно в коротком обзоре детально рассмотреть регуляторные механизмы трансляции, ниже описаны лишь те факты и закономерности, значение которых важно для ЭЗП как инструмента в функциональном скрининге мРНК и кДНК.

Заключение диссертации по теме «Биохимия», Александров, Александр Николаевич

5. Выводы.

1. Разработан метод получения бесклеточной системы трансляции с низкой активностью собственных протеаз и нуклеаз на основе экстракта зародышей пшеницы. Оптимизированы условия для синтеза нативных белковых библиотек и показано, что эффективность трансляции в такой системе повышается на 20%.

2. Показано, что в выбранной системе трансляции полипептидные продукты трансляции и кодирующая их полноразмерная мРНК образуют с рибосомами стабильные тройные комплексы. Предложена гипотеза, основанная на котрансляционной инактивации рибосом эндогенным фактором тритином, объясняющая существование таких комплексов в системе.

3. Впервые продемонстрировано, что рибосомы в составе тройных комплексов трансляционно неактивны, а синтезированный полипептид в составе комплекса способен образовывать димеры и проявлять ферментативную активность.

4. Предложен новый метод скрининга нативных белковых библиотек на основании данных о комплексах полипептид:рибосома:мРНК. С помощью этого метода синтезирована белковая библиотека печени мыши и проведен её скрининг, в модельных экспериментах измерена чувствительность предлагаемого метода отбора.

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