Бесклеточная система трансляции на основе представителя трипаносоматид Leishmania tarentolae. Создание оптимальной матрицы для трансляции in vitro тема диссертации и автореферата по ВАК РФ 03.00.03, кандидат биологических наук Муреев, Сергей Владимирович

• Муреев, Сергей Владимирович
• кандидат биологических наук
• 2007

Оглавление диссертации кандидат биологических наук Муреев, Сергей Владимирович

СПИСОК ИСПОЛЬЗУЕМЫХ СОКРАЩЕНИЙ.

1 ВВЕДЕНИЕ.

2 ОБЗОР ЛИТЕРАТУРЫ.

2.1 Характеристики бесклеточных систем трансляции.

2.1.1 Бесклеточная система трансляции Е. coli.

2.1.2 Проблема фолдинга.

2.1.3 Лизат Ретикулоцитов Кролика RRL (rabbit reticulocyte lysate).

2.1.4 Система Экстракта Зародышей Пшеницы WGE (wheat germ extract)

2.1.5 Трансляционно-активные экстракты трипаносоматид.

2.2 Бесклеточная трансляция в проточной системе.

2.3 АТР-регенерирующие системы.

2.4 Применение бесклеточной системы.

2.4.1 Включение модифицированных аминокислот.

2.4.2 IN VITRO продукция мембранных белков.

2.4.3 Продуктивность и автоматизация. Применение in vitro системы экспрессии в геномике и протеомике.

2.4.4 Бесклеточная система трансляции используется для молекулярной эволюции.

2.5 активность EIF2 определяет уровень трансляции in vivo & in vitro.

2.6 Сплайс-лидер в инициации трансляции у трипаносоматид.

2.7 Использование anti-sence подхода для избирательного подавления трансляции.

2.8 Инициация трансляции.

2.8.1 Проблемы инициации трансляции эукариот.

2.8.2 AUG контекст.

3 МАТЕРИАЛЫ И МЕТОДЫ.

3.1 Культивирование клеток Leishmania tarentolae.

3.1.1 Поддержание культуры клеток L. tarentolae.

3.1.2 Трансфекция Leishmania tarentolae.

3.2 Выделение митохондрий и анализ целостности внешней митохондриальной мембраны.

3.2.1 Способы разрушения клеток.

3.2.2 Процедура выделения митохондриальных фракций L. tarentolae.

3.2.3 Приготовление препаратов клеток и митохондрий для флуоресцентной микроскопии.

3.2.4 Измерение активности сукцинат-цитохром с дегидрогеназы и анализ целостности внешней митохондриальной мембраны.

3.3 Получение экстракта L. tarentolae и трансляция.

3.3.1 Процедура получения экстракта L. tarentolae.

3.3.2 Обработка экстракта для подавления эндогенной трансляции.

3.3.3 Постановка и проведение реакции in vitro трансляции.

3.3.4 Получение суммарных гомологичных мРНК и тРНК L. tarentolae.

3.4 Очистка EGFP и MALDI-TOF-macc спектроскопия.

3.5 Поиск пре-AUG триплетов для L. major.

3.6 Создание генноинженерных конструкций для геномной интеграции

3.6.1 Создание конструкций, содержащих ires-последовательность для геномной интеграции в тубулиновый локус Leishmania tarentolae.

3.6.2 Создание плазмид с 64 вариантами npe-ATG триплетов для геномной интеграции в SSU-локус.

3.6.3 Конструирование TET-R и DS-Red экспрессирующих плазмид.

3.7 Создание конструкций для in vitro экспрессии.

3.7.1 Конструкции содержащие IRES элемент в качестве 5 '-нетранслируемого участка.

3.7.2 Конструкции содержащие комбинацию обелинового 5 '-лидера с 3'-нетранслируемым участком сателлита вируса некроза табака.

3.7.3 Содание серии плазмид «ОКНА», содержащих дополнительный ATG в рамке с ОРС egfp, окруженный последовательностями полностью неструктурированных участков.

3.7.4 Содание серии плазмид «ОКНА», содержащих дополнительный ATG в рамке с ОРС egfp, окруженный последовательностями полностью (5') и частично (3') неструктурированных участков.

3.8 Методы Молекулярной Биологии.

3.8.1 Получение и трансформация компетентных клеток Е. coli.

3.8.2 Выделение плазмидной ДНК.

3.8.3 Расщепление ДНК эндонуклеазами.

3.8.4 Обработка продуктов эндонуклеазного расщепления ДНК полимеразой фага Т4 для достройки выступающих концов до «тупых».

3.8.5 ЛигированиеДНК.

3.8.6 Секвенирование ДНК.

3.8.7 ПЦР.

3.8.8 Норзерн-гибридизация.

3.8.9 Гибридизация по Вестерну.

3.8.10 Анализ продуктов трансляции.

3.8.11 Транскрипция in vitro.

3.8.12 Определение устойчивости РНК к нуклеазам VI &Т1.

РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ.

4.1 Оптимизация параметров получения клеточного экстракта.

4.1.1 Поиск наиболее щадящего метода разрушения клеток L. tarentolae

4.2 Трансляция в экстракте L. tarentolae (LiЕ).

4.2.1 Оценка способности экстракта L. tarentolae к эндогенной трансляционной активности и инициации трансляции на экзогенно-вводимой гомологичной РНК.

4.2.2 Применение антисмыслового олигонуклеотида к последовательности сплайс-лидера, для подавления трансляции SL-несущих РНК.

4.2.3 Трансляция гетерологичных РНК в aSL-обработанном экстракте L. tarentolae.

4.2.4 Сравнение эффективностей трансляции в LtE, после обработки aSL-олигонуклеотидом и микрококковой нуклеазой.

4.3 Экспрессия IRES-содержащих конструкций in vivo в составе генных кассет, интегрированных в геном L. tarentolae.

4.4 Создание оптимальных матриц для трансляции in vitro.

4.4.1 Влияние неструктурированного 5'- UTR на эффективность трансляции РНК-матрицы.

4.4.2 Трансляция РНК матриц, несущих оптимальный пре-А UG контекст и относительно легкоплавкий З'-участок «окна», кодирующий гидрофильную лидерную последовательность.

4.4.3 Сравнительный анализ степени структурных изменений, вызванных в РНК температурной денатурацией.

4.4.4 Оптимизация триплета, предшествующего A UG.

4.5 ВЫВОДЫ.

БЛАГОДАРНОСТИ.

Введение диссертации (часть автореферата) на тему «Бесклеточная система трансляции на основе представителя трипаносоматид Leishmania tarentolae. Создание оптимальной матрицы для трансляции in vitro»

С завершением многочисленных проектов по секвенированию геномов разных организмов, изучение генной-экспрессии выходит на новый уровень структурно-функциональной характеристики кодируемых геномом белков. В силу огромного набора открытых рамок считывания, обнаруженных в процессе секвенировния, прогресс в анализе соответствующих полипептидов напрямую зависит от их высоко-эффективной наработки в достаточном количестве для структурного и функционального анализа.

В настоящее время используются три основные стратегии получения белков для исследований: химический синтез, in vivo экспрессия и in vitro экспрессия

Химический синтез не применим для синтеза длинных полипептидов. Несмотря на более чем тридцатилетнюю историю, универсальная система экспрессии эукариотических белков так и не была разработана. Технические ограничения связанные с необходимостью быстрой и продуктивной наработки белков для анализа вынудили исследователей вернуться к наиболее эффективной системе на основе Е. coli, однако лишь 15% эукариотических белков могли быть проэкспрессированы в своей функционально-активной форме.

Эукариотические системы экспрессии in vivo основанные на культуре клеток млекопитающих, насекомых, трансгенных растениях и животных имеют значительные ограничения: дороговизна, трудоемкость. Дешевая и относительно гибкая система на основе Saccharomyces cerevisiae характеризуется относительно низким выходом и отсутствием желаемого типа пост-трансляционных модификаций. Использование других дрожжевых представителей (Pichia pastoris) наталкивается на препятствия связанные с активацией системы отрицательной обратной связи при гиперпродукции белка. При создании новой эукариотической системы разумным могла бы быть замена РНК-полимеразы 11 менее контролируемыми и более эффективными односубъединичными фаговыми полимеразами Т7, SP6 и т.д. Однако в данном случае проблематичным представляется кэпирование РНК, так как фаговые полимеразы не обладают свойством сопряжения транскрипции и процессинга (Moteki and Price, 2002).

Бесклеточные системы дают возможность работать с широким спектром РНК-матриц, лишенных основных регуляторных модификаций. Из множества прикладных задач in vitro система позволяет экспрессировать токсичные белки, включать модифицированные аминокислоты, вносить необходимые добавки для экспрессии мембранных и других труднорастворимых белков и селективно метить изотопами. Несмотря на видимую универсальность, бесклеточные системы имеют ряд ограничений: а) низкая эффективность экспрессии в batch-формате по сравнению с in vivo продукцией, b) дороговизна применения высокопродуктивной проточной системы, с) отсутствие универсальных регуляторных элементов (UTRs) для трансляции любых открытых рамок считывания в любой in vitro системе.

Наибольшее распространение получили системы на основе экстракта Е. coli, зародышей пшеницы (WGE) и лизата ретикулоцитов кролика (RRL) благодаря максимальному отношению выгодных свойств к стоимости получения белкового продукта. Под полезными свойствами понимают высокую эффективность синтеза белков в их функционально-активной форме, отсутствие эндогенного уровня трансляции.

Бесклеточная эукариотическая система трансляции на основе трипаносоматид могла бы обладать преимуществами по сравнению с вышеперечисленными системами благодаря нескольким факторам: а) легкой процедуре наработки клеточной биомассы и разрушения клеток Ь) присутствию идентичной 5-концевой последовательности сплайс-лидера (SL) на всех зрелых РНК трипаносоматид, допускающей использование анти-SL олигонуклеотида для подавления фонового уровня трансляции.

2 ОБЗОР ЛИТЕРАТУРЫ

Заключение диссертации по теме «Молекулярная биология», Муреев, Сергей Владимирович

4.5 ВЫВОДЫ

1. Бесклеточная система трансляции на основе представителя трипаносоматид L. tarentolae способна к инициации трансляции на гетерологичных РНК матрицах.

2. Применение anti6Z олигонуклеотида позволило эффективно подавить трансляцию эндогенной РНК, не прибегая к методике с использованием микрококковой нуклеазы.

3. Использование неструктурированного 5'-UTR в составе РНК матриц приводит к возрастанию выхода белкового продукта в системе экстракта L. tarentolae пропорционально увеличению количества вводимой матрицы. Привнесение структурных элементов как в 5'- так и в З'-область 5'UTR приводит к угнетению белкового синтеза.

4. Выявлена группа наиболее выгодных npe-AUG триплетов для экспрессии EGFP в клетках L. tarentolae, однако эти триплеты не оказались универсальными в сочетании с другими открытыми рамками считывания (Tet-R; DS-Red).

БЛАГОДАРНОСТИ

Автор считает своим долгом выразить глубокую благодарность своим научным руководителям, Александру Александровичу Колесникову и Александрову Кириллу Евгеньевичу за отзывчивость, помощь при постановке экспериментов, интерпретации результатов и написании статей и диссертации. Я безмерно признателен Мерзляк Екатерине за ее дружбу, отзывчивость, моральую поддержку в трудные минуты, неоценимую помощь в обсуждении и редактировании материалов диссертации. По прошествии нескольких лет, начиная с момента дипломной работы, я смело могу назвать ее одним из своих учителей. Слова глубокой благодарности я хотел бы адресовать Игорю Грановскому за помощь на начальном этапе работы и, в особенности, Шалойко Любови Анатольевне, без которой эта работа, вероятно, никогда не была бы сделана, за ее живое участие и интересные и полезные беседы. Я черезвычайно благодарен своим коллегам, Кушнир Сюзанне, за ценные практические советы в каждодневной экспериментальной работе, Райнхарду Брайтлингу за ценные советы, Кырстеа Иону, за помощь в проведении экспериментов и веселую дружескую атмосферу. Я очень признателен Малеевой Юлии Владимировне, Лупиловой Наталье, Эммериху Михаю Газдагу, Кристин Делон, Игнатьеву Александру, Васильевой Юле, Андрею Закальскому, Аймельту Ицену, Тиму Бергбредэ, Гохеан Нейлакши и By Тамми за помощь и идеальный климат для приятной и эффективной работы. Я также хотел бы поблагодарить аспирантов группы Юлиуса Лукаша: Жденека Париса, Сандеша Регми и Милана Йирку, часть результатов корторых, я использовал при написании диссертации, а так же, Карен Браунинг за любезное предоставление генноинженерных конструкций.

Я очень признателен коллективам кафедр молекулярной биологии и биоорганической химии за великолепное образование, полученное мной за время обучения и последующей работы на биологическом факультете. Хотелось также поблагодарить всех сотрудников отдела структурной биологии института Макса-Планка, Дортмунд, Германия, и, отдельно, его заведующего, Роджера Гуди за идеальные условия работы и доброжелательное отношение. И, в заключении, я выражаю глубочайшую благодарность своим родителям за все то, неоценимое, что они сделали для меня и продолжают делать сегодня.

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