Промоторы для высокоэффективной экспрессии и секреции проинсулина в Saccharomyces cerevisiae

ВВЕДЕНИЕ. Нехватка систематических сравнительных данных об активности промоторов в Saccharomyces cerevisiae является фактором, ограничивающим повышение эффективности биосинтеза рекомбинантного проинсулина человека. Настоящая работа направлена на экспериментальное сравнение активности ряда конститутивных промоторов в стандартных технологических условиях для определения наиболее продуктивного регуляторного элемента, применимого в промышленных штаммах-продуцентах.

ЦЕЛЬ. Оценить влияние промоторов TEF1, ADH2, ALD4, TDH3 (GPD), TPI1 на экспрессию рекомбинантного   проинсулина   человека   в   Saccharomyces   cerevisiae   и   ранжировать   их по силе для выбора оптимального промотора, обеспечивающего максимальную продуктивность.

МАТЕРИАЛЫ И МЕТОДЫ. Для экспрессии предшественника инсулина (проинсулина) человека использовали диплоидный штамм S. cerevisiae YS3, полученный слиянием гаплоидных клеток. Векторная система на основе плазмиды pRS425 включала в себя синтетические промоторы (TPI1, TEF1, ADH2, ALD4, TDH3) и сигнальную последовательность. Конструкции вводили в клетки методом электропорации. Сравнительный анализ экспрессии белка проводили при культивировании S. cerevisiae YS3 в колбах и биореакторах с использованием среды YPD. Определение содержания проинсулина и этанола осуществляли с помощью спектрофотометрии, высокоэффективной жидкостной хроматографии (ВЭЖХ) и газовой хроматографии.   Подтверждение    экспрессии    проводили    методом    ВЭЖХ,    совмещенной с масс-спектрометрией. Статистическая обработка данных выполнялась при помощи дисперсионного и корреляционного анализов.

РЕЗУЛЬТАТЫ. Получены штаммы-продуценты S. cerevisiae YS3/pF1145 (TPI1), YS3/pF1157 (TEF1), YS3/pF1199 (ADH2), YS3/pF1200 (ALD4), YS3/pF1201 (TDH3), и проведено сравнение уровня экспрессии рекомбинантного белка. Штамм YS3/pF1201, в котором экспрессия проинсулина находилась под контролем промотора TDH3, обладал максимальной продуктивностью (15,51±0,57 мг/л). В процессе ферментации в лабораторных биореакторах продуктивность штамма YS3/pF1201 (TDH3) достигла 139,17 мг/л через 72 ч культивирования при значении биомассы 154,50 г/л.

ВЫВОДЫ. Промоторы для экспрессии проинсулина в клетках S. cerevisiae можно распределить по их силе в следующем порядке: TDH3≈ALD4>ADH2>TEF1>TPI1. В процессе ферментации при использовании промотора TDH3 достигнута продуктивность, превышающая в 6,1 раза продуктивность штамма-продуцента с классическим промотором TPI1, который используется для экспрессии проинсулина.

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