biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2025-25-3-258-270biopreparat-676Research ArticleТЕМА НОМЕРА: РЕКОМБИНАНТНЫЕ ТЕРАПЕВТИЧЕСКИЕ БЕЛКИISSUE TOPIC RECOMBINANT THERAPEUTIC PROTEINSПромоторы для высокоэффективной экспрессии и секреции проинсулина в Saccharomyces cerevisiaePromoters for high-efficiency expression and proinsulin secretion in Saccharomyces cerevisiaehttps://orcid.org/0009-0000-4389-8605БуслаеваЕ. А.BuslaevaE. A.

Буслаева Евгения Александровна 

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Eugenia A. Buslaeva  

34 Svyazi St., Strelna, St Petersburg 198515

Evgeniia.Buslaeva@geropharm.comhttps://orcid.org/0000-0002-0065-1853ХасаншинаЗ. Р.KhasanshinaZ. R.

Хасаншина Зухра Рамилевна 

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Zukhra R. Khasanshina  

34 Svyazi St., Strelna, St Petersburg 198515

https://orcid.org/0000-0003-0207-5244КорнаковИ. А.KornakovI. A.

Корнаков Игорь Александрович  

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Igor A. Kornakov  

34 Svyazi St., Strelna, St Petersburg 198515

https://orcid.org/0009-0000-9807-7783КоробкинаМ. П.KorobkinaM. P.

Коробкина Мария Павловна 

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Мariya P. Korobkina  

34 Svyazi St., Strelna, St Petersburg 198515

https://orcid.org/0000-0003-0282-9843ШмуракВ. И.ShmurakV. I.

Шмурак Владимир Игоревич  

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Vladimir I. Shmurak  

34 Svyazi St., Strelna, St Petersburg 198515

https://orcid.org/0000-0003-4594-6097ДрайР. В.DraiR. V.

Драй Роман Васильевич, канд. мед. наук  

ул. Связи, д. 34, лит. А, пос. Стрельна, Санкт-Петербург, 198515

Roman V. Drai, Cand. Sci. (Med.)  

34 Svyazi St., Strelna, St Petersburg 198515

Закрытое акционерное общество «Фарм-Холдинг»РоссияPharmholding CJSCRussian Federation202529092025253258270Copyright © Буслаева Е.А., Хасаншина З.Р., Корнаков И.А., Коробкина М.П., Шмурак В.И., Драй Р.В., 20252025Буслаева Е.А., Хасаншина З.Р., Корнаков И.А., Коробкина М.П., Шмурак В.И., Драй Р.В.Buslaeva E.A., Khasanshina Z.R., Kornakov I.A., Korobkina M.P., Shmurak V.I., Drai R.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/676ВВЕДЕНИЕ

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

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

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

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

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.

INTRODUCTION

INTRODUCTION. The lack of systematic research comparing promoter activity in Saccharomyces cerevisiae limits approaches to improving the efficiency of recombinant human proinsulin biosynthesis. This study compared the activity of constitutive promoters under standardised technological conditions to identify the most productive regulatory elements for application in industrial strains.

AIM

AIM. This study aimed to compare the influence of TEF1, ADH2, ALD4, TDH3 (GPD), and TPI1 promoters on recombinant proinsulin expression in Saccharomyces cerevisiae and rank their effectiveness for selecting a highly productive promoter.

METERIALS AND METHODS

METERIALS AND METHODS. S. cerevisiae YS3 diploid strain obtained by haploid cells fusion was used for proinsulin expression. The vectors based on the pRS425 plasmid consisted of synthetic promoters (TPI1, TEF1, ADH2, ALD4, TDH3) and signal sequence. Plasmids were electroporated in the cell. Comparative expression analysis was performed by cultivating S. cerevisiae YS3 in flasks and bioreactors using YPD media. Proinsulin and ethanol concentration was measured using spectrophotometry, HPLC, and gas chromatography. The expression was confirmed using HPLC-MS. Statistical analysis included variance and correlation analysis.

RESULTS

RESULTS. Strains S. cerevisiae YS3/pF1145   (TPI1),   YS3/pF1157   (TEF1),   YS3/pF1199   (ADH2), YS3/pF1200 (ALD4), and YS3/pF1201 (TDH3) were obtained, then expressions of recombinant protein were compared. The strain YS3/pF1201 that had proinsulin expression controlled by the promoter TDH3 showed a maximum productivity of 15.51±0.57 mg/L. When fermented in laboratory bioreactors, the strain YS3/pF1201 achieved a productivity of 139.17 mg/L and a biomass of 154.5 mg/L after 72 hours of cultivation.

CONCLUSIONS

CONCLUSIONS. Analysis of experimental data ranked the promoters for proinsulin expression in S. cerevisiae cells by their effectiveness: TDH3≈ALD4>ADH2>TEF1>TPI1. During the fermentation, TDH3 promoter showed significantly higher productivity, which was 6.1 times more than in the classical strain with the TPI1 promoter for proinsulin expression.

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The authors declare that there are no conflicts of interest present.