pharmjournalРазработка и регистрация лекарственных средствDrug development & registration2305-20662658-5049LLC «CPHA»10.33380/2305-2066-2025-14-1-1825pharmjournal-2023Research ArticleПОИСК И РАЗРАБОТКА НОВЫХ ЛЕКАРСТВЕННЫХ СРЕДСТВRESEARCH AND DEVELOPMENT OF NEW DRUG PRODUCTSРазработка продуцентов терапевтических пептидов на основе Esherichia coli BL21 и технологии их культивированияDevelopment of therapeutic peptide producers based on Escherichia coli BL21 and their cultivation technologyhttps://orcid.org/0000-0002-0065-1853ХасаншинаЗ. Р.KhasanshinaZ. R.

198515, г. Санкт-Петербург, пос. Стрельна, ул. Связи, д. 34, литера А; 197101, г. Санкт-Петербург, Кронверкский пр., д. 49, литера А

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515; 49A, Kronverksky prospekt, Saint-Petersburg, 197101

zuhra.hasanshina@geropharm.comhttps://orcid.org/0000-0003-0207-5244КорнаковИ. А.KornakovI. A.

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

https://orcid.org/0009-0000-4389-8605БуслаеваЕ. А.BuslaevaE. A.

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

https://orcid.org/0009-0000-3314-2310КазаковаА. В.KazakovaA. V.

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

https://orcid.org/0000-0002-5754-7491ИщукС. А.IshchukS. A.

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

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

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

https://orcid.org/0000-0002-8445-1129СапароваВ. Б.SaparovaV. B.

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

https://orcid.org/0009-0009-0487-4227ЛатыповВ. Ф.LatypovV. F.

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

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

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

34A, Svyazi str., Strelna village, Saint-Petersburg, 198515

Закрытое акционерное общество «Фарм-Холдинг»; Федеральное государственное автономное образовательное учреждение высшего образования «Национальный исследовательский университет ИТМО»JSC "FARM-KHOLDING"; ITMO UniversityЗакрытое акционерное общество «Фарм-Холдинг»JSC "FARM-KHOLDING"2025270120251415466Copyright © Хасаншина З.Р., Корнаков И.А., Буслаева Е.А., Казакова А.В., Ищук С.А., Шмурак В.И., Сапарова В.Б., Латыпов В.Ф., Драй Р.В., 20252025Хасаншина З.Р., Корнаков И.А., Буслаева Е.А., Казакова А.В., Ищук С.А., Шмурак В.И., Сапарова В.Б., Латыпов В.Ф., Драй Р.В.Khasanshina Z.R., Kornakov I.A., Buslaeva E.A., Kazakova A.V., Ishchuk S.A., Shmurak V.I., Saparova V.B., Latypov V.F., 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.pharmjournal.ru/jour/article/view/2023Введение

Введение. В последнее десятилетие пептиды с молекулярной массой менее 5 кДа используется в медицине и биотехнологии для лечения различных заболеваний. Химический синтез пептидов имеет ограничения, такие как низкий выход и эффективность синтеза, сложность масштабирования. Альтернативой является использование Escherichia coli. Разработка эффективной технологии синтеза пептидов остается актуальной задачей в связи с низкой продуктивностью штаммов-продуцентов.

Цель

Цель. Разработка высокоэффективных штаммов Escherichia coli BL21, экспрессирующих терапевтические пептиды молекулярной массой менее 5 кДа, и технологии их культивирования.

Материалы и методы

Материалы и методы. Генетические конструкции получили с использованием рестриктазно-лигазного метода, подлинность подтвердили секвенированием по Сенгеру. Технологию культивирования разработали с использованием подхода Design of experiments. Валидацию условий культивирования проводили в биореакторе Biostat B. Гибридные белки очищали методом металл-хелатной хроматографии, целевые пептиды получали гидролизом с помощью ULP-протеазы. Количественное содержание целевого белка определяли капиллярным электрофорезом, подлинность белка – ВЭЖХ-МС и косвенным неконкурентным ИФА.

Результаты и обсуждение

Результаты и обсуждение. В ходе исследований разработаны высокоэффективные штаммы-продуценты пептидов и оптимальные условия культивирования: рН – 7,5 ± 0,5, температура культивирования – 37 °C, оптическая плотность индукции – 2,7 ± 0,3, концентрация ИПТГ – 0,05 мМ. Продуктивность штаммов-продуцентов достигла 4,82 ± 0,05 г/л. Получены образцы очищенных пептидов.

Заключение

Заключение. Разработанная технология позволяет получить пептиды с высоким выходом, не описанным ранее исследователями. Практическая значимость данной работы заключается в возможности применения данной технологии для получения пептидов с различными физико-химическими свойствами.

Introduction

Introduction. Peptides with a molecular weight of less than 5 kDa have been used in medicine and biotechnology over the past decade for the treatment of various diseases. However, chemical synthesis peptide has several disadvantages, including low yield, reduced efficiency, and high costs. An alternative approach to peptide production is the use of the Escherichia coli expression system. The development of effective peptide synthesis technology remains a critical task because of the low productivity of recombinant strains.

Aim

Aim. Developing highly efficient strains of Escherichia coli BL21 expressing therapeutic peptides with a molecular weight of less than 5 kDa in E. coli and their cultivation technology.

Materials and methods

Materials and methods. Genetic constructs were obtained using the restriction-ligase method, and their authenticity was confirmed by Sanger sequencing. Cultivation technology was developed using the Design of Experiments approach. The cultivation condition was validated in the Biostat B bioreactor. Hybrid proteins were purified by metal-chelate chromatography, followed by hydrolysis ULP proteas to obtain the target peptides. The quantitative content of the target protein was determined by capillary electrophoresis, and the authenticity of the protein was confirmed by HPLC-MS and ELISA.

Results and discussion

Results and discussion. Highly efficient peptide-producing strains were developed. Cultivation conditions were optimized: рН 7.5 ± 0.5, cultivation temperature 37 °C, induction optical density 28 ± 2, IPTG concentration 0.05 мМ. The productivity of the producer strains was up to 4.82 ± 0.05 g/L. Furthermore, samples of the target peptides were isolated and purified.

Conclusion

Conclusion. The productivity of peptides in this study were significantly higher than in previous research. The presented strategy for strain development, cultivation and purification technology can be used production of therapeutic peptides with diverse physical chemicals characteristics in the future.

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