vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2021-66-3-271-276vestib-756Research ArticleСтатьиБиосинтез аденозиндезаминазы Escherichia coli в системе бесклеточного синтеза белкаBiosynthesis of Escherichia coli adenosine deaminase using cell-free protein synthesisКазловскийИ. С.KazlouskiI. S.

Казловский Илья Сергеевич - магистр биологических наук, научный сотрудник.

ул. Купревича, 2, 220141, Минск.

Illia S. Kazlouski - Master of Biology, Researcher, Institute of Microbiology of the National Academy of Sciences of Belarus.

2, Kuprevich Str., 220141, Minsk.

leonardo_139@mail.ruЗинченкоА. И.ZinchenkoA. I.

Зинченко Анатолий Иванович - член-корреспондент, доктор биологических наук, профессор, заведующий лабораторией.

ул. Купревича, 2, 220141, Минск.

Anatoli I. Zinchenko - Corresponding Member, D. Sc. (Biol.), Professor, Head of the Laboratory, Institute of Microbiology of the National Academy of Sciences of Belarus.

2, Kuprevich Str., 220141, Minsk.

zinch@mbio.bas-net.byИнститут микробиологии НАН БеларусиInstitute of Microbiology of the National Academy of Sciences of Belarus202103082021663271276Copyright © Казловский И.С., Зинченко А.И., 20212021Казловский И.С., Зинченко А.И.Kazlouski I.S., Zinchenko A.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/756

Одним из новых перспективных направлений молекулярной биотехнологии является бесклеточный синтез белка (БСБ). Процедура БСБ основана на реконструкции in vitro всех этапов биосинтеза белка в клетке, включая транскрипцию, аминоацилирование тРНК и трансляцию мРНК рибосомами.

Ранее нами был сконструирован штамм Escherichia coli, продуцирующий гомологичную аденозиндезаминазу (АДазу). B настоящем исследовании в качестве варианта, альтернативного каноническому глубинному культивированию в ферментере, была изучена возможность синтеза АДазы в системе БСБ. Для синтеза этого фермента использовали SSO-клеточный экстракт E. coli, РНК-полимеразу бактериофага Т7 и высококопийный плазмидный вектор pET42mut со встроенным в него геном АДазы.

B результате выполнения работы нами впервые продемонстрирована возможность синтеза АДазы E. coli в системе БСБ. B частично оптимизированных условиях проведения процесса получен экспериментальный образец рекомбинантной АДазы с активностью 530 Ед/мл ферментного препарата.

One of the recent perspective trends of molecular biotechnology is cell-free protein synthesis (CFPS). The procedure of CFPS is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes.

Previously, we constructed a strain Escherichia coli that produces homologous adenosine deaminase (ADase). In the present study, as an alternative to canonical submerged cultivation in a fermenter, the possibility of the ADase synthesis in the system of CFPS was studied. For synthesis of this enzyme we used the E. coli-30 cell extract, T7 bacteriophage RNA polymerase, and high-copy plasmid vector pET42mut with gene ADase inserted into it.

As a result of the work we have demonstrated for the first time the possibility of synthesis of ADase E. coli in the CFPS system. In a partially optimized process conditions, an experimental sample of recombinant AD with an activity of 530 U/ml of enzyme preparation was obtained.

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