danДоклады Национальной академии наук БеларусиDoklady of the National Academy of Sciences of Belarus1561-83232524-2431The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1561-8323-2018-62-5-601-607dan-558Research ArticleБИОЛОГИЯBIOLOGYСоздание штамма-продуцента химерного белка, состоящего из РНК-полимеразы и ДНК-аффинного доменаConstruction of a strain-producer of the chimeric protein consisting of RNA polymerase and a DNA-affinity domainКазловскийИ. С.KazlovskiyI. S.

Казловский Илья Сергеевич - магистр биологических наук, младший научный сотрудник.

Kazlovskiy Il ’ya Sergeevich - Master of Biology, Junior researcher.

2, Kuprevich Str., 220141, Minsk

leonardo_139@mail.ruЗинченкоА. И.ZinchenkoM. A.

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

Ул. Купреви-ча, 2, 220141, Минск

Zinchenko Anatoliy Ivanovich - Corresponding Member, D. Sc. (Biology), Professor, Head of the Laboratory.

2, Kuprevich Str., 220141, Minsk

zinch@mbio.bas-net.byНациональная академия наук Беларуси, Институт микробиологииNational Academy of Sciences of Belarus, Institute of Microbiology201830102018625601607Copyright © Казловский И.С., Зинченко А.И., 20182018Казловский И.С., Зинченко А.И.Kazlovskiy I.S., Zinchenko M.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://doklady.belnauka.by/jour/article/view/558

Одним из новых перспективных направлений молекулярной биотехнологии является бесклеточный синтез белка. Процедура бесклеточного синтеза белка основана на реконструкции in vitro всех этапов биосинтеза белка в клетке, включая транскрипцию, аминоацилирование тРНК и трансляцию мРНК рибосомами. Для воспроизведения этапа транскрипции требуется участие специфических РНК-полимераз, которые инициируют процесс синтеза мРНК с определенных сайтов узнавания. Часто для этого применяется ДНК-зависимая РНК-полимераза бактериофага Т7 (Т7-РНК-полимераза). Для улучшения качественных характеристик Т7-РНК-полимеразы в настоящей работе создан новый штамм Escherichia coli, продуцирующий этот фермент, слитый с ДНК-аффинным доменом Sso7d термофильной бактерии Sulfolobus solfataricus. Продуцирующая способность полученного рекомбинантного штамма в отношении синтезируемого химерного белка достигает 625 ед/л культуральной жидкости, а удельная активность препарата очищенного фермента составила 80 ед/мкг белка. Полученный фермент предназначен для использования в качестве инструментария при синтезе белков в бесклеточной системе.

One of the recent perspective trends of molecular biotechnology is cell-free synthesis of protein. The procedure of cell-free synthesis of protein 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. Procreation of the transcription stage requires participation of specific RNA polymerase which initiates process of mRNA synthesis from the particular sites of recognition. Often the DNA-dependent RNA polymerase of a bacteriophage of T7 (T7 RNA polymerase) is for this purpose applied. For improvement of qualitative characteristics of the T7 RNA polymerase in the real work the new strain of Escherichia coli producing this enzyme fused with the DNA-affine Sso7d domain of a thermophilic bacterium Sulfolobus solfataricus is created. The producing ability of the received recombinant strain concerning synthesized chimera protein reaches 625 un/l of cultural liquid, and the specific activity of the purified enzyme preparation was 80 un/ μg of protein. The received enzyme is intended for use as tools at synthesis of proteins in cell-free system.

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