vestichИзвестия Национальной академии наук Беларуси. Серия химических наукProceedings of the National Academy of Sciences of Belarus, Chemical Series1561-83312524-2342The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"vestich-290Research ArticleБИООРГАНИЧЕСКАЯ ХИМИЯBIOORGANIC CHEMISTRYСАЙТ-СПЕЦИФИЧНОЕ БИОТИНИЛИРОВАНИЕ БЕЛКОВ И ПЕПТИДОВ, ИММОБИЛИЗОВАННЫХ НА МИКРОЧАСТИЦАХ, РЕКОМБИНАНТНОЙ БИОТИН ЛИГАЗОЙBIOTIN LIGASE FOR SITE SPECIFIC BIOTINYLATION OF PROTEINS AND PEPTIDES IMMOBILIZED ON MICROPARTICLESСвиридА. В.SviridA. V.

науч. сотрудник

ул. Акад. Купревича, 5/2, 220141

Researcher

5/2, Acad. Kuprevich Str., 220141

svirid.andrei@gmail.comЛущикА. Я.LushchykA. Ya.

науч. сотрудник

ул. Акад. Купревича, 5/2, 220141

Researcher

5/2, Acad. Kuprevich Str., 220141

sasha_l_88@mail.ruГилепА. А.GilepA. A.

канд. хим. наук, зав. отделом

ул. Акад. Купревича, 5/2, 220141

Ph. D. (Chemistry), Head of the Department

5/2, Acad. Kuprevich Str., 220141

agilep@iboch.bas-net.byУсановС. А.UsanovS. A.

член-корр., д-р хим. наук

ул. Акад. Купревича, 5/2, 220141

Corresponding Member of the National Academy of Sciences of Belarus, D. Sc. (Chemistry), Professor

5/2, Acad. Kuprevich Str., 220141

usanov@iboch.bas-net.byИнститут биоорганической химии НАН Беларуси, МинскInstitute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Minsk201714122017046273Copyright © Свирид А.В., Лущик А.Я., Гилеп А.А., Усанов С.А., 20172017Свирид А.В., Лущик А.Я., Гилеп А.А., Усанов С.А.Svirid A.V., Lushchyk A.Y., Gilep A.A., Usanov S.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestichem.belnauka.by/jour/article/view/290

С целью получения каталитически активной рекомбинантной биотин лигазы (rBirA) разработан подход к получению сшитого белка MBP-BirA путем гетерологической экспрессии в клетках E. coli, с последующей его очисткой и высвобождением rBirA с помощью высокоспецифичного протеолиза TEV протеазой. Рекомбинантный белок получен в гомогенном состоянии и обладает специфической каталитической активностью. Использование иммобилизации на магнитных микрочастицах позволило разработать подход к эффективному ферментативному биотинилированию, совмещенному с очисткой целевых белковых молекул на аффинной матрице. 

To obtain catalytically active recombinant biotin ligase (rBirA), we developed an approach for heterologous expression of BirA-MBP-fusion protein in E. coli cells, its purification and subsequent rBirA release by highly specific proteolysis using TEV protease. The recombinant protein is obtained in homogeneous state and retains specific catalytic activity. Enzyme immobilization on magnetic microparticles has allowed to develop an approach for effective enzymatic biotinylation combined with purification of target protein molecules on the affinity matrix.

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