Сибирский федеральный университет

Тип публикации: статья из журнала

Год издания: 2022

Идентификатор DOI: 10.1007/978-1-0716-2453-1_5

Ключевые слова: bioluminescent reporter, coelenterazine, disulfide bond formation, disulfide-rich protein, inclusion bodies, luciferase, oxidative refolding, protein purificationdisulfide, 16734-12-6, disulfides, luciferases, recombinant proteins

Аннотация: The small coelenterazine-dependent luciferase from Metridia longa (MLuc), in view of its high activity, simplicity of bioluminescent (BL) reaction, and stability, has found successful analytical applications as a genetically encoded reporter for in vivo assessment of cellular processes. However, the study on the biochemical and BL properties and the development of in vitro analytical applications of MLuc are hampered by the difficulties of obtaining a sufficient amount of the highly active recombinant protein due to the presence of multiple (up to five) disulfide bonds per molecule. Here, we present a protocol to obtain the recombinant disulfide-rich MLuc using a cheap and simple Escherichia coli expression system without any affinity tags in its native form by refolding from inclusion bodies. The method includes (i) purification of MLuc inclusion bodies, solubilization of the aggregated form with full reduction of disulfide bonds, and refolding to the native state using a glutathione redox system in the presence of arginine and Cu2+ ions and (ii) chromatographic purification of MLuc and its functional assessment in terms of activity. We introduce the empirical, optimal conditions for oxidative refolding and subsequent purification of MLuc, with its basic properties taken into account. We believe that this protocol is adaptable for a large-scale harvest of other natively folded copepod luciferases as well as other disulfide-rich recombinant proteins from E. coli inclusion bodies. © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Журнал: Methods in molecular biology (Clifton, N.J.)

Выпуск журнала: Vol. 2524

Номера страниц: 59-73

ISSN журнала: 19406029

• Markova S.V. (Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russian Federation, School of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk, Russian Federation)
• Larionova M.D. (Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russian Federation)
• Vysotski E.S. (Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, Russian Federation)

• Scopus