Production of Copepod Luciferases via Baculovirus Expression System.

Autor: Larionova MD; Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center 'Krasnoyarsk Science Center SB RAS', Krasnoyarsk, Russia., Markova SV; Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center 'Krasnoyarsk Science Center SB RAS', Krasnoyarsk, Russia., Vysotski ES; Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center 'Krasnoyarsk Science Center SB RAS', Krasnoyarsk, Russia. eugene.vysotski@gmail.com.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2524, pp. 75-89.
DOI: 10.1007/978-1-0716-2453-1_6
Abstrakt: Secreted copepod luciferases (CopLucs) represent highly homologous enzymes which catalyze the oxidation of a low molecular weight substrate, coelenterazine, with the emission of blue light (λ max  = 485-488 nm), that is called bioluminescence (BL). The well-studied Gaussia (GLuc) and Metridia (MLuc) luciferases originally cloned from the marine copepods Gaussia princeps and Metridia longa belong to the group of the smallest natural luciferases. Their minimal molecular weight, high luminescent activity, cofactor-independent BL, and the ability to be secreted due to the own signal peptide open up the horizons for genetic engineering of CopLuc-based sensitive biosensors for in vivo imaging and in vitro analytical applications. The "standard" soluble bacterial expression of the recombinant CopLucs and luciferase-based hybrid proteins is hampered by the presence of high amounts of intramolecular disulfide bonds (up to 5 per molecule). Here, we describe the universal protocol for highly effective secreted expression of disulfide-rich CopLucs using their own signal peptide in insect cells and their purification from serum-free culture medium. The suggested protocol allows obtaining high-purity CopLucs folded in their native form with the yield of up to 5 mg per liter.
(© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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