Abstract
The results of Luciola mingrelica firefly luciferase stabilization by genetic engineering methods are reviewed. The Cys62, Cys146, and Cys164 to Ser mutant enzymes with an enhanced thermostability and lower sensitivity to dithiothreitol were obtained by site-directed mutagenesis. The double mutant G216N, A217L was obtained, which displayed a higher thermostability and resistance to DMSO in comparison with WT luciferase. Random mutagenesis of the gene region encoding residues 1–225 and subsequent screening of the mutants resulted in the production of the mutant MT8 with a higher thermostability, as well as mutants MT3 and MT4 with higher resistance to dimethyl sulfoxide. The mutant 4TS was obtained by the method of directed evolution of the gene site encoding residues 130–390, which was shown to contain eight replacements after four cycles of mutagenesis and had two-fold higher specific activity, eight-fold lower K m value for ATP, and stability at 42°C, which was 65-fold higher that of WT luciferase. The stabilization mechanism of this mutant is discussed.
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Original Russian Text © N.N. Ugarova, 2010, published in Vestnik Moskovskogo Universiteta. Khimiya, 2010, No. 3, pp. 168–173.
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Ugarova, N.N. Stabilization of Luciola mingrelica firefly luciferase by genetic engineering methods. Moscow Univ. Chem. Bull. 65, 139–143 (2010). https://doi.org/10.3103/S0027131410030065
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DOI: https://doi.org/10.3103/S0027131410030065