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Moscow University Chemistry Bulletin Vol. 51, No. 3, P. 168 (2010)
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N. N. Ugarova

Stabilization of firefly luciferase Luciola mingrelica by genetic engineering methods

Abstract

The results of firefly luciferase Luciola mingrelica stabilization using genetic engineering methods are reviewed. Mutants Cys62,146,164Ser were obtained by site-specific mutagenesis, which posses higher thermostability and lower dependence on dithiothreitol additives, than WT-luciferase. Dual mutant G216N, A217L demonstrates increased thermostability and higher resistance to dimethylsulfoxide (DMSO) than WT-luciferase. Random mutagenesis of the luciferase 1–225 residues and screening of mutants allowed us to get thermostable mutant MT8 and DMSO–resistant mutants, MT3, MT4. Mutant 4TS was obtained by the method of directed evolution (four cycles of random mutagenesis) of 130–390 residues of luciferase, which contains 8 mutations. Its specific activity is two times and stability 65 times higher, than that of WT-luciferase, whereas the Km toATP is eight times lower. The mechanism of mutant 4TS stabilization is discussed.
Moscow University Chemistry Bulletin.
2010, Vol. 51, No. 3, P. 168
   

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