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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.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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