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Biochemistry (Moscow)

, 76:1147 | Cite as

Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase

  • Yu. A. Modestova
  • G. Yu. Lomakina
  • N. N. UgarovaEmail author
Article

Abstract

Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His6-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S, C62/164S, and the triple mutant C62/146/164S, the K m ATP and \(K_m^{LH_2 } \) values were increased by a factor of ∼1.5–1.9. Their expression level, specific activity, and thermal stability were significantly decreased. The other mutations had almost no effect on the K m ATP and \(K_m^{LH_2 } \) values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a factor of ∼2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties and structure of the enzyme is discussed.

Key words

firefly luciferase Luciola mingrelica site-directed mutagenesis cysteine residues polyhistidine tag kinetic parameters thermal stability 

Abbreviations

DTT

dithiothreitol

kin

inactivation rate constant

λex

fluorescence excitation wavelength

LH2

firefly luciferin

PCR

polymerase chain reaction

RLU

relative light units

WT

initial recombinant luciferase

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Copyright information

© Pleiades Publishing, Ltd. 2011

Authors and Affiliations

  • Yu. A. Modestova
    • 1
  • G. Yu. Lomakina
    • 1
  • N. N. Ugarova
    • 1
    Email author
  1. 1.Faculty of ChemistryLomonosov Moscow State UniversityMoscowRussia

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