The PCR Random Mutagenesis Kit (Takara, Shiga, Japan) was used to generate a random mutant library of the Photinus pyralis luc genes. Random mutations were introduced at the rate of one mutation per gene. PCR was conducted using the primers 5′-GACTCCATGGAAGACGCCAAAAAC-3′ and 5′-GACACTCGAGCAATTTGGACTTTCCGCC-3′ to generate mutant insert containing restriction sites NcoI and XhoI. All mutant genes were cloned into the pET-28a vector (Merck, Darmstadt, Germany) to generate recombinant luciferases containing a C-terminal His-tag.
The vectors containing the mutant luc genes were introduced into Escherichia coli HMS174 (DE3) (Merck), spread onto Luria-Bertani agar plates containing kanamycin, and incubated at 37 ℃. The colonies were inoculated into a 2YT liquid medium containing kanamycin and induced with 0.1 mM isopropyl-ß-D-thiogalactoside (IPTG) in deep-well plates (Watson, Tokyo, Japan) to produce the luciferase protein.
Crude extracts containing mutant luciferase proteins were prepared by freezing and thawing the recombinant bacteria. Each extract was divided into two portions to measure the luminescence intensities in the absence or presence of 140 mM sodium chloride in a reaction mixture containing 50 µl each of 1 µM ATP and 0.1 µM D-luciferin in the Tris-HCl buffer.
The vectors containing the mutant luc genes were introduced into Escherichia coli HMS174 (DE3) (Merck), spread onto Luria-Bertani agar plates containing kanamycin, and incubated at 37 ℃. The colonies were inoculated into a 2YT liquid medium containing kanamycin and induced with 0.1 mM isopropyl-ß-D-thiogalactoside (IPTG) in deep-well plates (Watson, Tokyo, Japan) to produce the luciferase protein.
Crude extracts containing mutant luciferase proteins were prepared by freezing and thawing the recombinant bacteria. Each extract was divided into two portions to measure the luminescence intensities in the absence or presence of 140 mM sodium chloride in a reaction mixture containing 50 µl each of 1 µM ATP and 0.1 µM D-luciferin in the Tris-HCl buffer.