Increase in bioluminescence intensity of firefly luciferase using genetic modification.
Firefly luciferase is widely used for enzymatic measurement of ATP, and its gene is used as a reporter for gene expression experiments. From our mutant library, we selected novel mutations in Photinus pyralis luciferase with higher luminescence intensity. These included mutations at Ile423, Asp436, and Leu530. Luciferase is structurally composed of a large N-terminal active site domain (residues 1-436), a flexible linker (residues 436-440) peptide, and a small C-terminal domain (residues 440-550) facing the N domain. Thus, the mutations are located at the junction of the N-terminal domain and the flexible linker, in the flexible linker peptide, and in the tip of the C-terminal domain, respectively. Substitution of Asp436 with a nonbulky amino acid such as Gly remarkably increased the substrate affinity for ATP and d-luciferin. Substitution of Ile423 with a hydrophobic amino acid such as Leu and that of Leu530 with a positively charged amino acid such as Arg increased the substrate affinity and the turnover rate. Combining these mutations, we obtained luciferases that generate more than 10-fold higher luminescence intensity than the wild-type enzyme.
Fujii
Hiroya
H
Bussan Nanotech Research Institute Inc., Tsukuba, Ibaraki 305-0074, Japan.
Noda
Kenichi
K
Asami
Yasuo
Y
Kuroda
Akio
A
Sakata
Minoru
M
Tokida
Akihiko
A
D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D048888:Fireflies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049449:Luminescence; D008163:Luminescent Measurements; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D007532:Isoleucine; D001224:Aspartic Acid; D049409:Luciferases, Firefly; D007930:Leucine