Creating a mutant luciferase resistant to HPV chemical inhibition by random mutagenesis and colony-level screening.
Firefly luciferase covers a wide range of applications. One common usage of the bioluminescence assay is the measurement of intracellular concentration of adenosine triphosphate (ATP) for cell viability. However, inhibition of the enzyme reaction by chemicals in the assay has so far limited the application of luciferase for high production volume (HPV) chemical testing. The objective of this research was to obtain a mutant luciferase with increased stability to inhibition by HPV chemicals, yet retaining specific activity comparable to, or better than, wild-type luciferase. The enzymatic properties of the wild-type luciferase were improved by random mutagenesis and colony-level screening. In this paper, the detailed process of creating mutant luciferases for testing the toxicity of HPV chemicals is described. As a result, two mutant luciferases were created, with different degrees of improved tolerance to inhibition by chloroform and other HPV chemicals.
Kim-Choi
Eileen
E
Cambrex North Brunswick, North Brunswick, NJ 08902, USA.
Danilo
Christiane
C
Kelly
Jeffrey
J
Carroll
Ronnie
R
Shonnard
David
D
Rybina
Irina
I
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002725:Chloroform / Q000494:pharmacology; D001517:Coleoptera / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D016296:Mutagenesis; D009154:Mutation
D002725:Chloroform; D000255:Adenosine Triphosphate; D008156:Luciferases