Construction of a fused LuxAB gene by site-directed mutagenesis.
Bacterial luciferases are heteropolymetric enzymes consisting of two non-identical subunits (alpha and beta). The two polypeptides are produced by transcription in the same direction of two genes, luxA and luxB, located immediately adjacent to each other and separated by only 29 base pairs in the Vibrio harveyi genome. Using site-directed mutagenesis, stop codons after luxA were eliminated and the luxB gene was placed in-frame with luxA, resulting in a fused luxAB gene. Transcription of two luxAB mutant genes from the bacteriophage T7 promoter and translation in Escherichia coli resulted in the synthesis of fused polypeptides containing the alpha and the beta subunits of luciferase linked by either a single amino acid residue or a decapeptide. E. coli synthesizing the latter fusion protein with the decapeptide linker expressed a level of luminescence comparable to E. coli containing the wild type genes while E. coli synthesizing the polypeptide with a single amino acid as a linker expressed about 2000-fold lower light. These results provide the basis for generating a bacterial luciferase system that can be expressed under the control of a single promoter in both eukaryotic and prokaryotic systems.
Boylan
M O
MO
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Pelletier
J
J
Dhepagnon
S
S
Trudel
S
S
Sonenberg
N
N
Meighen
E A
EA
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D014733:Vibrio / Q000235:genetics
D004269:DNA, Bacterial; D008156:Luciferases