Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi.
Luciferase (Lux)-encoding sequences are very useful as reporter genes. However, a drawback when applying Vibrio harveyi Lux as a reporter enzyme in eukaryotic cells, is that it is a heterodimeric enzyme, thus requiring simultaneous synthesis of both Lux subunits to be active. To overcome this disadvantage, luxA and luxB genes encoding the A and B subunits of this light-emitting heterodimeric Lux, were fused and expressed in Escherichia coli. Comparative analysis of four fused monomeric Lux enzymes by in vivo enzyme assay, immunoblotting and partial enzyme purification, showed that the fused Lux were active both as AB or as BA monomers, albeit at different levels (up to 80% activity for AB and up to 2% for BA, as compared with the wild type binary A + B construct). One of the LuxAB fusion proteins was stably expressed in calli of Nicotiana tabacum, and displayed very high Lux activity, thus demonstrating its potential as a reporter enzyme in eukaryotic systems.
Olsson
O
O
Department of Plant Physiology, University of Umeå, Sweden.
Escher
A
A
Sandberg
G
G
Schell
J
J
Koncz
C
C
Szalay
A A
AA
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D008156:Luciferases / Q000235:genetics*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D014026:Nicotiana / Q000235:genetics; D014733:Vibrio / Q000235:genetics*
D008156:Luciferases