The construction of a glucose-sensing luciferase.
A novel luminescence-based glucose-sensing molecule was created by combining a galactose-/glucose-binding protein (GGBP) with luciferase. The glucose-sensing luciferase (GlcLuc) was constructed using a GGBP fused with a large domain and a small domain of Firefly luciferase (Lluc and Sluc). The luminescence intensity-based analysis with E. coli recombinant protein showed that the GlcLuc had luciferase activity in glucose or galactose in a concentration-dependent manner (K(d)=3.9 microM for glucose and 11 microM for galactose), and that the increase in the activity saturated within one minute after the injection of the ligands. These results indicated that the conformation change of the GGBP moiety following the ligand binding effectively induced the reconstitution of the GGBP-fused split luciferase. The Asp459Asn mutation, which was expected to lead to a glucose specific binding ability, was then introduced into the GlcLuc. The GlcLuc mutant showed the luciferase activity increasing only with the increase of glucose concentration, but not with that of galactose. Our results demonstrate that the GGBP fused with a split luciferase, which is reconstituted rapidly and specifically in the presence of glucose, provides a novel glucose-sensing system based on luminescence and may also contribute to the construction of luminescence-based sensing molecules for other substrates using other PBPs.
Taneoka
Atsushi
A
Graduate School of Technology, Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Sakaguchi-Mikami
Akane
A
Yamazaki
Tomohiko
T
Tsugawa
Wakako
W
Sode
Koji
K
D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000032:analysis; D005947:Glucose / Q000032:analysis*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C509064:mglB protein, E coli; D008156:Luciferases; D005947:Glucose; D005690:Galactose