Taneoka Atsushi, Sakaguchi-Mikami Akane, Yamazaki Tomohiko, Tsugawa Wakako, Sode Koji
Graduate School of Technology, Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Biosens Bioelectron. 2009 Sep 15;25(1):76-81. doi: 10.1016/j.bios.2009.06.004. Epub 2009 Jun 10.
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.
通过将半乳糖/葡萄糖结合蛋白（GGBP）与荧光素酶相结合，构建了一种新型的基于发光的葡萄糖传感分子。葡萄糖传感荧光素酶（GlcLuc）是利用与萤火虫荧光素酶（Lluc和Sluc）的一个大结构域和一个小结构域融合的GGBP构建而成。对大肠杆菌重组蛋白进行基于发光强度的分析表明，GlcLuc在葡萄糖或半乳糖中具有浓度依赖性的荧光素酶活性（葡萄糖的K(d)=3.9 microM，半乳糖的K(d)=11 microM），并且在注入配体后一分钟内活性增加达到饱和。这些结果表明，配体结合后GGBP部分的构象变化有效地诱导了GGBP融合的分裂荧光素酶的重组。然后将预期会导致葡萄糖特异性结合能力的Asp459Asn突变引入GlcLuc。GlcLuc突变体仅随着葡萄糖浓度的增加而显示荧光素酶活性增加，而不随半乳糖浓度增加而增加。我们的结果表明，与分裂荧光素酶融合的GGBP在葡萄糖存在下能快速且特异性地重组，提供了一种基于发光的新型葡萄糖传感系统，也可能有助于使用其他PBPs构建用于其他底物的基于发光的传感分子。
