CSIRO Food Futures National Research Flagship & CSIRO Ecosystem Sciences, Australia, Canberra, ACT 2601, Australia.
Anal Biochem. 2012 May 15;424(2):206-10. doi: 10.1016/j.ab.2012.02.028. Epub 2012 Mar 1.
Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP(2)) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET(2) fusion protein increased the light output by a factor of ~10. Substitution of native RLuc with either of the RLuc mutants resulted in a decrease in BRET(2) ratio by a factor of ~2 when BRET(2) components were separated by the thrombin cleavage sequence. BRET(2) ratios changed by factors of 18.8±1.2 and 18.2±0.4 for GFP(2)-RG-RLuc2 and GFP(2)-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8±0.20 for GFP(2)-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET(2) systems, respectively, and 15 pM for GFP(2)-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET(2).
生物发光能量转移（BRET）是研究蛋白质-蛋白质相互作用和蛋白质构象变化的有力工具。我们使用体外凝血酶测定法直接比较了两种最近开发的海肾荧光素酶（RLuc）变体，RLuc2 和 RLuc8，作为 BRET 供体。通过在供体荧光酶和绿色荧光蛋白（GFP(2)）受体之间放置凝血酶特异性切割序列来进行比较。在 BRET(2)融合蛋白中用 RLuc 突变体 RLuc2 和 8 替代天然 RLuc，使光输出增加了约 10 倍。当用凝血酶切割序列分离 BRET(2)成分时，用 RLuc 突变体中的任一种替代天然 RLuc 会导致 BRET(2)比率降低约 2 倍。对于 GFP(2)-RG-RLuc2 和 GFP(2)-RG-RLuc8 融合蛋白，BRET(2)比率分别变化了 18.8±1.2 和 18.2±0.4 倍，与 GFP(2)-RG-RLuc 相比，分别变化了 18.8±0.20 倍。RLuc2 和 RLuc8 BRET(2)系统检测凝血酶的检测限分别为 0.23 和 0.26 nM，而 GFP(2)-RG-RLuc 的检测限为 15 pM。然而，总体而言，突变 BRET 系统仍然比 FRET 更灵敏，比标准 BRET(2)更亮。
