Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor

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• 作者：Helen Dacres ; ^{Helen.dacres@csiro.au} ; Michelle Michie ; Alisha Anderson ; Stephen C. Trowell
• 关键词：RLuc2 ; RLuc8 ; Maltose binding proteins ; Beer analysis ; Conformational change
• 刊名：Biosensors and Bioelectronics
• 出版年：2013
• 出版时间：15 March, 2013
• 年：2013
• 卷：41
• 期：Complete
• 页码：459-464
• 全文大小：462 K

文摘

A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP²) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer² (BRET²) system showed a 30 % increase in the BRET ratio upon maltose binding, compared with a 10 % increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET² provides a better matched F?rster distance to the known separation of the N and C termini of MBP than FRET. The sensor responded to maltose and maltotriose and the response was completely abolished by introduction of a single point mutation in the BRET² tagged MBP protein. The half maximal effective concentration (EC₅₀) was 0.37 ¦ÌM for maltose and the response was linear over almost three log units ranging from 10 nM to 3.16 ¦ÌM maltose for the BRET² system compared to an EC₅₀ of 2.3 ¦ÌM and a linear response ranging from 0.3 ¦ÌM to 21.1 ¦ÌM for the equivalent FRET-based biosensor. The biosensor¡¯s estimate of maltose in beer matched that of a commercial enzyme-linked assay but was quicker and more precise, demonstrating its applicability to real-world samples. A similar BRET²-based transduction scheme approach would likely be applicable to other binding proteins that have a ¡°venus-fly-trap¡± mechanism.