Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
Biosens Bioelectron. 2016 Mar 15;77:901-6. doi: 10.1016/j.bios.2015.10.063. Epub 2015 Oct 26.
NADP(+), the oxidized form of nicotinamide adenine dinucleotide phosphate, plays an essential role as a coenzyme in cellular electron transfer reactions. The concentration of NADP(+) in cytoplasm or organelles is dynamic due to its conversion to many important derivatives. To track the NADP(+) concentration in single living cells, we developed a genetically encoded NADP(+) biosensor by inserting a reporter element, ketopantoate reductase (KPR), between the Förster resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). This recombinant sensor showed a NADP(+) concentration-dependent decrease in the fluorescence ratio in vitro assay. In order to optimize this biosensor, we performed peptide-length optimization and site-directed mutagenesis in the binding pocket of KPR guided by predictions from computational protein redesign. This modified biosensor showed a 70% Δratio increase compared to the wild type and was found to be highly specific to NADP(+), with a detection limit of 1 μM. The sensor also reported NADP(+) real-time cellular dynamics in Escherichia coli (E. coli) after the addition of its precursor, nicotinic acid (NA). Altogether, these results demonstrate the feasibility of the biosensor for visualizing NADP(+) both in vitro and in vivo.
NADP(+)，即烟酰胺腺嘌呤二核苷酸磷酸的氧化形式，作为细胞电子转移反应中的辅酶，起着至关重要的作用。由于 NADP(+)可转化为许多重要的衍生物，其在细胞质或细胞器中的浓度是动态变化的。为了在单个活细胞中追踪 NADP(+)浓度，我们通过在Förster 共振能量转移（FRET）对，即青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）之间插入报告元件酮戊二酸还原酶（KPR），开发了一种遗传编码的 NADP(+) 生物传感器。在体外实验中，该重组传感器的荧光比值随着 NADP(+)浓度的变化而降低。为了优化该生物传感器，我们根据计算蛋白质重新设计的预测，对 KPR 结合口袋中的肽长度进行了优化和定点突变。与野生型相比，这种改良的生物传感器的Δ比值增加了 70%，并且对 NADP(+)具有高度特异性，检测限为 1 μM。该传感器还在向大肠杆菌（E. coli）添加其前体烟酸（NA）后，报告了 NADP(+) 的实时细胞动力学。总之，这些结果证明了该生物传感器在体外和体内可视化 NADP(+) 的可行性。
