Liang Guo-Teng, Lai Cuixin, Yue Zejun, Zhang Hanbin, Li Danyang, Chen Zhong, Lu Xingyu, Tao Liang, Subach Fedor V, Piatkevich Kiryl D
School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China.
Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China.
Front Bioeng Biotechnol. 2022 Oct 17;10:1039317. doi: 10.3389/fbioe.2022.1039317. eCollection 2022.
Engineered light, oxygen, and voltage (LOV)-based proteins are able to fluoresce without oxygen requirement due to the autocatalytic incorporation of exogenous flavin as a chromophore thus allowing for live cell imaging under hypoxic and anaerobic conditions. They were also discovered to have high sensitivity to transition metal ions and physiological flavin derivatives. These properties make flavin-binding fluorescent proteins (FPs) a perspective platform for biosensor development. However, brightness of currently available flavin-binding FPs is limited compared to GFP-like FPs creating a need for their further enhancement and optimization. In this study, we applied a directed molecular evolution approach to develop a pair of flavin-binding FPs, named miniGFP1 and miniGFP2. The miniGFP proteins are characterized by cyan-green fluorescence with excitation/emission maxima at 450/499 nm and a molecular size of ∼13 kDa. We carried out systematic benchmarking of miniGFPs in and cultured mammalian cells against spectrally similar FPs including GFP-like FP, bilirubin-binding FP, and bright flavin-binding FPs. The miniGFPs proteins exhibited improved photochemical properties compared to other flavin-binding FPs enabling long-term live cell imaging. We demonstrated the utility of miniGFPs for live cell imaging in bacterial culture under anaerobic conditions and in CHO cells under hypoxia. The miniGFPs' fluorescence was highly sensitive to Cu(II) ions in solution with K values of 67 and 68 nM for miniGFP1 and miniGFP2, respectively. We also observed fluorescence quenching of miniGFPs by the reduced form of Cu(I) suggesting its potential application as an optical indicator for Cu(I) and Cu(II). In addition, miniGFPs showed the ability to selectively bind exogenous flavin mononucleotide demonstrating a potential for utilization as a selective fluorescent flavin indicator. Altogether, miniGFPs can serve as a multisensing platform for fluorescence biosensor development for and in-cell applications.
基于工程化光、氧和电压（LOV）的蛋白质能够在无需氧气的情况下发出荧光，这是由于外源性黄素作为发色团的自催化掺入，从而允许在缺氧和厌氧条件下进行活细胞成像。人们还发现它们对过渡金属离子和生理性黄素衍生物具有高敏感性。这些特性使黄素结合荧光蛋白（FPs）成为生物传感器开发的一个有前景的平台。然而，与类绿色荧光蛋白（GFP）相比，目前可用的黄素结合FPs的亮度有限，因此需要对其进行进一步增强和优化。在本研究中，我们应用定向分子进化方法开发了一对黄素结合FPs，命名为miniGFP1和miniGFP2。miniGFP蛋白的特征是发出青绿色荧光，激发/发射最大值在450/499nm，分子大小约为13kDa。我们在原代和培养的哺乳动物细胞中对miniGFP进行了系统的基准测试，与光谱相似的FPs进行比较，包括类GFP、胆红素结合FP和明亮的黄素结合FPs。与其他黄素结合FPs相比，miniGFP蛋白表现出改善的光化学性质，能够进行长期活细胞成像。我们证明了miniGFP在厌氧条件下的细菌培养和缺氧条件下的CHO细胞中的活细胞成像应用。miniGFP的荧光对溶液中的Cu（II）离子高度敏感，miniGFP1和miniGFP2的K值分别为67和68nM。我们还观察到miniGFP被还原形式的Cu（I）淬灭荧光，表明其作为Cu（I）和Cu（II）光学指示剂的潜在应用。此外，miniGFP显示出选择性结合外源性黄素单核苷酸的能力，表明其有潜力用作选择性荧光黄素指示剂。总之，miniGFP可作为用于原代和细胞内应用的荧光生物传感器开发的多传感平台。
