Heng Yu Chyuan, Foo Jee Loon
NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, Singapore, Singapore.
Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Biotechnol Notes. 2022 Dec 9;3:108-112. doi: 10.1016/j.biotno.2022.12.001. eCollection 2022.
Fluorescent proteins are widely used molecular reporters in studying gene expression and subcellular protein localization. To enable the monitoring of transient cellular events in the model yeast , destabilized green and cyan fluorescent proteins have been constructed. However, their co-utilization is limited by an overlap in their excitation and emission spectra. Although red fluorescent protein is compatible with both green and cyan fluorescent proteins with respect to spectra resolution, a destabilized red fluorescent protein is yet to be constructed for applications in . To realize this, we adopted a degron-fusion strategy to prompt destabilization of red fluorescent protein. Specifically, we fused two degrons derived from Cln2, a G-specific cyclin that mediates cell cycle transition, to the N- or C-terminus of mCherry to generate four destabilized fluorescent proteins that are soluble and functional in . . Importantly, the four mCherry fluorescent proteins are highly differential with regards to fluorescence half-life and intensity, which provides a greater choice of tools available for the study of dynamic gene expression and transient cellular processes in the model yeast.
荧光蛋白是研究基因表达和亚细胞蛋白定位时广泛使用的分子报告物。为了能够监测模式酵母中的瞬时细胞事件，已构建了不稳定的绿色和青色荧光蛋白。然而，它们的共同使用受到其激发光谱和发射光谱重叠的限制。尽管就光谱分辨率而言，红色荧光蛋白与绿色和青色荧光蛋白都兼容，但尚未构建用于[具体应用场景未提及]的不稳定红色荧光蛋白。为了实现这一点，我们采用了降解子融合策略来促使红色荧光蛋白不稳定。具体而言，我们将源自介导细胞周期转换的G1特异性细胞周期蛋白Cln2的两个降解子融合到mCherry的N端或C端，以生成四种不稳定的荧光蛋白，它们在[具体环境未提及]中是可溶且有功能的。重要的是，这四种mCherry荧光蛋白在荧光半衰期和强度方面有很大差异，这为研究模式酵母中的动态基因表达和瞬时细胞过程提供了更多的工具选择。
