美国威尔康奈尔医学院Benjamin C. Campbell等研究人员共同合作近期取得重要工作进展，他们研究开发了用于高级显微镜的化学稳定型荧光蛋白。相关论文2022年11月7日在线发表于《自然—方法学》杂志上。

研究人员报道了一种非常稳定的黄色荧光蛋白（YFP）“超折叠YFP”（hfYFP，hyperfolder YFP）的合理工程，该蛋白能够承受在数秒内变性大多数生物结构的离体条件，包括超折叠绿色荧光蛋白（GFP）。hfYFP不含半胱氨酸，对氯化物不敏感，比普通荧光蛋白更好地耐受醛和四氧化锇的固定，使其能够用于扩增和电子显微镜。研究人员分析了hfYFP的晶体结构（分辨率为1.7-Å），一种单一变体，单体超折叠YFP（1.6Å）和mGreenLantern突变体（1.2Å），然后从这些结构中合理设计高度稳定的405nm可激发GFP、大斯托克斯位移（LSS）单体GFP（LSSmGFP）和LSSA12。

最后，研究人员通过设计荧光辅助蛋白纯化策略，直接利用hfYFP和LSSmGFP的化学稳定性，使变性亲和层析的所有步骤都可以使用紫外线或蓝光进行可视化。hfYFP和LSSmGFP是为先进生物技术应用开发的新一代稳定型荧光蛋白。

附：英文原文

Title: Chemically stable fluorescent proteins for advanced microscopy

Author: Campbell, Benjamin C., Paez-Segala, Maria G., Looger, Loren L., Petsko, Gregory A., Liu, Ce Feng

Issue&Volume: 2022-11-07

Abstract: We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), ‘hyperfolder YFP’ (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7- resolution), a monomeric variant, monomeric hyperfolder YFP (1.6) and an mGreenLantern mutant (1.2), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

DOI: 10.1038/s41592-022-01660-7

Source: https://www.nature.com/articles/s41592-022-01660-7