Fluorescent Leaf-Like Structures Formed by Self-Assembly of Green Fluorescent Protein

Green fluorescent protein (GFP) was originally obtained from Aequorea victoria and was successfully synthesized through recombinant technology. In this study, we revealed novel, fascinating insights into the properties of this well-known GFP by examining a traditional GFP separation process involving aqueous two-phase systems and several liquid chromatographs. We investigated the impact of organic solvents and salts on the conformation of GFP by measuring its fluorescence intensity and circular dichroism spectrum, and then proposed an optimized process to improve the yield of fluorescent GFP. We have noticed that fluorescent GFP migrates anomalously to ~37 kDa on non-heating SDS-PAGE due to retention of its compact β-barrel conformation, whereas the commonly reported 27 kDa migration corresponds to the fully heat-denatured, non-fluorescent form. Nevertheless, the most exciting discovery is that the purified GFP exhibited a remarkable ability to self-assemble into an intriguing fluorescent leaf-like structure upon evaporation-concentration under ambient conditions. To our knowledge, this represents the first direct visualization of such macroscopic fluorescent leaf-like assemblies formed by recombinant GFP, with fluorescence retained throughout the assembled morphology. This captivating phenomenon may have promising implications for protein-based nanomaterials and will inspire further exploration of protein self-assembly nanotechnology.