Split Fluorescent Protein Engineering and Applications

Self-associating split fluorescent proteins (SAsFPs) have been widely used for labeling proteins, visualization of subcellular protein localization, scaffolding protein assembly and detecting cell-cell contacts. This PhD thesis research has been focused on expanding the toolbox of SAsFPs by adding more colors, increasing the brightness and empowering them with various biophysical properties. Utilizing a screening platform for the direct engineering of SAsFPs, we have generated a yellow–green split-mNeonGreen21–10/11, a 10-fold brighter red-colored split-sfCherry21–10/11 and demonstrated dual-color endogenous protein tagging using sfCherry211 and GFP11. However, the newly developed SAsFPs have suffered from sub-optimal fluorescence signal. By investigating the complementation process, we have employed two approaches to improve the overall brightness of SAsFPs: assistance through SpyTag/SpyCatcher covalent interaction and directed evolution for complementation-enhancing mutations. The latter strategy has then yielded two red-colored split sfCherry3 variants with substantially enhanced endogenous protein labeling performance. Based on sfCherry3, we have further developed a red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living animals, demonstrating the broad applications of SAsFPs