Soluble preparation and characterization of tripartite split GFP for In Vitro reconstitution applications

Tripartite split GFP can be easily fused with the protein of interest (POI) and does not require any substrate for protein-protein interaction (PPI) analysis. However, split GFP1–9 in tripartite GFP causes problems owing to its low solubility and suboptimal expression levels. In particular, for in vitro applications, an elaborate refolding process is currently needed to prepare split GFP1–9. Here, we prepared split GFP1–9 with improved expression levels and solubility using Escherichia coli as host, for in vitro reconstitution applications. We designed the expression and purification systems based on the intein-splicing method, involving an optimized linker. Intein-mediated purified split GFP1–9 (ipGFP1–9) with a GRA linker showed over 95% solubility with a 14-fold improved total expression yield, and the purification yield of ipGFP1–9 was 52-fold higher than refolded split GFP1–9. ipGFP1–9 had 49-times higher fluorescence recovery yield than refolded split GFP1–9, when it was reconstituted with partial beta-strand 10–11 of GFP (S10–11). Using ipGFP1–9, we also investigated the effects of pH and ionic strength on the in vitro reconstitution of GFP. Finally, we probed the effect of the molar ratio of tripartite split GFP on the reconstitution process. These results will enable to use the split GFP system for in vitro reconstitution-based applications, in particular the design of PPI-based assays or biosensing systems.