Development of reporter system using split green fluorescent protein in escherichia coli

Abstract

Green fluorescent protein (GFP) is autofluorescent protein widely used for bioimaging, intracellular cell tracking and small molecule sensing. In early 2000s, split green fluorescent protein (split GFP) complementation, two (GFP1-10 OPT and GFP 11 M3) or three (GFP1-9 OPT, GFP10 M1, GFP11 M4) of split GFP fragments that reassemble in vivo and in vitro, was also successfully demonstrated. And split GFP systems were utilized in many research fields as a sensor to assay solubility of target protein, protein-protein interaction and engineering novel protein scaffolds. The typical bacterial host for protein expression, Escherichia coli, is generally used as the host to develop these fluorescent reporters and also it can be engineered in itself by using these fluorescent protein-based biosensors. In this regard, the more sophisticate tools working in E. coli are essential to engineer peptide pairs or E. coli itself. In this study, two reporter systems working respectively in each of E. coli compartment were developed by using split GFP complementation. First, the split GFP reporter system acting in periplasm was developed for the purpose of signal recognition particle pathway (SRP pathway) engineering with introduction of several secretory pathways and cytoplasmic protein degradation system. Moreover, the cytoplasmic tripartite split GFP system was reconstructed, so it can emit fluorescence in sufficient intensities to be detected by fluorescence activated cell sorter (FACS). Then, the reconstructed tripartite split GFP system was evaluated with introduction of several affinity pairs showing different affinities (from 1μM to 1nM), and it was also confirmed that this system has ability to detect a strong affinity pair of which Kd value is approximately 1nM. Here, along with the previously confirmed systems, it is expected that reconstructed tripartite split GFP system can be applied to engineer peptide pairs with remarkably high affinity below 1nM in $K_d$ value.