Improvement Of The Tripartite Superfolder GFP Method For Protein-protein Interaction Assay

Objective : Detecting the interaction between protein molecules in living cells is very important to reveal the role and mechanism of these molecules in physiological or pathological processes.Bimolecular fluorescence complementary based on fluorescent proteins is a new method developed in recent years to detect protein-protein interaction in vivo or in vitro.As an improved bimolecular fluorescence complementary,tripartite superfolded GFP(sfGFP)system has the advantages of small fusion fragment and low background fluorescence.However,when we used the tripartite sfGFP system to detect the protein-protein interaction,we found that the fluorescence signal of the tripartite sfGFP system was very weak.In order to solve this problem,we improved the fluorescence signal and signal-to-noise ratio of the tripartite sfGFP system by improving the affinity between the two fragments(G1-9 and G11)in the tripartite system.Methods: The interaction model of HBc/HBc was used to test the performance of the original tripartite sfGFP;FKBP12/FRB interaction model was used to compare the signal-to-noise ratio between the tripartitesfGFP system and the original bipartite sfGFP system.Two strategies were proposed to enhance the affinity between G1-9 and G11 to enhance the fluorescence signal of the tripartite sfGFP system:(1)Mutate G1-9 or G11 to screen the mutants with enhanced affinity;(2)Fuse high affinity molecular pairs to G1-9 and G11 to promote their affinity;Covalent connection of G1-9 and G11 can be considered an extreme condition of this strategy and the tripartite GFP system becomes a new bipartite system.We first tried strategy(1)by using HBc/HBc interaction as a screening model.We mutated G11 and observed whether the mutations improved the fluorescence signal of the system by fluorescence microscope.For strategy(2),we used FKBP12/FRB interaction model to screen the affinity pairs that improve the signal-to-noise ratio of the system,and then compared the two improved tripartite sfGFP systems with the original tripartite sfGFP system in parallel by flow cytometry analysis.We also compared the performance of the new systems and original system by HBc/HBc interaction model.Results: The original tripartite sfGFP system showed a weak fluorescence signal difficult to be observed by fluorescent microscopy in testing the interaction between HBc and HBc.In the analysis of the interaction of FKBP12/FRB,the original tripartite sfGFP system was better than the bipartite sfGFP system in background and signal-to-noise ratio.Among the 227 G11 mutants constructed,no mutant showed significantlyenhanced fluorescence compared with that of wild type.A preliminary screening showed that LgBiT-HiBiT increased S/N of the tripartite system to 57,and covalent fusion of G1-9 and G11 increased S/N to 111.A comparison in parallel demonstrated that the optimal LgBiT-HiBiT tripartite system and the new bipartite system had approximately 2-and7-fold increases in the S/N respectively compared with the original system.For the assay of the interaction between HBc monomers,flow cytometry analysis showed that only the LgBiT-HiBiT tripartite sfGFP system increased the S/N by 3-fold,while the new bipartite sfGFP system increased the proportion of fluorescent cells,but not S/N.The results of confocal microscopy were consistent with the results of FACS.Conclusion: The fluorescence signal and the signal-to-noise ratio of the original tripartite sfGFP system are significantly improved by introducing a pair of affinity molecules LgBiT-HiBiT,or by covalent fusion of G1-9 and G11.The new strategy derived from this new bipartite sfGFP system may be extended to the development of new BiFC from other fluorescent proteins with similar structures.