Strategies to improve the fluorescent signal of the tripartite sfGFP system.

Strategies to improve the fluorescent signal of the tripartite sfGFP system.
Acta Biochim Biophys Sin (Shanghai). 2020 Jun 24;:
Authors: Shen J, Zhang W, Gan C, Wei X, Li J, Sun Y, Yuan Y, Cai X, Long Q, Cui J, Guo H, Huang A, Hu J
Abstract
Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein-protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1-9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1-9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC s...

Source: Acta Biochimica et Biophysica Sinica - June 23, 2020 Category: Biochemistry Authors: Shen J, Zhang W, Gan C, Wei X, Li J, Sun Y, Yuan Y, Cai X, Long Q, Cui J, Guo H, Huang A, Hu J Tags: Acta Biochim Biophys Sin (Shanghai) Source Type: research