Abstract
Characterizing protein–protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein–protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.
Highlights
Characterizing protein–protein interactions (PPIs) is an effective method to help explore protein function
The Split Protease-E. coli ClpXP (SPEC) system consists of two sub-parts, the ClpXP-SsrA protein degradation machinery mediated super-folder GFP (sfGFP) fluorescence reporting sub-system and the split-human Rhinovirus (HRV) 3 C protease-mediated protein interacting sub-system (Fig. 1a)
We compared the functionality of Tobacco Etch Virus (TEV) and human Rhinovirus 3 C (HRV 3 C) proteases in SPEC system
Summary
Characterizing protein–protein interactions (PPIs) is an effective method to help explore protein function. Quantitative proteomics is a newly developed method, which was recently used to map receptor-ligand interactions in the activation mechanism of class B G-protein-coupled receptors (GPCRs) upon interaction with peptide hormones in signal-transduction pathways of mammalian cells and elucidate the protein interactome in chromatin biology[9] Besides these methods, split-protein assays, termed protein fragment complementation assays (PCAs), are another set of methods for characterizing PPIs, including split-GFP10, splitluciferase[11], split-ubiquitin[12], and split-TEV protease[13] et al PCAs have been widely applied in both prokaryotic and eukaryotic species to characterize transient and irreversible PPIs, and manipulate signal pathways, which consist of modular elements to prompt the system flexibility and functionality under in vivo environment. Using SPEC method, we further analyzed the interactions of effects of type I-E and I-F CRISPR/Cas to understand its possible complex structure and functions
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