A split protease-E. coli ClpXP system quantifies protein–protein interactions in Escherichia coli cells
| Autor: | Guimin Zhang, Yueli Yun, Ting Wang, Shengchen Wang, Shihui Yang, Meng Mei, Li Yi, Faying Zhang |
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| Rok vydání: | 2021 |
| Předmět: |
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Molecular Histidine Kinase Protein Conformation QH301-705.5 medicine.medical_treatment Green Fluorescent Proteins Medicine (miscellaneous) Computational biology Protein degradation medicine.disease_cause Article General Biochemistry Genetics and Molecular Biology Protein–protein interaction Green fluorescent protein Viral Proteins 03 medical and health sciences 0302 clinical medicine Bacterial Proteins Escherichia coli medicine Humans CRISPR Protein Interaction Maps Biology (General) Enterovirus 030304 developmental biology 0303 health sciences Protein function Protease Molecular engineering Chemistry Effector Escherichia coli Proteins Serine Endopeptidases Endopeptidase Clp Proteolysis CRISPR-Cas Systems General Agricultural and Biological Sciences Microbiology techniques 030217 neurology & neurosurgery Protein Binding |
| Zdroj: | Communications Biology, Vol 4, Iss 1, Pp 1-9 (2021) Communications Biology |
| ISSN: | 2399-3642 |
| Popis: | 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. Wang et al. developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to characterise protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. Their method can quantify these interactions with high sensitivity, easy manipulation, and broad temperature adaptability |
| Databáze: | OpenAIRE |
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