PloS one, 2015-04, Vol.10 (4), p.e0123646-e0123646
2015
Details
- Autor(en) / Beteiligte
- Titel
- A gateway-based system for fast evaluation of protein-protein interactions in bacteria
- Ist Teil von
- PloS one, 2015-04, Vol.10 (4), p.e0123646-e0123646
- Ort / Verlag
- United States: Public Library of Science
- Erscheinungsjahr
- 2015
- Link zum Volltext
- Quelle
- EZB Free E-Journals
- Beschreibungen/Notizen
- Protein-protein interactions are important layers of regulation in all kingdoms of life. Identification and characterization of these interactions is one challenging task of the post-genomic era and crucial for understanding of molecular processes within a cell. Several methods have been successfully employed during the past decades to identify protein-protein interactions in bacteria, but most of them include tedious and time-consuming manipulations of DNA. In contrast, the MultiSite Gateway system is a fast tool for transfer of multiple DNA fragments between plasmids enabling simultaneous and site directed cloning of up to four fragments into one construct. Here we developed a new set of Gateway vectors including custom made entry vectors and modular Destination vectors for studying protein-protein interactions via Fluorescence Resonance Energy Transfer (FRET), Bacterial two Hybrid (B2H) and split Gaussia luciferase (Gluc), as well as for fusions with SNAP-tag and HaloTag for dual-color super-resolution microscopy. As proof of principle, we characterized the interaction between the Salmonella effector SipA and its chaperone InvB via split Gluc and B2H approach. The suitability for FRET analysis as well as functionality of fusions with SNAP- and HaloTag could be demonstrated by studying the transient interaction between chemotaxis response regulator CheY and its phosphatase CheZ.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1932-6203eISSN: 1932-6203DOI: 10.1371/journal.pone.0123646Titel-ID: cdi_plos_journals_1672093519
- Format
- –
- Schlagworte
- Analysis, Bacteria, Bacterial Proteins - genetics, Bacterial Proteins - metabolism, Chemotaxis, CheY protein, CheZ protein, Cloning, Cloning, Molecular, Deoxyribonucleic acid, DNA, DNA sequencing, DNA, Bacterial - genetics, E coli, Energy transfer, Escherichia coli, Escherichia coli - genetics, Escherichia coli - metabolism, Expression vectors, Fluorescence Resonance Energy Transfer, Fragments, Gene expression, Genetic Vectors, Gram-positive bacteria, Identification methods, Luciferases - genetics, Medical research, Membrane Proteins - genetics, Membrane Proteins - metabolism, Methyl-Accepting Chemotaxis Proteins, Microfilament Proteins - genetics, Microfilament Proteins - metabolism, Molecular biology, Nucleotide sequencing, Physiological aspects, Plasmids, Protein interaction, Protein Interaction Maps - genetics, Protein-protein interactions, Proteins, Salmonella, Salmonella - genetics, Salmonella - metabolism