A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway
| Autor: | Emilie Gauliard, Macy G. Olson, Megan Goldammer, Scot P. Ouellette, Daniel Ladant |
|---|---|
| Přispěvatelé: | University of South Dakota [Vermillion] (USD), University of Nebraska Medical Center, University of Nebraska System, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Springer, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Bacterial two-hybrid Bordetella pertussis BACTH MESH: Operon Operon [SDV]Life Sciences [q-bio] 030106 microbiology Genetic Vectors Protein interaction assay Adenylate kinase MESH: Adenylyl Cyclases MESH: Two-Hybrid System Techniques Cyclase Article 03 medical and health sciences Gateway® cloning MESH: Genetic Vectors Two-Hybrid System Techniques Protein Interaction Mapping Cyclic AMP Escherichia coli Adenylate cyclase MESH: Protein Binding MESH: Cloning Molecular Cloning Molecular Gene MESH: Cyclic AMP Cloning biology Chemistry MESH: Escherichia coli MESH: Protein Interaction Mapping cyaA biology.organism_classification MESH: Adenylate Cyclase Toxin cAMP signaling Cell biology Cytosol Adenylate Cyclase Toxin Adenylyl Cyclases Protein Binding |
| Zdroj: | Methods Mol Biol Methods in Molecular Biology Springer. Methods in Molecular Biology, 1794, Humana Press, pp.75-96, 2018, Two-Hybrid Systems, 978-1-4939-7870-0. ⟨10.1007/978-1-4939-7871-7_6⟩ Methods in Molecular Biology ISBN: 9781493978700 |
| ISSN: | 1940-6029 |
| Popis: | International audience; The bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure. |
| Databáze: | OpenAIRE |
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