title: An in vivo crosslinking system for identifying mycobacterial protein-protein interactions.
abstract: The analysis of protein-protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His-Strep-Strep tag. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex.
authors.affiliation: Department of Life Sciences, Imperial College London, SW7 2AZ, United Kingdom.
authors.forename: Kathryn E A
authors.initials: KE
authors.lastname: Lougheed
authors.affiliation: Department of Life Sciences, Imperial College London, SW7 2AZ, United Kingdom.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Bennett
authors.affiliation: Department of Life Sciences, Imperial College London, SW7 2AZ, United Kingdom. Electronic address: h.d.williams@imperial.ac.uk.
authors.forename: Huw D
authors.initials: HD
authors.lastname: Williams
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D011768:Pyruvate Dehydrogenase Complex; D005557:Formaldehyde
keywords: AceE; Formaldehyde crosslinking; Mycobacterium tuberculosis; Pyruvate dehydrogenase; Tandem affinity purification