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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.

SUBMITTER: Lougheed KE

PROVIDER: S-EPMC4169665 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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An in vivo crosslinking system for identifying mycobacterial protein-protein interactions.

Lougheed Kathryn E A KE Bennett Mark H MH Williams Huw D HD

Journal of microbiological methods 20140714

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-t ...[more]

PMID: 25034228

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