In vitro investigation of the MexAB efflux pump from Pseudomonas aeruginosa.
J Vis Exp
; (84): e50894, 2014 Feb 17.
Article
em En
| MEDLINE
| ID: mdl-24638061
ABSTRACT
There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.
Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Membrana Transportadoras
/
Pseudomonas aeruginosa
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Proteínas da Membrana Bacteriana Externa
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article