Single copy gene expression system as a tool for the purification of membrane proteins from Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen known to cause a variety of infections that are difficult to treat due to extremely high resistance to almost all antibiotics currently in clinical use. One of the major contributors to this resistance is the active efflux of antibiotics from the cell, primarily by action of the Resistance Nodulation Division (RND) family of efflux pumps. These pumps are composed of three proteins; an inner membrane RND pump, a periplasmic membrane fusion adaptor protein, and an outer membrane protein. The mechanism by which the three proteins interact to form a functional complex is largely unknown and the methods currently available for their study involves expression systems geared for high levels of expression. In the case of membrane proteins which play a role in clinically relevant activities, such as multidrug resistance, an expression system which does not always reflect biologically relevant levels of protein in the cell is not ideal for studying their interactions as correlation of conclusions from interaction studies to true interactions may not be possible. In this study a single copy gene expression system was designed and demonstrated to better reflect clinically relevant levels of overexpression compared to a multi-copy expression system. Quantitative-real time PCR analysis of C-terminally hexa-histidine tagged outermembrane protein, OpmH, expression shows approximately 100-fold and 20-fold overexpression from multi-copy and single-copy expression systems respectively. OpmH-H6 was successfully purified from both multi copy and single copy expression systems with proportionate purification schemes indicating the feasibility of single copy expression systems for the study of membrane bound protein complexes.