Forceful large-scale expression of “problematic” membrane proteins

• Published in: Biochemical and biophysical research communications Vol. 327; no. 3; pp. 650 - 655
• Main Authors: Mokhonova, Ekaterina I., Mokhonov, Vladislav V., Akama, Hiroyuki, Nakae, Taiji
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.02.2005
• Subjects: Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Bicistronic plasmid; Drug Resistance, Multiple; Efflux pump; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Expression vector; Gene Expression Regulation, Bacterial; Gene Transfer Techniques; Genetic Vectors; Large-scale expression; Membrane protein; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Plasmids - genetics; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - metabolism; Xenobiotic transporter; Membrane protein; Large-scale expression; Bicistronic plasmid; Efflux pump; Xenobiotic transporter; Expression vector
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2004.12.059

We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host. This enabled us to enhance the expression level of a toxic membrane protein 30–50 times compared with expression in the native state and to obtain 3–5 mg of a highly purified functionally active protein per liter of culture. We describe the method for the amplified expression of membrane proteins, using the Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example. The amplified MexY was correctly folded in the cytoplasmic membrane of the E. coli without forming inclusion bodies. This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce.