Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Summary
In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.
Abstract
Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.
Protocol
Discussion
In addition to multidrug resistance, many vital cellular activities, including ion transport, cell-cell communication, vesicle transport, maintenance of cellular structure, and host-pathogen interactions, involve proteins that are embedded in the cell membrane. Transmembrane proteins make up over 30% of known proteins and are the targets for the majority of pharmaceuticals in use today. The improper folding or activity of transmembrane proteins lead to important genetic diseases, including cystic fibrosis and diabetes. I…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This project was supported by grants to CJJ from the National Science Foundation and the Society for Biomolecular Sciences.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| SDS sample buffer | Biorad | 161-0737 | ||
| C43(DE3) E. coli strain | Lucigen | 60345-1 | ||
| kanamycin sulfate | Sigma-Aldrich | K4378 | ||
| 2XYT media | Fisher | BP2466-2 | ||
| LB media | Fisher | AC61189-5000 | ||
| IPTG | Sigma-Aldrich | I6758 | ||
| DNaseI | Fisher | BP3226-1 | ||
| Lysozyme | Sigma-Aldrich | L7651 | ||
| Complete EDTA-free protease inhibitor tablets | Roche | 11 873 580 001 | ||
| NaP monobasic | Sigma-Aldrich | S6566 | ||
| NaP dibasic | Sigma-Aldrich | S5136 | ||
| NaCl | Sigma-Aldrich | S6191 | ||
| MgCl2 | Sigma-Aldrich | M1028 | ||
| Glycerol | Fisher | BP229-1 | ||
| n-dodecyl-β-D-maltopyranoside | Anatrace | D310 | ||
| 15ml tubes | Corning | 430052 | ||
| See-Saw Rocker | Fisher | SSL 4 | ||
| Talon metal affinity resin | Clontech | 635503 | ||
| imidazole | Sigma-Aldrich | I5513 | ||
| 10% polyacrylamide SDS PAGE gels | BioRad | 161-1454 | ||
| Tris/glycine/SDS PAGE running buffer | BioRad | 161-0732 | ||
| Kaleidascope prestained molecular weight markers | BioRad | 161-0324 | ||
| Superose 12 30/10 column | GEHealthcareSuperose | 12 10/300 GL | ||
| Amicon centrifugal concentrator | Millipore | UFC801024 | ||
| Syringe filter | Fisher | SLFG R04 NL | ||
| Fernbach flasks | Fisher | 09-552-39 | ||
| Shaker to hold Fernbach flasks | Fisher Scientific | |||
| Akta system | GE Healthcare | |||
| J6 Large scale centrifuge with JLA-8.1000 rotor | Beckman | |||
| 1 l centrifuge bottles | Beckman | 969329 | ||
| RC-5 centrifuge | ThermoScientific | |||
| SS34 fixed-angle rotor and tubes | ThermoScientific | |||
| Sorvall floor model Ultracentrifuge | ThermoScientific | |||
| T647.5 rotor and tubes with caps | ThermoScientific | 08322 | ||
| French Pressure Cell | ThermoScientific | FA-032 |
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