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.
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.
1. Day 1:
MexB from Pseudomonas aeruginosa is encoded by pFB101. The MexB gene was amplified from P. aeruginosa genomic DNA and inserted in the NdeI and XhoI restriction sites of the pET30b+ vector. The construct contains a C-terminal hexahistidine tag.
2. Day 2: Overnight Cultures:
3. Day 3: Growing 6 Liter Cultures:
4. Day 4: Harvesting Cells and Purifying the Protein:
5. TM Protein Solublization:
6. IMAC:
7. Gel Filtration Column:
8. Representative Results:
Figure 1 includes a polyacrylamide gel with pooled column fractions from the IMAC column and individual fractions from the gel filtration column. After the gel filtration column the protein appears pure by Coomassie stained polyacrylamide gel. Figure 2 includes a trace from the gel filtration column showing the main peak of the protein detergent complex eluting from the column. The average yield of MexB protein is approximately 2 mg per 6 liters of 2XYT culture.
Figure 1. SDS-PAGE gel of purification of MexB PDCs. Lane 1, Molecular weight markers. 2, Pooled IMAC fractions. 3-7, Gel filtration column fractions.
Figure 2. Example of Gel Filtration Results for MexB protein detergent complexes (PDC).
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. In spite of the vast importance of transmembrane proteins, there is far less known about their structures and molecular mechanisms than for soluble proteins. The presence of hydrophobic sequences can make it difficult to express and isolate large amounts of these proteins and makes them refractory to many biochemical and structural methods.
This protocol demonstrated the expression, detergent solubilization and purification of an MDR membrane protein as a soluble protein detergent complex. These methods can be used with some modification for many recombinantly expressed transmembrane proteins. The resulting purified protein detergent complexes are soluble and can be used for crystallization trials for X-ray crystallographic structure determination and for other biophysical or biochemical characterization, including reconstitution into liposomes or crosslinking studies.
During the purification procedures, it is important to be careful that protein detergent complexes do not precipitate out during the spin concentration steps. Different methods might also be used to help concentrate the PDC before or after the gel filtration step, such as repeating the IMAC step with a very small column and small elution volume.
The authors have nothing to disclose.
This project was supported by grants to CJJ from the National Science Foundation and the Society for Biomolecular Sciences.
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 |