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

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.

1. The plasmid is used to transform E. coli strain C43(DE3) ¹², and the transformants are plated on LB agar containing 30 ug/mL kanamycin.

2. Day 2: Overnight Cultures:

2. In the evening, 4 X 3 mL LB cultures containing 30 ug/mL kanamycin are inoculated from the fresh transformant colo....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SDS sample buffer	Bio-Rad	161-0737
C43(DE3) E. coli strain	Lucigen	60345-1
kanamycin sulfate	Sigma-Aldrich	K4378
2XYT media	Fisher Scientific	BP2466-2
LB media	Fisher Scientific	AC61189-5000
IPTG	Sigma-Aldrich	I6758
DNaseI	Fisher Scientific	BP3226-1
Lysozyme	Sigma-Aldrich	L7651
Complete EDTA-free protease inhibitor tablets	Roche Group	11 873 580 001
NaP monobasic	Sigma-Aldrich	S6566
NaP dibasic	Sigma-Aldrich	S5136
NaCl	Sigma-Aldrich	S6191
MgCl2	Sigma-Aldrich	M1028
Glycerol	Fisher Scientific	BP229-1
n-dodecyl-β-D-maltopyranoside	Anatrace	D310
15ml tubes	Corning	430052
See-Saw Rocker	Fisher Scientific	SSL 4
Talon metal affinity resin	Clontech Laboratories	635503
imidazole	Sigma-Aldrich	I5513
10% polyacrylamide SDS PAGE gels	Bio-Rad	161-1454
Tris/glycine/SDS PAGE running buffer	Bio-Rad	161-0732
Kaleidascope prestained molecular weight markers	Bio-Rad	161-0324
Superose 12 30/10 column	GE Healthcare	12 10/300 GL
Amicon centrifugal concentrator	EMD Millipore	UFC801024
Syringe filter	Fisher Scientific	SLFG R04 NL
Fernbach flasks	Fisher Scientific	09-552-39
Shaker to hold Fernbach flasks	Fisher Scientific
Akta system	GE Healthcare
J6 Large scale centrifuge with JLA-8.1000 rotor	Beckman Coulter Inc.
1 l centrifuge bottles	Beckman Coulter Inc.	969329
RC-5 centrifuge	Thermo Fisher Scientific, Inc.
SS34 fixed-angle rotor and tubes	Thermo Fisher Scientific, Inc.
Sorvall floor model Ultracentrifuge	Thermo Fisher Scientific, Inc.
T647.5 rotor and tubes with caps	Thermo Fisher Scientific, Inc.	08322
French Pressure Cell	Thermo Fisher Scientific, Inc.	FA-032