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Updated: Oct 6, 2025
Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
Published on: December 3, 2010
Expression, purification and characterization of the suppressor of copper sensitivity (Scs) B membrane protein from
Russell J Jarrott1, Emily J Furlong2, Guillaume A Petit1
1Griffith Institute for Drug Discovery, Griffith University, Building N75, 46 Don Young Rd, Nathan, QLD, 4111, Australia.
Abstract:
Suppressor of copper sensitivity (Scs) proteins play a role in the bacterial response to copper stress in many Gram-negative bacteria, including in the human pathogen Proteus mirabilis. Recently, the ScsC protein from P. mirabilis (PmScsC) was characterized as a trimeric protein with isomerase activity that contributes to the ability of the bacterium to swarm in the presence of copper. The CXXC motif catalytic cysteines of PmScsC are maintained in their active reduced state by the action of its membrane-bound partner protein, the Proteus mirabilis ScsB (PmScsB). Thus, PmScsC and PmScsB form a redox relay in vivo. The predicted domain arrangement of PmScsB comprises a central transmembrane β-domain and two soluble, periplasmic domains, the N-terminal α-domain and C-terminal γ-domain. Here, we provide a procedure for the recombinant expression and purification of the full-length PmScsB protein. Using Lemo21 (DE3) cells we expressed PmScsB and, after extraction and purification, we were able to achieve a yield of 3 mg of purified protein per 8 L of bacterial culture. Furthermore, using two orthogonal methods - AMS labelling of free thiols and a scrambled RNase A activity assay - PmScsB is shown to catalyze the reduction of PmScsC. Our results demonstrate that the PmScsC and PmScsB redox relay can be reconstituted in vitro using recombinant full-length PmScsB membrane protein. This finding provides a promising starting point for the in vitro biochemical and structural characterization of the P. mirabilis ScsC and ScsB interaction.
Insights
Researchers purified the Proteus mirabilis ScsB (PmScsB) protein and demonstrated its ability to reduce PmScsC in vitro. This establishes a functional redox relay for copper stress response in bacteria.
Area of Science:
- Biochemistry
- Microbiology
- Structural Biology
Background:
- Suppressor of copper sensitivity (Scs) proteins are crucial for bacterial copper stress response.
- Proteus mirabilis ScsC (PmScsC) is a trimeric protein with isomerase activity essential for copper-induced swarming.
- PmScsC's catalytic cysteines require reduction by its partner protein, Proteus mirabilis ScsB (PmScsB), forming an in vivo redox relay.
Purpose of the Study:
- To develop a method for recombinant expression and purification of full-length PmScsB.
- To demonstrate the in vitro reconstitution and function of the PmScsC-PmScsB redox relay.
Main Methods:
- Recombinant expression of PmScsB in Lemo21 (DE3) cells.
- Protein purification yielding 3 mg of PmScsB per 8 L of culture.
- In vitro assays including AMS labeling and scrambled RNase A activity assay to confirm PmScsB's reduction of PmScsC.
Main Results:
- Successfully expressed and purified full-length PmScsB.
- Confirmed PmScsB's ability to catalyze the reduction of PmScsC using orthogonal methods.
- Established the reconstitution of the PmScsC-PmScsB redox relay in vitro.
Conclusions:
- The study provides a reliable method for obtaining functional PmScsB.
- The in vitro reconstitution of the PmScsC-PmScsB redox relay is achieved.
- This work lays the foundation for future biochemical and structural studies of this important bacterial protein interaction.
