Expression, purification and characterization of the suppressor of copper sensitivity (Scs) B membrane protein from Proteus mirabilis

• Published in: Protein expression and purification 2022-05, Vol.193, p.106047-106047, Article 106047
• Main Authors: Jarrott, Russell J., Furlong, Emily J., Petit, Guillaume A., Drew, David, Martin, Jennifer L., Halili, Maria A.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Copper - metabolism; Disulfide bond; Humans; Isomerase; Membrane proteins; Membrane Proteins - metabolism; Periplasm - metabolism; Proteus mirabilis - chemistry; Proteus mirabilis - genetics; Proteus mirabilis - metabolism; Suppressor of copper sensitivity
• ISSN: 1046-5928; 1096-0279; 1096-0279
• DOI: 10.1016/j.pep.2022.106047

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. •The bacterial membrane protein ScsB was purified using an E. coli expression system.•ScsB contains two periplasmic domains (α- and γ-) connected by a transmembrane β-domain.•ScsB forms a redox relay with its partner protein ScsC to mediate disulfide isomerization.