CpxR Binding to hcp2B Promoter by EMSA

The cpxR gene was cloned into the pET28a(+) plasmid (Novagen, Madison, WI, USA), and the recombinant proteins were expressed in E. coli BL21 (DE3) cells by addition of 1 mM isopropyl-β-d-thiogalactopyranoside. The purification of CpxR fusion protein was performed with a HisTrap high-performance column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) as previously described [14 (link)]. The CpxR protein was phosphorylated with acetyl phosphate (Sigma, St. Louis, MO, USA) as previously described [51 (link)]. Then, EMSA were performed to determine the binding of phosphorylated CpxR (CpxR-P) to the hcp2B promoter. Briefly, the sequence of the hcp2B promoter region with or without the putative CpxR binding site was amplified and labeled with biotin. The biotin-labeled DNA probe (40 ng) was incubated with increasing concentrations of CpxR-P protein in EMSA binding buffer (10 mM Tris, 50 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM MnCl₂, 2.5% glycerol and 50 ng/μL poly[dI-dC]). After incubation for 30 min at room temperature, the reactions were subjected to electrophoresis and transferred to a nylon membrane. The biotin-labeled DNA was detected with a chemiluminescent substrate (Amersham Pharmacia Biotech). A competitive EMSA was performed by simultaneously incubating the biotin-labeled and unlabeled hcp2B promoter region with CpxR-P protein.