Mse soniprep 150

Two-dimensional protein gel electrophoresis was conducted as described previously (Shaw et al., 2012 (link)). C. jejuni cells were harvested from broth culture (50 ml) by centrifugation at 3220 × g for 15 min at room temperature. Cell pellets were resuspended in 500 μl lysis buffer (50 mM Tris (pH 7.5), 0.3% sodium dodecyl sulfate (SDS), 0.2 M dithiothreitol, 3.3 mM MgCl₂, 16.7 μg of RNase ml⁻¹, and 1.67 U of DNase ml⁻¹) and lysed (Soniprep 150 MSE, Sanyo) on ice until clear. The samples were then centrifuged (20,000 × g, 20 min, 4°C) to remove any unlysed cells. Total cell protein was quantified using a 2D Quant kit (GE Healthcare, UK) as per the manufacturer's instructions.

Expression plasmids were transformed into E. coli BL21 (DE3) cells (NEB). For induced protein expression, 50 ml of LB supplemented with 30 μg ml⁻¹ kanamycin was inoculated with 50 μl overnight broth culture and grown at 37°C with 200 rpm shaking. The A₆₀₀ of the cultures was monitored; when the culture reached an A₆₀₀ of ~0.5, IPTG was added to a final concentration of 0.4 mM. Cultures were grown with shaking for a further 3 h (37°C, 200 rpm). Un-induced, and hourly induction samples were collected to monitor protein expression. Cell were harvested by centrifugation (3200 × g, 15 min, 4°C) and lysed by sonication (Soniprep 150 MSE, Sanyo). His-tagged proteins were purified using Ni-NTA Spin Columns (QIAGEN). Columns were equilibrated with NPI-10 (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole), and washed with NPI-20 (NPI + 20 mM imidazole). Protein was eluted in NPI-500 (NPI + 500 mM imidazole). For SDS-PAGE analysis, samples were mixed with 4x LDS buffer (Invitrogen, Life Technologies) with or without β-mercaptoethanol (± reducing conditions) and resolved on 4–20% precast 10 × 10 cm gels (Expedeon, UK) in 1 × MOPS at 120 V. To visualize proteins, gels were rinsed with water and stained in InstantBlue (Expedeon) overnight. Gels were imaged using a GS800 Calibrated Densitometer (BioRad) at 63.5 μm resolution.

Positive E. coli BL21 colonies, containing pGEX6p-2/Cpn 0810, were cultured in Luria-Bertani (LB) solid medium (with ampicillin) at 37°C overnight, after which the culture was transferred to fresh LB liquid medium (with ampicillin). When the optical density reached a wavelength of 600 nm, isopropyl β-D-1-thiogalactopranoside (IPTG) was added with a final concentration of 0.2 mM, and the culture was shaken at 30°C for 4 h. The bacteria were then collected, and phosphate-buffered saline (8 ml/g cells) and lysozyme (4.0 g/l) were added to the cell pellet. Following incubation at room temperature for 2 h, the cells were subjected to sonication (10 sec on, 10 sec off) 30 times using a MSE Soniprep 150 (SANYO, Osaka, Japan). Following centrifugation at 10,000 × g for 20 min at 4°C, the supernatant was purified using a glutathione S-transferase (GST) purification resin column (Novagen; Merck KGaA, Darmstadt, Germany), according to the manufacturer’s instructions. The GST-Cpn 0810 recombinant protein was identified by western blot analysis using a mouse anti-Cpn AR39 primary antibody (1:2,000 dilution; ab190064, Abcam, Cambridge, MA, USA), and the protein concentration was detected using bicinchoninic acid kits (Pik-day Bio Co., Ltd., Beijing, China).