The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Teoh, Kim-Tat; Siu, Yu-Lam; Chan, Wing-Lim; Schlüter, Marc A.; Liu, Chia-Jen; Peiris, J. S. Malik; Bruzzone, Roberto; Margolis, Benjamin; Nal, Béatrice

For GST-fusion protein production, a single colony of transformed BL21 bacteria was amplified according to standard procedures. Expression of GST-fusion proteins was induced by addition of 1–2 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG; Sigma Aldrich, St. Louis, MO) for 1–2 h at 30°C. Bacteria were harvested and incubated on ice for 30 min in 10 ml lysis buffer (400 mM NaCl, 50 mM Tris-HCl pH7.5, 0.3% Triton X-100) supplemented with 2% N-lauroylsarcosine, 100 μg/ml lysozyme (Roche Diagnostic GmbH), 0.6 mM PMSF (Sigma Aldrich), and 1X Complete Protease Cocktail inhibitor (Roche Diagnostic GmbH). The suspension was sonicated and the lysate was clarified by centrifugation at 12,000 × g for 30 min at 4°C. Subsequently, GST fusion proteins were purified by overnight incubation at 4°C with 200 μl 50% sepharose glutathione 4B bead slurry (Amersham Biosciences, Uppsala, Sweden). On the next day, beads were centrifuged at 500 × g for 5 min at 4°C to remove the unbound fraction and washed (5×) with 800 μl of modified lysis buffer (200 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.15% Triton X-100, 0.6 mM PMSF, 1X Complete Protease Cocktail inhibitor, 1% N-lauroylsarcosine) on a rocking platform for 5 min, and eventually centrifuged at 500 × g, for 1 min at 4°C. Five microliters of each purified GST fusion protein were run on 4–12% acrylamide SDS-PAGE in parallel to serial dilution of bovine serum albumin of known concentration (BSA, Sigma-Aldrich). Gels were stained with Coomassie blue, and the protein concentration, ranging between 0.08–0.40 μg/μl, was estimated in comparison to the band intensity of the BSA control. The purified GST fusion proteins were stored at 4°C for further usage.