Complete protease inhibitors

As reported in Bayburt et al. (51 (link)), E. coli BL21 (DE3) were transformed with MSP1D1 plasmid DNA in vector pET28a. Cells were grown in LB medium, induced by 1 mM IPTG at an optical density of 0.7 and incubated 5–6 h at 37°C, then pelleted down. Cells were resuspended in 50 mM Tris-HCl pH 8, 500 mM NaCl (buffer B) supplemented with 6 M Gdn-HCl and EDTA-free Complete protease inhibitors (Macherey-Nagel, Düren, Germany) lysed by sonication (Sonopuls MS72 probe; Bandelin, Berlin, Germany), centrifuged at 17,000 × g for 1 h (Cat. No. J2-21 rotor JA-20.1; Beckman Coulter, Brea, CA) and incubated 1 h with previously equilibrated 2.5 mL Ni-NTA agarose resin/3L culture (Macherey-Nagel). The column was washed subsequently with 4 CV buffer B; 4 CV buffer B supplemented with 1% Triton X-100; 4 CV buffer B + 60 mM Na-cholate; and 4 CV buffer B, 4 CV buffer B + 20 mM imidazole. Four fractions of 1 CV were eluted with 250 mM imidazole. The whole process was kept at 4°C in a cold room. The elution fractions were pooled and dialyzed against 100-fold 200 mM Tris-HCl pH 7.5, 100 mM NaCl. N-terminal His-tag was cleaved using TEV protease incubated overnight at 4°C. ΔHis MSP was separated from MSP by immobilized metal ion affinity chromatography and concentrated to the desired molarity using a Vivaspin centrifugal device (Vivaproducts, Littleton, MA) of 10 kDa MWCO.