Expression and purification of the BiP SBD

The BiP SBD was obtained from the ²H, ¹⁵N, Ileδ1-[ ¹³CH₃]-labeled FL BiP using the BiP-linker specific protease SubA. 400 μl of 80 μM BiP was incubated for 4 hr at 30°C with 100 μl of 0.1 mg/ml SubA in the presence of 5 mM ATP. SBD was purified using one-step Ni-NTA affinity purification, dialyzed against the final buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl₂, pH 7.6) and concentrated to 20 μM.

SubA was expressed and purified using a standard protocol from Prof David Ron’s laboratory (University of Cambridge). The protein was expressed in E. coli Origami 2 (DE3) cell (Novagen). A single colony was resuspended in 2 ml LB broth (Fisher Scientific) supplemented with ampicillin and grown overnight at 37°C. The small amount of the overnight culture were transferred into 500 ml LB broth media to reach starting optical density at a wavelength of 600 nm (OD₆₀₀) ~0.1. The culture was incubated at 37°C with shacking until OD₆₀₀ reached ~0.9. Them protein expression was induced with IPTG (final concentration 1 mM) and the culture was grown for another 3 hr. Cells were harvested by centrifugation, resuspended in the binding buffer (50 mM HEPES, 300 mM NaCl, 2 M Urea, 5% glycerol, pH 7.4) and frozen at 80°C until purification step.

Thawed cells were incubated for 30 min on ice with 0.5 ml lysozyme (50 mg/ml, Sigma-Aldrich), 0.1 ml DNaseI (10 mg/ml, Sigma-Aldrich) and Protease Inhibitor Cocktail (Roche). After sonication, lysates were spun down at 20000 rpm for 45 min and filtered to remove cell debris. Supernatants were loaded onto a HisTrap HP nickel column (GE Healthcare) pre-equilibrated with the binding buffer (50 mM HEPES, 300 mM NaCl, 2 M urea, 5% glycerol, pH 7.4). A gradual refolding of the protein was performed on-column using a linear urea gradient from 2M to 0M. Then, the protein was eluted using a linear imidazole gradient from 10 to 500 mM. Finally, the protein sample was dialysed against 50 mM HEPES, 300 mM NaCl, and 5% glycerol (pH 7.4).