Nickel complexed chelating sepharose

Complex IV was accidentally copurified from mitochondrial membranes of N. crassa (Künkele et al., 1998 ▸ ; Ahting et al., 1999 ▸ ) by immobilized metal-affinity chromatography (IMAC) without introducing an affinity tag, most likely on account of the internal histidine-rich region in the Cox4 subunit (Supplementary Fig. S3). Isolated N. crassa mitochondria (2 g) were solubilized in 20% glycerol, 20 mM Tris pH 8.5, 300 mM NaCl, 20 mM imidazole, 1%(w/v) DDM and four tablets of cOmplete (EDTA-free) protease inhibitor (Roche) for 1 h at 4°C. The solution was centrifuged at 150 000g for 40 min at 4°C and the resulting supernatant was incubated with 10 ml Nickel-complexed Chelating Sepharose (GE Healthcare) for 30 min. The resin was washed with 10% glycerol, 50 mM HEPES pH 7.2, 0.1%(w/v) DDM, 1 mM PMSF. The protein was eluted with 300 mM imidazole in the same buffer. The protein concentration of the yellow protein fraction was 1.9 mg ml⁻¹. A 5 ml volume was used for reconstitution into nanodiscs with the membrane-scaffold protein MSP2N2.

The gene for MSP2N2 was cloned into a pET-28a vector with an N-terminal His tag and a TEV cleavage site. The protein was expressed in Escherichia coli BL21(DE3) cells for 3 h in TB medium at 37°C after induction with IPTG. The cells were disrupted in 50 mM Tris pH 8, 150 mM NaCl, 1 mM PMSF, 1% Triton X-100. After high-speed centrifugation, the supernatant was incubated with Nickel-complexed Chelating Sepharose (GE Healthcare) and washed in three steps with 40 mM Tris pH 8, 300 mM NaCl, 1% Triton X-100, then with 40 mM Tris pH 8, 300 mM NaCl, 50 mM sodium cholate, 20 mM imidazole and finally with 40 mM Tris pH 8, 300 mM NaCl, 40 mM imidazole. MSP2N2 was purified by IMAC using the N-terminal His tag. The protein was eluted with 40 mM Tris pH 8, 300 mM NaCl, 400 mM imidazole. After dialysis against 20 mM Tris pH 8, 100 mM NaCl overnight, the His tag was cleaved off using TEV protease and separated from the cleaved MSP2N2 by a second IMAC step (His-Select, Sigma) using the unbound flowthrough.