Ni nta agarose resin

As reported before^{59 (link)}, E. coli BL21 (DE3) were transformed with MSP1D1 or MSP1D1∆H5 plasmid DNA in vector pET28a. Cells were grown in lysogeny broth (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 GdnHCl and EDTA-free Complete protease inhibitors (Macherey–Nagel) lysed by sonication (Bandelin Sonopuls MS72 probe), centrifuged at 17,000 × g for 1 h (Beckman J2-21 rotor JA-20.1) and incubated 1 h with previously equilibrated 2.5 ml Ni-NTA agarose resin/3 l culture (Macherey–Nagel). Column was washed with 4 column volumes (CV) buffer B, 4 CV buffer B supplemented with 1% Triton X-100, 4 CV buffer B + 60 mM Na-cholate, 4 CV buffer B, and 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 dialysed against 100-fold 200 mM Tris-HCl pH 7.5, 100 mM NaCl. N-terminal His-tag was cleaved using tobacco etch virus (TEV) protease incubated overnight at 4 °C. ΔHis-MSP was separated by immobilized metal affinity chromatography (IMAC) and concentrated to the desired molarity using a Vivaspin centrifugal device of 10 kDa molecular weight cutoff (MWCO).

The E.coli F₁ ATPase double cysteine mutants were expressed in E.coli BL21-CodonPlus-RP cells (Aligent Technologies) grown in LB medium and purified via affinity chromatography using a Ni-NTA agarose resin (Macherey-Nagel, Düren, Germany) as described previously (Greene & Frasch, 2003 (link)). The presence of the α, β and γ subunits in the purified samples indicates the correct folding and assembly of the F₁ complex.