Bl21 de3

For the generation of cell lysates, E. coli strain BL21(DE3) (Agilent) was grown in MMB (LB supplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂) at 37°C. Whenever applicable, media were supplemented with chloramphenicol (30 μg mL⁻¹) or kanamycin (50 μg mL⁻¹), to ensure the maintenance of plasmids. For protein purification, E. coli strain BL21(DE3) (Agilent) was grown in 2YT media (1.6% Bacto-tryptone, 1% yeast extract, 0.5% NaCl) at 37°C in the presence of ampicillin (100 μg mL⁻¹) to ensure maintenance of plasmids.

E. coli BL21(DE3) (Agilent Technologies) carrying pGGE6 derivatives were grown in TB (terrific broth) medium [45 ]. Expression was induced at an OD₆₀₀ of 0.6 to 0.9 in presence of 0.5 μM IPTG for 18 h at 16°C. Cells were harvested by centrifugation, resuspended in ice-cold lysis buffer (50 mM Tris/HCl, 10 mM NaCl, 10 mM imidazol, 0.1% Tween20, pH 8.0), supplemented with protease inhibitors (Roche), and lysed by three freeze-thaw cycles. Protein extracts were separated by Ni-NTA sepharose column (QIAGEN), equilibrated with lysis buffer. After washing with lysis and wash buffer (50 mM Tris, 10 mM NaCl, 40 mM imidazol, 0.1% Tween20, pH 8.0), respectively, His₆-tagged proteins were eluted with elution buffer (50 mM Tris, 10 mM NaCl, 250 mM imidazol, 0.1% Tween20, pH 8.0) at 4°C.
For protein purification under reducing conditions, buffers were supplemented with 1 mM Tris (2-carboxyethyl) phosphine (TCEP). Elution fractions containing enriched protein were pooled and dialysed 2× 5 h with 200× volumes of storage buffer (50 mM Tris, 10 mM NaCl, 10% glycerol, pH 8.0). Protein concentrations were determined at 280 nm using the molar absorption coefficient calculated according to Pace et al. [46 (link)], or by Bradford assay (BioRad, Hercules, USA). Protein aliquots were frozen in liquid nitrogen and stored at -80°C.

F. trinervia and F. pringlei PEPC were cloned, expressed and purified as described previously4 (link). Briefly, the full-length ppcA gene of PEPC from F. trinervia (EMBL-Bank X61304) and F. pringlei (EMBL-Bank Z48966) were cloned in pETEV16b (Novagene). The proteins were expressed in Escherichia coli strain BL21(DE3) (F. trinervia) or BL21-Gold(DE3) (F. pringlei) (Agilent Technologies) at 16 °C upon induction with 0.5 mM isopropyl-β-D-thiogalactoside. The proteins were purified by affinity chromatography with nickel column and changed to a final buffer containing 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 10 mM MgCl₂ using PD10 columns (GE Healthcare).