Spectra por membrane

A preculture of 90 ml of E. coli BL21 harboring the pMSP1D1 plasmid (Addgene #20061) in LB medium containing 2 mM MgSO₄ and kanamycin sulfate (50 μg/ml) was grown at 37°C and stored overnight at 4°C after reaching an OD_600nm (optical density at 600 nm) of 0.7. The preculture was used to inoculate 6 liters of TB buffer with 2 mM MgSO₄ and kanamycin sulfate (50 μg/ml). Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM at an OD_600nm of 2. Cells were harvested after 3 hours of protein overexpression.
The cell pellet (13 g) was resuspended in 100 ml of 40 mM KH₂PO₄/K₂HPO₄ (pH 7.4) with one tablet of cOmplete EDTA-free Protease Inhibitor Cocktail (Roche #05056489001), lysozyme (1 mg/ml), and 1 mg of deoxyribonuclease (DNase) I and passed through a Constant Systems T5 cell disruptor. Centrifugation was followed by addition of 1% (w/v) Triton X-100 and imidazole to a final concentration of 20 mM. MSP1D1 was purified using a 5-ml HisTrap FF column essentially according to Bayburt et al. (48 ). The relevant fractions were pooled, concentrated, and dialyzed against 20 mM tris-HCl, 0.1 M NaCl, and 0.5 mM EDTA (pH 7.4; volume ratio, 1:300) using 6 to 8 MWCO (molecular weight cutoff) Spectra/Por membranes (Thermo Fisher Scientific #132650), yielding 5.2 ml of MSP1D1 (7.6 mg/ml).

A preculture of 50 ml of E. coli BL21 harboring the pMSP1E3D1 plasmid (Addgene #20066) in LB medium containing kanamycin sulfate (50 μg/ml) was grown at 37°C and stored overnight at 4°C after reaching an OD_600nm of 0.7. The preculture was used to inoculate 6 liters of TB buffer with 2 mM MgSO₄ and kanamycin sulfate (50 μg/ml). IPTG was added to a final concentration of 1 mM at an OD_600nm of 2. Cells were harvested after 3 hours of protein overexpression.
The cell pellet (9.4 g) was resuspended in 100 ml of 40 mM KH₂PO₄/K₂HPO₄ (pH 7.4) with one tablet of cOmplete EDTA-free Protease Inhibitor Cocktail (Roche #05056489001), lysozyme (1 mg/ml), and 1 mg of DNAse I and passed through a Constant Systems T5 cell disruptor. Centrifugation was followed by addition of 1% (w/v) Triton X-100 and imidazole to a final concentration of 20 mM. MSP1E3D1 was purified using a 5-ml HisTrap FF column essentially according to Bayburt et al. (48 ). The relevant fractions were pooled, concentrated, and dialyzed against 20 mM tris-HCl, 0.1 M NaCl, and 0.5 mM EDTA (pH 7.4; volume ratio, 1:300) using 6 to 8 MWCO Spectra/Por membranes (Thermo Fisher Scientific #132650), yielding 7 ml of MSP1E3D1 (10.9 mg/ml).