Amicon ultracel 10k centrifugal filters

His-tagged MdbA or TsdA proteins were purified according to a published procedure with some modification (18 (link)). Briefly, E. coli BL21 (DE3) cells harboring recombinant plasmids were cultured in LB medium supplemented with ampicillin (100 µg/mL) at 37 °C. When cells reached OD₆₀₀ of 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM for overnight induction at 18 °C. Cells were harvested by centrifugation and lysed by sonication, and clear lysates were obtained by centrifugation. Recombinant proteins were purified by affinity chromatography using a Ni-NTA epharose column (Qiagen), followed by an Econo-Pac 10DG column (Bio-Rad) in 100 mM potassium acetate buffer, pH 7.5 and stored at −20 °C for further experimentation.
For crystallization, purified TsdA proteins were digested with 0.15 mg TEV protease per 20 mg of purified protein for 16 h at 4 °C, and then passed through a Ni-NTA column to remove both the TEV protease and cleaved N-terminal tags. The final step of purification was gel-filtration on HiLoad 16/60 Superdex 200 pg column (GE Healthcare) in 10 mM HEPES buffer pH 7.5, 200 mM NaCl and 1 mM DTT. The protein was concentrated on Amicon Ultracel 10K centrifugal filters (Millipore) up to 60 mg/mL concentration.

E. coli BL21(DE3) clpP::cm^r strain, transformed with appropriate plasmids was used to overproduce ΔN-HtrA3 (residues 130–453–His₆), proteolytically inactive ΔN-HtrA3_S305A, ΔN-HtrA3-ΔPDZ (residues His₆–122–349), ΔN-HtrA3-ΔPDZ_S305A, ΔN-HtrA3S (residues 130–357–His₆) and ΔN-HtrA3S_S305A in a pET System (Novagen, San Diego, CA, USA). The proteins were purified by affinity chromatography on Ni-NTA columns according to the manufacturer’s instructions (Qiagen, Germany). The purity of the proteins was estimated to be more than 95% as judged by SDS-polyacrylamide gel electrophoresis. Prior to crystallization, the ΔN-HtrA3_S305A protein was further purified by gel-filtration on a HiLoad 16/60 Superdex 200pg column (GE Healthcare) in 10 mM HEPES buffer pH 7.5, 500 mM NaCl and 1 mM DTT. The protein was concentrated on Amicon Ultracel 10K centrifugal filters (Millipore) up to 16 mg/ml concentration. The concentration of the HtrA3 preparations was estimated by staining with Amido Black as described previously [40 (link)], or with the Bradford method [41 (link)] and, prior to protein crystallization, by using an ND-1000 Spectrophotometer System (Nanodrop Technologies).