His spintrap column

The barnase constructs in the pTriEx based vectors were expressed in T7 Express E. coli (NEB) using ampicillin as the selection antibiotic. 10 mL of an overnight starter culture (grown in 2 × YT at 37 °C in a shaking incubator), was inoculated in 100 mL of 2 × YT and grown to an OD_600 nm of 0.5 AU at 37 °C in a shaking incubator. The culture was cooled to 18 °C and expression was induced with 0.4 mM Isopropyl β-D-1-thiogalactopyranoside with cells grown overnight at 18 °C in a shaking incubator. Cells were pelleted (5000 × g; 4 °C) and resuspended in phosphate buffered saline (PBS) supplemented with Complete EDTA-free protease inhibitor cocktail (Roche). Hen egg white lysozyme was added to a concentration of 1 mg mL⁻¹ and the lysate was frozen at −20 °C. The lysate was thawed, Benzonase nuclease was added according to the manufacturer’s instructions (EMD-Millipore). The debris was pelleted and removed by centrifugation (16,000 × g; 15 min; 4 °C). Imidazole was added to the supernatant to a concentration of 20 mM and the solution was applied to a His SpinTrap column (GE Life Sciences) pre-equilibrated in binding buffer (PBS and 20 mM imidazole). Proteins were purified as per the manufacturer’s directions, using PBS and 200 mM imidazole as the elution buffer. Proteins were used immediately.

Selected colonies on the M9 plates were inoculated in LB medium and cultivated overnight at 37 °C and 200 rpm. Plasmids were extracted and transformed into E. coli BL21 (DE3) using electroporation (2.5 kV, 25 µF, 200 Ω). On the next day, colonies were incubated in LB medium at 37 °C and 200 rpm and used for inoculation of 50 mL fresh LB medium in 300 mL baffled Erlenmeyer flasks and cultivated at 37 °C and 200 rpm. When OD₆₀₀ reached 0.5, 1 mM IPTG was added to induce protein expression. After cultivation overnight, cells were centrifuged and the cell pellet was resuspended in 1 mL lysis buffer (500 mM NaCl, 20 mM imidazole, 20 mM NaH₂PO₄, pH 7.4). Lysis of the cells was achieved by using three rounds (3 × 45 sec, at a speed of 6.0 m/sec) of FastPrep homogenization (MP Biomedicals™, USA). After centrifugation, the target enzyme was purified from the cell extract via His-tag purification by using a His SpinTrap column (GE Healthcare, USA) with an elution buffer (300 mM NaCl, 250 mM imidazole, 50 mM NaH₂PO₄, pH 8). Finally, the enzyme buffer was changed to 100 mM HEPES buffer (pH 7.5) using an Amicon® Ultra 0.5 mL Centrifugal Filter (Merck, Germany).

First, the reaction mixture (60 μL) was incubated with 20 μL of Flag M2 affinity gel. After incubation at room temperature for 15 min, the column was washed three times with wash buffer (20 mM phosphate, 0.5 M NaCl, 0.1% polyoxyethylene(23)lauryl ether, pH 7.4). The bound proteins were subsequently eluted with two 200-μL volumes of wash buffer containing 100 μg/mL of Flag peptide. The eluted proteins were then applied to a His Spin Trap Column (GE Healthcare, Piscataway, NJ, USA). After incubation at room temperature for 15 min, the column was washed three times with wash buffer containing 60 mM imidazole. The bound proteins were then eluted with two 200-μL volumes of wash buffer containing 0.5 M imidazole. The eluate was subsequently passed through an UltraFree-0.5 centrifugal device (Millipore, Billerica, MA) and equilibrated with phosphate buffered saline supplemented with Tween-20 (PBST, 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween-20, pH 7.4) to concentrate the protein in the buffer. The concentration of the labeled Fab protein was determined by comparing the fluorescence intensities of a known concentration of free TAMRA- (Anaspec) and the sample under denaturing conditions in 7 M GdnHCl, 100 mM DTT, pH 7.4.