Expression and Purification of SNAP-His6 Constructs

Plasmids encoding each His-tagged SNAP-tag conjugate were individually used to transform E. coli BL21(DE3) cells. Individual colonies were selected on the basis of Ampicillin (Amp) resistance and used to inoculate 5 mL of lysogeny broth (LB) media supplemented with Amp (100 mg/L). The primary cultures were used to inoculate 1 L of LB medium supplemented with Amp, which was then allowed to grow at 37 °C with shaking at 200 rpm. When the OD₆₀₀ reached 0.6–0.8, the culture was cooled to 18 °C. Protein expression was induced by the addition of IPTG to a final concentration of 1 mM. After 16 h, the cells were harvested by centrifugation and lysed by sonication in 20 mM Tris pH 8.0, 150 mM NaCl, and 10% glycerol, supplemented with one cOmplete, Mini EDTA-free protease inhibitor cocktail tablet. The cleared lysate was obtained by centrifugation at 15000g for 30 min. Next, the cleared lysate was incubated with 2 mL of Ni-NTA resin for 1 h at 4 °C. After the resin/lysate mixture was transferred to a column, the resin was washed with high-salt wash buffer (20 mM Tris pH 8.0, 1 M NaCl, 30 mM imidazole, and 10% glycerol, 2 × 20 mL) followed by a low-salt wash (20 mM Tris pH 8.0, 150 mM NaCl, and 10% glycerol, 2 × 20 mL). The proteins were eluted in eight 1 mL portions of 20 mM Tris pH 8.0, 150 mM NaCl, 250 mM imidazole, and 10% glycerol. Elution fractions were analyzed by SDS-PAGE, combined, and dialyzed into 20 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT, and 10% glycerol overnight at 4 °C. ZiF-SNAP-His₆ and ZF5.3-SNAP-His₆ were dialyzed into the same buffer supplemented with 100 μM of ZnCl₂. Following dialysis, each protein was analyzed by mass spectrometry. We observed that a portion of the expressed ZF5.3-SNAP-His₆ contained an oxidative modification corresponding a mass increase of 80 Da. Purified ZF5.3-SNAP-His₆ was buffer exchanged into Zn-free SNAP-tag buffer (20 mM Tris pH 8.0, 150 mM NaCl, containing 10% glycerol). Bond-breaker tris(2-carboxyethyl) (TCEP) solution was added to 3 mL of 50 μM ZF5.3-SNAP-His₆ to a final concentration of 50 mM. The protein solution containing the added reducing agent was incubated overnight at 37 °C. Removal of the 80 Da adduct was confirmed by mass spectrometry. After removal of the 80 Da adduct, TCEP was removed from the reaction mixture using a PD-10 column. The protein was then redialyzed into SNAP-tag buffer supplemented with 100 μM ZnCl₂ overnight at 4 °C. Protein concentrations were assessed using the Pierce 660 nm protein assay and stored at −80 °C until further use.