Scab E1 Expression and Purification by Immobilized Metal Ion Affinity Chromatography (IMAC)

ScAb E1 expression and purification by Immobilized Metal Ion Affinity Chromatography (IMAC) and gel filtration (GF)

Briefly, single colonies were selected from LB-Agar supplemented with 25 μg/mL carbenicillin and grown overnight in 5 mL Super Broth (SB) medium supplemented with 25 μg/mL carbenicillin and 1% (w/v) glucose at 37oC, with shaking at 220 rpm. This starter culture was used to inoculate 100 mL SB with 25 μg/mL carbenicillin and was grown to O.D600 = 0.6–0.8 before sub-culturing into 10 x 500 mL SB with 25 μg/mL carbenicillin (2 L flasks). At O.D600 = 0.6–08 the cultures were induced with 0.2 mM IPTG, at 30oC with shaking at 230 rpm overnight.

The bacteria were harvested by centrifugation (3,220 x g) at 4oC for 20 minutes. Soluble scAb was released from the periplasmic space by osmotic shock in a two-step process. The pellet was first thoroughly resuspended in 1X TBS (25 mM Tris, pH 8.0, 150 mM NaCl) and an equal volume of 2X shock buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 1 M sucrose and 2 mM EDTA) was added before incubation at room temperature (RT) for 15 minutes. Shocked cells were recovered by centrifugation at 12,400 x g, at 4oC for 20 minutes, followed by resuspension in ice-cold 5 mM MgSO4 and incubation on ice for 15 minutes.

The periplasmic-stripped cells were collected by centrifugation (27,200 x g at 4oC for 20 minutes) and 0.2 times the volume of 5X binding buffer (125 mM Tris, pH 8.0, 750 mM NaCl, 50 mM imidazole, 0.02% (w/v) NaN3) was added to the supernatant. HisBind (Novagen) resin (1 mL equilibrated in 30 mL 1X binding buffer) was added and scAb recovered by batch binding overnight at 4oC on an end-over-end roller. The resin was collected by gravity flow and washed with 30 mL binding buffer followed by a second wash with 30 mL wash buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 20 mM imidazole, 0.05% (v/v) Tween® 20, 0.02% (w/v) NaN3). Bound protein was eluted with 10 mL elution buffer (1X running buffer with 300 mM Imidazole) in 1 mL fractions.

Eluted protein fractions from the IMAC purification were further purified by chromatography on a size exclusion column. An S75 (16/60) gel filtration column (GE Healthcare) was equilibrated with 25 mM Tris, pH 7.4, 150 mM NaCl, 0.02% (w/v) NaN3 (1 column volume [CV] = 50 mL). Protein-containing fractions of scAb E1 previously purified by IMAC were applied to the column at a flow rate of 1 mL/minute. Fractions were collected after 0.4 CV until 1 CV was reached. Protein was monitored online by A280 using the dedicated Unicorn control and evaluation software (GE Healthcare). Protein peaks were analysed by SDS-PAGE.