Purification of DcpS Enzyme from E. coli

Human or C. elegans DcpS was expressed
from the respective
pET vectors in the Rosetta 2(DE3) Escherichia coli strain. Protein expression was induced with 0.4 mM IPTG when OD₆₀₀ reached 0.5–0.8, and then, cells were further incubated
at 18 °C overnight. After expression, the bacterial pellet was
collected by centrifugation (7000g, 10 min) and was
frozen at −80 °C. The frozen pellet was thawed on ice
and then resuspended in ice-cold lysis buffer: 50 mM phosphate buffer
pH 7.2, 150 mM NaCl, 1% Triton X-100, 20 mM imidazole. Suspension
was sonicated and then centrifuged for 2 h. The supernatant was loaded
on the HisTrapHP column (GE Healthcare Life Sciences). All separation
by affinity chromatography was done with a gradient of imidazole 20–600
mM in 50 mM phosphate buffer pH 7.2. The DcpS protein was further
purified and buffer exchanged: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol
pH 7.5, by gel filtration (“Superdex 200 10/300GL” GE
Healthcare Life Sciences) using an ÄKTA protein purification
system (GE Healthcare Life Sciences) and stored in −80 °C. A. suum DcpS was prepared according to the procedure
described previously.^{23 (link)}