4.13. Recombinant protein expression and purification

E. coli cells harboring recombinant UGTs were grown at 37°C in LB medium + 50 mg/l carbenicillin to OD = .8 and induced with 1 mM IPTG (isopropyl β‐D‐1‐thiogalactopyranoside). The cultures were placed at 18–20°C for 20 h, then centrifuged and the pellets placed at −20°C overnight. The bacterial pellets were thawed on ice and resuspended in PBS pH 7.4 and freeze/thawed three times. The cells were sonicated for 15 min on ice and centrifuged. The soluble protein sonicate was transferred to GE Sepharose 4B glutathione agarose beads and incubated on and end‐over‐end rotator for 2 h at 4 °C. Following incubation the beads were washed three times with PBS. The protein was eluted three times with 50 mM glutathione and 50 mM Tris pH 8. Eluted proteins were pooled and concentrated on an Amicon Ultra‐4 centrifugal filter. The concentrated eluted protein was aliquoted and stored at −80°C.