Urea → L-Pyrrolysine

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coli BL21(DE3) cells were co-transformed with pAra-pylSTBCD and the respective mutant mEGF Tyr10TAG and Tyr29TAG genes on a pET22b plasmid vector. Both constructs were expressed in the presence of 5 mM D-ornithine in TB medium at 37° C. with addition of 1 mM IPTG and 0.2% (w/v) arabinose when the OD600 reached 0.5. The temperature was then reduced to 30° C., and cells were harvested 16 hours after induction. The cell pellet was resuspended in 20 mL of 20 mM Tris/HCl (pH 8.5) and sonicated for 5 minutes. After centrifugation at 30,000×g for 20 minutes, the supernatant was discarded. The pellet was resuspended by sonication into 20 mL of 20 mM Tris/HCl (pH 8.5) containing 2% (v/v) Triton-X100. After another round of centrifugation at 30,000×g, for 20 minutes, the pellet was solubilized by sonication into 10 mL of 8 M urea, 20 mM Tris/HCl, 10 mM β-mercaptoethanol, pH 8.5. Insoluble cell debris was removed by centrifugation (30,000×g for 20 minutes) and the supernatant was diluted 2-fold with refolding buffer (100 mM Tris/HCl, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 20% (v/v) ethanol, pH 8.5). The diluted sample was then dialyzed against refolding buffer overnight at 4° C. by using a slide-a-lyzer dialysis cassette (3500 Da molecular weight cut off, Pierce). Insoluble protein was removed by centrifugation at 30,000×g for 20 minutes, and the supernatant was supplemented with β-mercaptoethanol to a final concentration of 2 mM. 1 ml of 50% Ni-NTA slurry (Qiagen) was added to the refolded protein and gently mixed by shaking at 4° C. for 60 minutes. The protein-Ni-NTA mixture was loaded into a column and the flow-though was collected. After washing the resin with 20 mL of 25 mM imidazole in PBS (pH 8.0), the protein was eluted with 2.5 ml of 250 mM imidazole in PBS (pH 8.0). Finally, the protein was buffer-exchanged into PBS (pH 7.4) by using a PD-10 column (GE Healthcare). The purity of the protein preparation was investigated by SDS-PAGE (FIG. 21B). In FIG. 21B, Lane 1 is the molecular weight standard SeeBlue Plus2 Pre-Stained Standard and Lane 2, mEGF Tyr10TAG mutant protein after Ni-NTA purification.

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