Bl21 de3 ril cells

The scATE1 was cloned into a pGEX 6p-1 vector with an N-terminal GST-tag and PreScission cleavage site and expressed in BL21(DE3)-RIL cells (Agilent). This RIL strain contains a rare codon plasmid encoding extra copies of argU, ileY, and leuW and overexpresses tRNA^Arg. Protein production was induced with 0.4 mM IPTG overnight at 16 °C in Luria broth (LB). The GST-tagged scATE1 proteins were purified on glutathione Sepharose 4B beads (GE Healthcare) in 50 mM Tris (pH 7.5), 0.5 M NaCl, 1 mM MgCl₂, and 5 mM DTT. The GST tag was cleaved with PreScission protease overnight at 4 °C. The pET21a ΔNHsArgRS plasmid with an N-terminal His-tag is a generous gift from Aaron Smith lab at University of Maryland. The His-tagged proteins were purified on Ni-NTA beads (Qiagen) in 50 mM Tris-HCl (pH 7.5) buffer, supplemented with 0.5 M NaCl, 5 mM β-ME, and 10 mM Imidazole. The His-tag was cleaved overnight at 4 °C with TEV protease. Proteins were further purified by size exclusion chromatography and concentrated in Millipore concentrators. All mutants were generated by site-directed mutagenesis using the Q5 polymerase mutagenesis protocol, grown, and purified as WT proteins.

Skd3 variants were expressed as an N-terminally MBP-tagged protein in BL21 (DE3) RIL cells (Agilent). Cells were lysed via sonication in 40 mM HEPES-KOH pH = 7.4, 500 mM KCl, 20% (w/v) glycerol, 5 mM ATP, 10 mM MgCl₂, 2 mM β-mercaptoethanol, 2.5 µM PepstatinA, and cOmplete Protease Inhibitor Cocktail (one tablet/250 mL, Millipore Sigma). Lysates were centrifuged at 30,597xg and 4°C for 20 min and the supernatant was applied to amylose resin (NEB). The column was washed with 15 column volumes (CV) of wash buffer (WB: 40 mM HEPES-KOH pH = 7.4, 500 mM KCl, 20% (w/v) glycerol, 5 mM ATP, 10 mM MgCl₂, 2 mM β-mercaptoethanol, 2.5 µM PepstatinA, and cOmplete Protease Inhibitor Cocktail) at 4°C, 3 CV of WB supplemented with 20 mM ATP at 25°C for 30 min, and 15 CV of WB at 4°C. The protein was then exchanged into elution buffer (EB: 50 mM Tris-HCl pH = 8.0, 300 mM KCl, 10% glycerol, 5 mM ATP, 10 mM MgCl₂, and 2 mM β-mercaptoethanol) with 8 CV and eluted via TEV cleavage at 34°C. The protein was then run over a size exclusion column (GE Healthcare HiPrep 26/60 Sephacryl S-300 HR) in sizing buffer (50 mM Tris-HCl pH = 8.0, 500 mM KCl, 10% glycerol, 1 mM ATP, 10 mM MgCl₂, and 1 mM DTT). Peak fractions were collected, concentrated to >5 mg/mL, supplemented with 5 mM ATP, and snap frozen. Protein purity was determined to be >95% by SDS-PAGE and Coomassie staining.