Q sepharose hp

The gene for XcPrxQ, codon-optimized for expression in E. coli and synthesized by GenScript (Piscataway, NJ), was cloned between the NcoI and HindIII sites of pTHCm(Nelson et al., 2008 (link)) to express a non-His-tagged protein. Mutations were created using the QuikChange II method (Agilent). Expression was carried out in strain B834, using ZYM-5052 auto-induction medium at 37 °C (Studier, 2005 (link)). Purifications were done at 4 °C. Cells were disrupted using an Avestin C5 homogenizer, nucleic acids removed using streptomycin sulfate, and after overnight dialysis against 10 mM Tris-Cl, pH 8.0, 0.5 mM EDTA and centrifugation, the supernatant was loaded onto a 75 mL Q-Sepharose HP (GE Healthcare) column. XcPrxQ was eluted using a 0→1 M NaCl gradient, with XcPrxQ-containing fractions precipitated at 75 % saturating ammonium sulfate. The protein pellet was dissolved in a minimal volume of 25 mM potassium phosphate, pH 7.0, 1 mM EDTA and 0.1 M NaCl, and loaded onto a 250 mL Superose 12PG (GE Healthcare) column equilibrated with the same buffer. For assays, pure protein was buffer-exchanged into 20 mM Tris-Cl, pH 8.0, 1 mM EDTA, and concentrated to 10 mg ml⁻¹ by ultrafiltration, and frozen at −80 °C in aliquots. E. coli thioredoxin 1 (TrxA) and thioredoxin reductase (TrxR) were expressed and purified as previously described (Reeves et al., 2011 (link)).