Bl21 de3 rosetta2 plyss cells

Recombinant retromer protein (wildtype or 3KE mutant) was expressed and purified from Escherichia coli as previously described (39 (link)). Briefly, retromer plasmids were transformed into BL21(DE3) Rosetta2 pLysS cells (Millipore). Cells were grown to an absorbance at 600 nm between 0.8 and 1.0 and induced for 16 to 20 h at 22 °C with 0.4 mM IPTG. Cells were lysed by a disruptor (Constant Systems). The protein was purified in 10 mM Tris–HCl (pH 8.0), 200 mM NaCl, 2 mM β-mercaptoethanol using Glutathione Sepharose (GE Healthcare). Protein was cleaved overnight using thrombin (Recothrom; The Medicines Company) at room temperature and batch eluted in buffer. Retromer was further purified by gel filtration on a Superdex S200 16/60 analytical column (GE Healthcare) into 10 mM Tris–HCl (pH 8.0), 200 mM NaCl, or dialyzed into 20 mM Hepes (pH 8.2), 50 mM NaCl, and 2 mM DTT prior to vitrification.