Affinity Purification of Human Proteasomes

Human proteasomes were purified through affinity chromatography on a large scale from a stable HEK293 cell line expressing HTBH (hexahistidine, TEV cleavage site, biotin, and hexahistidine) tagged hRPN11 (a gift from L. Huang, Departments of Physiology and Biophysics and of Developmental and Cell Biology, University of California, Irvine, CA 92697)^{52 (link)}. The cells were homogenized with a Dounce tissue grinder in a lysis buffer (50 mM NaH₂PO₄, pH 7.5, 100 mM NaCl, 10% glycerol, 5 mM MgCl₂, 0.5% NP-40, 5 mM ATP and 1 mM DTT) containing protease inhibitor cocktail (Roche, Germany). The lysates were cleared, and incubated with the NeutrAvidin agarose beads (Thermo Fisher Scientific, MA, USA) overnight at 4 °C. The beads were washed by excess lysis buffer and then by the wash buffer (50 mM Tris-HCl pH 7.5, 1 mM MgCl₂ and 1 mM ATP). Usp14 was removed from the proteasomes using the wash buffer containing 150 mM NaCl for 30 min. The proteasome holoenzymes were eluted from the beads through cleavage by TEV protease (Invitrogen, CA, USA). The doubly capped proteasome was further purified by gel filtration on a Superose 6 10/300 GL column (GE Healthcare, PA, USA) at a flow rate of 0.15 ml/min in the running buffer (30 mM Hepes pH 7.5, 60 mM NaCl, 1 mM MgCl₂, 10% Glycerol, 0.5 mM DTT, 0.8 mM ATP). The gel-filtration fractions were concentrated to about 2 mg/ml. Immediately before cryo-EM sample preparation, the proteasome sample was buffer-exchanged into 50 mM Tris-HCl pH 7.5, 1 mM MgCl₂, 1 mM ATP-γ-S, 0.5 mM TCEP, and 0.005% NP-40.