Purification of AviTag-20S Proteasome Core

A sequence encoding for an AviTag followed by a 3xFLAG tag was cloned into the C terminus of the endogenous Pre1 gene of a W303 yeast strain. The purification of AviTag-20S core was performed similar to a procedure previously described (56 (link)). Briefly, the yeast strain was grown in yeast extract, peptone, and dextrose at 30°C for 3 days. The cells were pelleted, resuspended in lysis buffer [60 mM Hepes (pH 7.6), 500 mM NaCl, 1 mM EDTA, and 0.2% NP-40], popcorned into liquid nitrogen, and then lysed in a Cryomill 6875D (SPEX SamplePrep). The lysate was allowed to return to room temperature and clarified by centrifugation, and the 20S core particle was purified using anti-Flag M2 affinity resin (Sigma-Aldrich). The elution was concentrated using an Amicon Ultracel-100K, and the concentration was determined by absorbance at 280 nm. After biotinylation through incubation with 25 μM E. coli biotin ligase BirA and 100 μM biotin in the presence of 10 mM ATP and 10 mM MgCl₂ overnight at 4°C, the core was further purified by size exclusion chromatography with a Superose 6 Increase 10/300 column (Cytiva), equilibrated in GF buffer [30 mM Hepes (pH 7.6), 50 mM NaCl, 50 mM KCl, 10 mM MgCl₂, 5% glycerol, and 0.5 mM TCEP]. The extent of biotinylation was determined in a gel shift assay by incubation with NeutrAvidin (Thermo Fisher Scientific).