Purification of 20S Proteasome and Recombinant FAT10

The 20S proteasome was purified as described previously (Groettrup et al, 1995 (link); Schmidtke et al, 1996 (link); Basler & Groettrup, 2012 ). In brief, cell pellets from LCL721.45 cells were collected and lysed with 100 mM KCl + 0.1% Triton X-100. The suspension was homogenized with a Dounce homogenizer and subsequently centrifuged for 30 min at 30,600g and 4°C. The supernatant was mixed with the anion exchanger DEAE Sephacel (GE Healthcare) and incubated overnight at 4°C while rotating. The next day, the DEAE mix was column purified and eluted with 500 mM KCl. (NH₄)₂SO₄ was added (35% of total volume), and samples were centrifuged twice for 20 min at 17,211g at 4°C. The resulting pellet was resolved in 100 mM KCl and incubated on ice for 60 min. Afterward, the dissolved pellet was added on a sucrose gradient (15–40%) and centrifuged for 16 h at 274,355g at 4°C.
After centrifugation, the sucrose gradient was separated into fractions of 700 μl and tested for proteasomal activity and further purified via a Resource Q column and fast protein liquid chromatography. Successful isolation of the 20S proteasome was confirmed with activity assay and SDS–PAGE.
Recombinant FAT10 was purified from transfected Escherichia coli BL21(DE3) CodonPlus cells (Stratagene) as described before using Ni-affinity chromatography and size-exclusion gel filtration (Aichem et al, 2018 (link)).