Culture supernatants were collected and TREM2 (a.a 19-131) was purified by affinity chromatography (Ni-NTA, Qiagen) in buffers containing 20 mM Tris-HCl pH 8, 500 mM NaCl, 5% glycerol followed by size exclusion chromatography (Superdex 75 10/30GL, GE Healthcare). The purified protein was deglycosylated with EndoH (PE019, Novoprotein) in 50 mM sodium citrate pH 5.5 overnight at 4°C, at the ratio of 1 mg protein: 2000 U EndoH. EndoH was removed using a second Ni-NTA affinity column, and the purified protein was stored in buffer containing 50 mM Tris pH 8.5, 200 mM NaCl, and 5% glycerol at 10 mg/mL concentration.
The longer TREM2 (a.a. 19-174) for crystallisation was expressed and purified similarly but transfection was performed with DNA to L-PEI 1:3 ratio and cells were grown in 2 L roller bottles (Greiner Bio-one). Affinity chromatography was performed in base buffer: 20 mM HEPES 7.4, 300 mM NaCl, 5% glycerol and proteins were immediately deglycosylated in solution adjusted with sodium citrate (final buffer pH 6). Size exclusion was performed in 20 mM HEPES 7.4, 150 mM NaCl, 5% glycerol on Superdex 200 16/60 (GE Healthcare).
The longer TREM2 (a.a. 19-174) for crystallisation was expressed and purified similarly but transfection was performed with DNA to L-PEI 1:3 ratio and cells were grown in 2 L roller bottles (Greiner Bio-one). Affinity chromatography was performed in base buffer: 20 mM HEPES 7.4, 300 mM NaCl, 5% glycerol and proteins were immediately deglycosylated in solution adjusted with sodium citrate (final buffer pH 6). Size exclusion was performed in 20 mM HEPES 7.4, 150 mM NaCl, 5% glycerol on Superdex 200 16/60 (GE Healthcare).