PP2A:B55 complex purification

Expi293F cell pellets expressing StrepII–PP2Ac and eGFP–B55 constructs were resuspended in ice-cold lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 1 mM MnCl₂, 0.1% Triton X-100, EDTA-free protease inhibitor tablet (ThermoFisher)), lysed by high-pressure cell homogenization (Avestin Emulsiflex C3). Purified PP2Aa was added to the cell lysate. Cell debris was pelleted by centrifugation (42,000g, 45 min, 4 °C), and the supernatant was filtered with 0.22-µm syringe filters. Lysates were loaded onto a GFP–nanobody-coupled agarose bead (see preparation in ‘eGFP–nanobody protein expression, purification, and immobilization onto agarose beads’ in Methods) column, pre-equilibrated with wash buffer 1 (20 mM Tris pH 8.0, 500 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP) and slowly rocked at 4 °C for 2 h. After 2 h, the flow through (FT1) was collected and the column was washed 3 times with 25 ml of wash buffer (washes 1–3). The GFP–B55 resin was resuspended in 20 mM Tris pH 8.0, 250 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP, and TEV was added for on-column cleavage rocking overnight at 4 °C. The flow through was again collected (FT2) and the resin was washed with 20 ml of wash buffer 2 (20 mM Tris pH 8.0, 250 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP) (wash 4) and 2× 20 ml with the wash buffer 1 (washes 5 and 6). The flow through 2 (FT2) and washes 4–6 were collected, diluted to ~100 mM salt concentration (with 0 mM NaCl Wash buffer), and loaded onto Mono Q column (Cytiva) for further purification. The proteins were eluted with a 100 mM–1 M salt gradient (buffer A: 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP; buffer B: 20 mM Tris pH 8.0, 1 M NaCl, 1 mM MnCl₂ and 0.5 mM TCEP). PP2A:B55 complex and B55 fractions were pooled, concentrated and further purified using SEC (Superdex 200 26/60 (Cytiva)) in NMR buffer (20 mM Na₂HPO₄/NaH₂PO₄ pH 6.3, 150 mM NaCl and 0.5 mM TCEP) or assay buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM MnCl₂ and 0.5 mM TCEP).