Purification of RAD51AP1-N88 Protein

The purification procedures for full-length human RAD51AP1, the RAD51AP1 fragments and mutants, and for human RAD54, RAD51 and NAP1 have been described elsewhere (5 (link), 6 (link), 11 (link), 24 (link), 41 (link), 42 ). Purified human histone octamer was purchased from The Histone Source at Colorado State University (https://www.histonesource.com/) and purified as described (43 (link)).
For purification of the RAD51AP1-N88 protein, E. coli Rosetta cells harboring the plasmid were grown and induced for protein expression as described earlier (42 ). RAD51AP1-N88 was purified by tandem purification using Ni-NTA resin (Thermo Scientific) first, as described (42 ). Eluted protein was incubated with pre-equilibrated Strep-Tactin Superflow Plus (Qiagen) and gentle rotation at 4 °C for 1 h in binding buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM DTT. The resin was washed 4 × in three bed volumes of binding buffer, bound protein was eluted with binding buffer containing 2.5 mM d-Desthiobiotin (Millipore) and dialyzed into dialysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.05% Triton X-100, 2 mM DTT and 20% glycerol) at 4 °C overnight before snap-freezing, and stored at –80 °C.