Purification of Fusion Proteins from E. coli

Fusion proteins were expressed in Escherichia coli strain BL21(pLysS) and induced overnight with 0.1 mM isopropyl-β-d-thiogalactoside (IPTG) at 16°C. Cells were harvested and resuspended in buffer containing 500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM DTT and protease inhibitor cocktail (Roche), except for TIRR-His where DTT were omitted. Cells were lysed by sonication and cleared extracts were incubated for 1 h at 4°C with anti-FLAG- (Sigma) or Ni-NTA agarose resin (Life Technologies) to purify FLAG- or His-fusion proteins, respectively. Proteins were eluted in lysis buffer containing 0.4 mg/ml of FLAG peptide (Sigma) (FLAG proteins) or 250 mM imidazole (His proteins) and dialyzed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 10% glycerol. For NMR spectroscopy and ITC measurements, TIRR-His was treated with benzonase (EMD Millipore) after cell lysis and upon binding to Ni-NTA resin, was extensively washed with 50 mM sodium phosphate (pH 7.5), 20 mM imidazole and 1 M NaCl to remove nucleic acids bound to the protein. TIRR was further purified by size-exclusion chromatography using a HiLoad 16/60 Superdex 75 column (GE healthcare) and 50 mM sodium phosphate (pH 7.5), 500 mM NaCl running buffer. 53BP1 was purified as previously reported^{15 (link),38 (link)}.