Pandemic influenza A/California/04/09 H1N1 PA endonuclease motif (residues 1–204) was expressed and purified as described previously.[22 (link)] BL21 (RIL) cells (Stratagene) were grown to an OD600 of 0.8 and induced with 0.15 mM IPTG at 17 °C for 17 h. Cells were harvested by centrifugation and purified on Ni-NTA (Qiagen) according to manufacturer’s recommendations except the final elution was completed with 500 mM imidazole-containing buffer. The dual hexa-histidine (6xHis) tag was then removed by HRV14 3C protease cleavage. The endonuclease was further purified by size exclusion chromatography using HiLoad 26/60 Superdex 75 (GE Healthcare). The buffer used for size exclusion and the final buffer for storage of the purified protein was 100 mM NaCl and 20 mM Tris pH 8.0. The protein was concentrated to 5 mg/ml using an Ultrafree 10K (Millipore), aliquoted and stored at −80 °C.
Crystals were formed by mixing in a 1:1 ratio the purified endonuclease protein (5 mg/ml) with crystallization buffer containing 200 mM MES pH 6.7, 27% PEG8000, 200 mM ammonium sulfate, 1 mM manganese chloride, 10 mM magnesium acetate, 10 mM taurine, and 50 mM sodium fluoride. Crystals matured to full size in one to two weeks at 20 °C.
Crystals were formed by mixing in a 1:1 ratio the purified endonuclease protein (5 mg/ml) with crystallization buffer containing 200 mM MES pH 6.7, 27% PEG8000, 200 mM ammonium sulfate, 1 mM manganese chloride, 10 mM magnesium acetate, 10 mM taurine, and 50 mM sodium fluoride. Crystals matured to full size in one to two weeks at 20 °C.
