Hiload 26 60 superdex 75

The pET vectors for over-expression of the full- length mammalian CaM (CaM_1–148) and the corresponding domain fragments (CaM_1–80 and CaM_76–148) were described previously.[29 (link)–32 (link)] Recombinant CaM proteins were purified on phenyl sepharose CL-4B columns.[33 (link)] To precipitate contaminating proteins with a lower thermal stability than calcium-saturated CaM, eluate was incubated at 80 °C for 10 min in the presence of 10 mM CaCl₂ (total) before it was purified on a phenyl sepharose column pre-equilibrated with CaCl₂-containing buffer. In some instances, the proteins required further purification via DEAE-Sephacel anion exchange and/or HiLoad 26/60 Superdex 75 (GE Healthcare) chromatography. The purified recombinant proteins were shown to be 95–99% pure, as judged by reversed-phase HPLC. Protein concentrations were determined by UV absorbance in the presence of 0.1 N NaOH, using published extinction coefficients for Phe and Tyr[34 (link)] (CaM does not contain Trp).

The DNA encoding human Zα_DLM-1 (6–74) was cloned into an NESG-modified pET15 expression vector derivative with a short N-terminal hexaHis affinity purification tag (MGHHHHHHSH) (Acton et al. 2005 (link)). Following NESG standard protocols (Acton et al. 2011 (link)), Zα_DLM-1 was expressed and purified to prepare U-¹³C, ¹⁵N (NC) and U-¹⁵N, 5% biosynthetically-directed ¹³C (NC5) samples. NMR samples were purified using an ÄKTAxpress (GE Healthcare) with a Ni-affinity column (HisTrap HP IMAC column, 5 ml) followed by gel filtration chromatography (HiLoad 26/60 Superdex 75). The protein was concentrated to 1.0 mM in the NMR buffer containing 10% v/v D₂O: 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), 100 mM NaCl, 5 mM CaCl₂, 10 mM DTT, 50 μM DSS, and 0.02% NaN₃ at pH 6.5. A D₂O-exchanged sample was prepared for HD exchange experiments by freezing the NC sample followed by lyophilization and resuspension in 99.9% D₂O (Acros Organics).

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.