Jla8.1 rotor

DNA encoding for the core MBL-β-CASP domains of SNM1B (aa 1/2–335) were cloned for expression via ligation independent cloning (LIC) into baculoviral expression vectors pFB-CT10HF-LIC (GenBank EF199842), except for the D35A/H36A SNM1B mutant, which was cloned in pFB-LIC-Bse (33 (link)). Mutants were generated using site-directed mutagenesis or a megaprimer PCR protocol (34 (link)). The transfer vectors containing the SNM1B constructs were transposed into bacmids by recombination in DH10Bac (Life Technologies) for viral infection (33 (link)). Baculoviruses were generated by transfection and amplification in Sf9 insect cells (24 (link)). SNM1B proteins were produced in 2 L Sf9 cells at a density of 2 × 10⁶ cells/ml infected with 6 ml of P2 virus stock and cultures were grown for 72 h at 27°C before being harvested by centrifugation in a JLA8.1 rotor (Beckmann-Coulter) at 900 x g for 30 min. Cell pellets were resuspended in 50 ml cold lysis buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol (v/v), 1 mM TCEP, supplemented with protease inhibitors (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Roche)).

The protein expression and purification procedure was reported previously^{13 (link),22 (link)} with some modifications. Briefly, the construct corresponding to the first 19-residue deleted (∆19) NaK was cloned into pQE60 vector. Plasmids were transformed into the XL1-Blue Escherichia coli competent cells to allow growth in the presence of 100 μg ml⁻¹ ampicillin at 37 °C. When the OD₆₀₀ = 0.8, protein expression was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25 °C for 20 h. Cells were harvested by spinning at 4000 rpm using a JLA-8.1 rotor (Beckman Coulter). The cell pellet was suspended in 50 mM Tris buffer (pH 8.0), 150 mM NaCl, 1 mM PMSF, 2 μg ml⁻¹ DNase, 10 μg ml⁻¹ lysozyme, and Pierce Protease Inhibitors. After passing through a Microfluidizer (Microfluidics Corporation) at 15,000 psi, the cell lysate was spun at 42,000 rpm using Ti45 rotor in Optima L-90K Ultracentrifuge (Beckman Coulter) for 1 h. The pelleted membrane was re-suspended in 50 mM Tris buffer (pH 8.0) and 150 mM NaCl. Homogenized membranes were kept at −80 °C before use.