Baculoviral Expression of SNM1B Mutants

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)).