Sp ff column

Map1 containing an N-terminal His₈-tag followed by a linker and a HRV 3C cleavage site was expressed from a pET28a vector. E. coli cells were harvested by centrifugation in a SCL6000 rotor (Sorvall) for 10 min at 4,500 rpm and 4°C. After washing with 1× PBS, the pellet was resuspended in cold lysis buffer (50 mM Tris (pH 8.0), 500 mM NaCl, 1 mM PMSF, 1 EDTA-free protease inhibitor cocktail pill/50 ml (Roche)) and subjected to mechanical lysis using a Microfluidizer (Micro Fluidics). Clarified lysate was obtained after spinning for 15 min at 15,000 rpm in a SS34 rotor (Sorvall) at 4°C. After filtering the lysate, it was applied on a HisTrap HP column (GE Healthcare) equilibrated with HT-20 buffer (50 mM Tris (pH 7.5), 500 mM NaCl, 20 mM imidazol, 10 mM β-mercaptoethanol). After elution with HRV 3C protease under high salt conditions, the main fractions were loaded onto a SP FF column (GE Healthcare) equilibrated with 20 mM HEPES at pH 7.0. Elution was performed by a linear salt gradient up to 1 M NaCl, the main fractions showing purified Map1 protein were pooled and the buffer was adjusted to 20 mM HEPES (pH 7.5), 100 mM KOAc, 2.5 mM Mg(OAc)2, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml cycloheximide including a protease inhibitor cocktail tablet (Roche).

Full-length UrCas13d gene was expressed in E. coli Rosetta (DE3) cells (Novagen). After the cells were harvested and lysed in buffer A, the target protein was purified with a Ni-NTA Superflow (QIAGEN) column using buffer A and then with the SP FF column (GE Healthcare) using a salt gradient between buffer B3 (25 mM Tris-HCl (pH 7.5), 250 mM NaCl, 2 mM DTT) and buffer B4 (25 mM Tris-HCl (pH 7.5), 1 M NaCl, 2 mM DTT). Fractions of UrCas13d were dialyzed against buffer B3, concentrated, aliquoted and stored for later pre-crRNA or target RNA cleavage assays.