Hitrap heparin

eIF2B was purified from yeast strain GP5949, using Flag affinity gel and a high salt buffer containing 1 M KCl to ensure purification away from eIF2 as previously described (Mohammad-Qureshi et al., 2007b (link)). eIF2B with K66 mutated eIF2Bγ variants were similarly purified from strains GP7050 and GP7051. eIF2 was purified by successive chromatography steps of Nickel affinity (Qiagen), HiTrap heparin and HiTrap Q sepharose (Cytiva) from strain GP3511 as described (Jennings et al., 2017 (link)).

The LbCas12a recombinant proteins were produced as previously described [5 (link)]. Briefly, DNA sequences encoding the C′-terminal 6 × His-tagged LbCas12a proteins were cloned into pET28a vector. Transformed E. coli BL21 (DE3) cells (EMD Millipore) were grown in TB medium with 50 μg/mL Kanamycin until OD₆₀₀ reaches 0.6–0.8. Cells were chilled at 4 °C for 30 min, and the protein expression was induced using 1 mM IPTG for 12–16 h at 4 °C. Cells were harvested by centrifugation (4000 × g, 20 min, 4 °C) and resuspended in the lysis buffer (20 mM NaPO₄, pH 6.8, 0.5 M NaCl, 15 mM imidazole, 10 mM CaCl₂, and 10% glycerol) supplemented with protease inhibitor cocktail (Sigma: 11,873,580,001), DNaseI and lysozyme. The resuspended cells were lysed by passing through Avestin Emulsiflex C3 three times at 15,000 psi, 4 °C. The cell lysate was centrifuged at 14,000 × g for 40 min, and the soluble fraction was sequentially purified by Nickle affinity (HisTrap HP, 5 mL, Cytiva) and cation exchange chromatography (HiTrap Heparin, 5 mL, Cytiva). Purified protein was concentrated (Amicon centrifugal filter, 10 kDa) and dialyzed against storage buffer overnight (20 mM TrisHCl, pH 7.4, 0.3 M NaCl, 0.1 mM EDTA, 50% Glycerol, and 1 mM DTT). The protein concentration was determined by Nanodrop using an extinction coefficient at 167,780 M⁻¹ cm⁻¹.