Hitrap q sepharose

For msfGFP expression, E. coli BL21-CodonPlus(DE3)-RIPL cells were transformed with pSUMO-msfGFP [msfGFP was cloned into homemade pSUMO vector (30 (link))] and cultivated in LB medium with ampicillin at 37°C. The protein was expressed by 0.1 mM IPTG at OD₆₀₀ = 0.4, and the cells were further cultivated for 3 hours. Cell collection, cell disruption, and purification by Ni column were the same as for msfGFP-MinC purification except the NaCl in buffers was 500 mM. After elution, SUMO-tag was digested by homemade Ulp1 (30 (link)) and was removed by using Ni column after 1:20 dilution with 20 mM Hepes-KOH (pH 7.6). msfGFP in the flowthrough of the Ni column was further purified by using HiTrap Q Sepharose (Cytiva) by elution using KCl [20 mM Hepes-KOH (pH 7.6) and 125 mM KCl]. The purified msfGFP was concentrated by ultrafiltration using an AmiconUltra-4 10K to 300 μM. The concentration of msfGFP was quantified by 280 nm. mCherry protein used in this study is purified in our previous study (56 (link)). Expression and purification procedures were similar to those of MinE. After Ni column purification, the elution buffer was exchanged to 20 mM Hepes-KOH (pH 7.6) by using AmiconUltra-4 10K and concentrated into 300 μM. Both proteins were stored at −30°C.

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)).