Protino ni nta agarose resin

For overexpression of fre, a protocol based on Zeng et al.83 (link) was used. The transformant (BL21-CodonPlus(DE3)-RP-pET28a-fre) was grown in LB medium supplemented with 25 μg ml⁻¹ chloramphenicol and 35 μg ml⁻¹ kanamycin at 30 °C and 200 r.p.m. orbital shaking to an OD₆₀₀ of 0.4–0.6. Subsequently, cultures were induced with IPTG at a final concentration of 0.2 mM and incubated at 20 °C for 16 h. After collecting of cells, the pellet was resuspended in cold Fre lysis buffer (up to 50 ml for 1.6 l culture; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT) and lysed by means of Sonopuls HD 2,200 (BANDELIN). Separated soluble fraction was subjected to 3 ml of Protino Ni-NTA agarose resin (Macherey-Nagel GmbH & Co. KG), and the mixture was shaken at 4 °C for 30 min before it was loaded into a gravity-flow column. Fre was eluted with 25–250 mM imidazole in Fre storage buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 2 mM DTT). Fractions containing Fre protein were concentrated using Amicon Ultra-15 Centrifugal Filter Units (MWCO 3,000 Da) (Merck KGaA) and dialysed with Fre storage buffer at 22 °C. The purity of His-tagged Fre (28.4 kDa) was checked on SDS–PAGE (Supplementary Fig. 1) and confirmed using in-gel tryptic digestion with MALDI-TOF-MS(MS). For long-term storage, protein was frozen at −80 °C in 50% glycerol.