5 ml ni nta column

Purification was entirely performed at 4 °C. Pellets were homogenized in lysis buffer containing immobilized metal affinity chromatography (IMAC) loading buffer (500 mM NaCl, 30 mM Tris-HCl, 5 mM MgCl2, 20 mM Imidazole, pH 7.5), complete EDTA-free protease inhibitors (Roche) and bovine pancreas DNase I (Roche). Cell lysis was performed using French-press (3 passes, 10,000 psi). The lysate was ultracentrifuged at 31,000 RCF for 1 h (Thermo Fisher Scientific centrifuge), to remove cell debris and suspended particles. The clear supernatant was applied on a 5 mL Ni-NTA column (GE Healthcare) pre-equilibrated with IMAC loading buffer. Bound protein was eluted with a linear gradient of IMAC elution buffer (500 mM NaCl, 30 mM Tris-HCl, 5 mM MgCl₂, 500 mM Imidazole, pH 7.5) using an ÄKTA Purifier system (GE Healthcare). Fractions containing the desired protein were pooled and concentrated to a volume of 5 mL. The concentrated protein was centrifuged at 16,000 × g at 4 °C for 15 min and loaded onto a Superdex 75 gel filtration column (Amersham Biosciences) equilibrated with 30 mM Tris/HCl pH 7.5, 5 mM MgCl₂, 100 mM NaCl, 1 mM DTT. The concentrations of the collected samples were quantified by UV absorption with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and either used freshly (e.g. for crystallization) or stored at −80 °C.

A pET28b plasmid containing the cry5B(1–772) gene was transformed into E. coli BL21(DE3) and the recombinant protein was overexpressed in Luria–Bertani (LB) media supplemented with 50 μg/mL kanamycin and induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20 °C and grown overnight. The harvested cells were lysed by sonication in buffer A (20 mM Tris pH 8.0, 500 mM NaCl, 0.1% phenylmethylsulfonyl fluoride (PMSF) and 0.1% benzydamine) and loaded on to a 5 mL Ni-NTA column (GE Healthcare), which was then washed with buffer containing a 0 to 500 mM imidazole gradient. The recombinant Cry5B(1–772) protein was eluted at ~150 mM imidazole and further purified by size-exclusion chromatography (GE Healthcare) with buffer B (20 mM HEPES pH 8.0, 50 mM NaCl). The fractions were analyzed by SDS-PAGE and checked by mass spectrometry-based protein identification.

Wet cells were resuspended in buffer A (100 mM NaCl, 100 mM HEPES, 20 mM imidazole, pH 7.5) (Lichman et al. 2017b (link)), and cell suspensions were lysed in an ice bath using an JY92-II ultrasonic oscillator (Scientz Biotech Co.) with 4 s pulses and 6 s pauses at 20% amplitude for 15 min. Cell lysates were centrifuged (12,000 rpm, 1 h, 4 °C) to obtain clarified supernatants. The filtered supernatant was passed through a 5 mL Ni–NTA column (GE Healthcare, America) previously equilibrated with deionised water and buffer A (1.5-fold column volumes). Bound TfNCS protein and its mutants were eluted with 70% buffer B (100 mM NaCl, 100 mM HEPES, 500 mM imidazole, pH 7.5) and collected in 10 kDa ultrafiltration tubes. The eluent buffer containing pure enzyme was exchanged for assay buffer (50 mM NaCl, 20 mM Tris, pH 7.5). The protein concentration of purified enzyme was determined by a Nanodrop 2000c spectrophotometer (Thermo Scientific, America) at 280 nm, and the extinction coefficient was calculated using the ExPaSy ProtParam Tool (Gasteiger et al. 2005 ).