Hitrap q xl column

Following expression of human ncOGT (full length, 1–1046) in E.coli, cells were resuspended in a buffer containing 25 mM imidazole, 10 % glycerol, 250 mM NaCl, and 25 mM Hepes, pH 7.5, 5 mM β-mercaptoethanol. DNaseI and an EDTA-free protease inhibitor tablet (Sigma) were also added to the lysis buffer. Following lysis by sonication and French press, the protein was purified by nickel affinity chromatography and eluted in the same buffer with 250 mM instead of 25 mM imidazole. Fractions containing pure protein were then dialyzed in 25 mM Hepes, pH 7.5, 40 mM NaCl, 0.5 mM EDTA, and 5 mM β-mercaptoethanol and then loaded onto a 5 ml HiTrap Q-XL column (Cytiva) and purified at 4 °C using a gradient from 0.05 to 1.0 M NaCl. Although the protein appeared pure after this anion exchange step, we further purified the protein using a HiLoad Superdex 200 HR16/600 size exclusion column using a buffer containing 40 mM KPO₄, pH 7.5, 125 mM NaCl, 0.5 mM EDTA, 0.5 mM benzamidine, and 5 mM β-mercaptoethanol to ensure that no contaminating proteases remained.

To capture GbpA from the culture
media of Vmax, the media were first dialyzed overnight at 4 °C
against 20 mM Tris-HCl pH 8.0, 100 mM NaCl (volume ratio 1:20 sample:buffer)
using 10K MWCO SnakeSkin dialysis tubing (ThermoScientific). Dialyzed
supernatant was subsequently loaded onto an equilibrated 5 mL HiTrap
Q XL column (Cytiva) for anion-exchange chromatography (AEX). For
the protein produced in E. coli, the fractions resulting
from the osmotic shock were directly loaded onto the AEX column. After
a washing step with 20 CV binding buffer, the protein was eluted over
a 12 CV 0–100% linear gradient with elution buffer (20 mM Tris-HCl
pH 8.0, 400 mM NaCl). Fractions were analyzed by SDS-PAGE and those
containing the target protein were pooled and concentrated with Amicon
Ultra Centrifugal Filter Units 10K MWCO (Merck). GbpA was further
purified by SEC using a Superdex 200 Increase 30/100 GL column (Cytiva)
equilibrated with 20 mM Tris-HCl pH 8.0, 100 mM NaCl. For perdeuterated
GbpA, a Superdex 75 Increase 30/100 GL column (Cytiva) equilibrated
in the same buffer was used instead.