Ni histrap column

The filtered lysate (see Experimental Model and Subject Details: Bacterial Culture) was loaded onto a 5 mL Ni HisTrap column (GE Healthcare; Chicago, IL). The protein was eluted with a linear imidazole gradient (5 – 250 mM imidazole), concentrated and loaded onto a HiLoad 16/60 Superdex 200 size exclusion chromatography column (GE Healthcare) that had been equilibrated with 150 mM NaCl, 50 mM Na₂HPO₄/NaH₂PO₄ (pH 7.4). Fractions containing 25QP-GFP were pooled and dialysed overnight with TEV protease to remove the tag. The tag was separated from 25QP-GFP by Ni affinity chromatography with a 5 mL Ni HisTrap column (GE Healthcare). Pure, concentrated 25QP-GFP was flash frozen in liquid nitrogen and stored at −80°C until use. 43QP-GFP was purified in the same way as 25QP-GFP except that an MBPTrap (GE Healthcare) chromatography step was added at the end to ensure complete removal of the cleaved tag.