Mbptrap

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.

Each of GII.3 (TCH04-577) and GII.4 Sydney 2012 P-domain sequence was cloned into the expression vector pMal-C2E (New England BioLabs). The recombinant P-domain was expressed with an N-terminal His6-maltose-binding protein (MBP) tag, and a tobacco etch virus (TEV) cleavage site between the MBP and P-domain in E. coli BL21(DE3) and purified using His-Trap (GE Healthcare). The His-MBP tag was then removed using TEV protease and separated from the P-domain by purifying it once again using His-Trap (GE Healthcare), MBPTrap (GE Healthcare) affinity columns, and size exclusion chromatography as previously described^{61 (link)}. The purified P-domain was concentrated and stored in a buffer containing 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, and 2.5 mM MgCl₂. The recombinant M4 was expressed in E. coli WK6 strain. The periplasmic proteins were extracted by osmotic shock using Tris/EDTA/Sucrose (TES) buffer, and His-tagged M4 was purified from the periplasmic extract using a High-Trap HP Ni-chelating column (GE Healthcare, US).