High trap hp ni chelating column

VHH cDNA of 11 clones that scored positive in phage ELISA for NoV VLPs of Norwalk strain (GI.1) or MD2004 strain (GII.4) were subcloned using the restriction enzymes SfiI and NotI into the expression vector pHEN6 [62 (link)]. Expression of recombinant monovalent VHH was performed as previously described [45 (link)]After the expression the bacteria were pelleted, the periplasmic proteins were extracted by osmotic shock [63 (link)] and the VHH were purified from this periplasmic extract by using a High-Trap HP Ni-chelating column (GE Healthcare, U.S.).
The VHH nucleotide sequences of the obtained VHH clones were aligned by ClustalW with Mega 6.06 and the alignment was edited with BioEdit.

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).