TEV protease cleavage

For TEV protease cleavage of fusion tags after IMAC chromatography, 1 mg of His₆-tagged TEV protease from in-house stock was added to every 45 mg of protein of a size of 68 kDa. The mixture was dialyzed against 5 l of TEV dialysis buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 1 mM MgCl₂, 1 mM ß-Me for all CbFic2 versions; 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM ß-Me for TS-H3 peptides) at 4 °C oN in dialysis tubing with a molecular weight cut-off (MWCO) of 12,000–14,000 Da (CbFic2 versions) or MWCO of 6000–8000 Da (TS-H3 peptides) and 29 mm diameter (Serva Electrophoresis). Afterward, the protein solution was submitted a second time to IMAC. Unlike the first run, no imidazole was added to the protein before loading, and the protein eluted in the flow-through or 40 mM imidazole wash step due to the lack of His tag, while the cleaved off fusion tag and the TEV protease bound to the column. Protein-containing fractions were analyzed by SDS PAGE, combined, and concentrated for injection onto preparative size exclusion chromatography. For H3 peptides from pGATEV, concentrated GST-H3 peptides were cleaved by TEV in a 1.5 ml reaction tube oN at 4 °C, before being centrifuged through 0.5 ml Amicon Ultra centrifugal filter units (Merck Millipore) with a MWCO of 10 kDa. The peptide containing flow-through was collected, and concentration determined by UV/Vis analysis at 205 nm with an extinction coefficient of ε^{1 mg ml−1} = 31⁸².