Histrap ff 5 ml affinity column

The pET28b plasmid was used as expression vector with the cDNA of either DNJ-13, CDC-37 and the respective fragments of CDC-37 (ΔN & ΔC), subcloned after the N-terminal His₆ tag. For expression, transformed E. coli BL21-CodonPlus(DE3)RIL cells were grown to an OD₆₀₀ of 0.6 at 37 °C. Protein production was induced by adding 1 mM isopropyl 1-thio-β-D-galactopyranoside and further incubation at 20 °C. Cells were harvested and subsequently disrupted in a TS 0.75 cell disruption instrument (Constant Systems Ltd., Northants, UK). The His₆-tagged proteins were trapped on a HisTrap FF 5-mL affinity column (GE Healthcare) in 40 mM HEPES/KOH, pH7.5, 20 mM KCl, 1 mM DTT and eluted with buffer containing 300 mM imidazole. ResourceS ion-exchange chromatography and size exclusion chromatography on a 16/60 Superdex 75 HiLoad column (GE Healthcare) were subsequently performed. Proteins were stored and measured in a buffer containing 40 mM HEPES/KOH, pH7.5, 20 mM KCl, 1 mM DTT. The quality of each purified protein was confirmed by SDS-PAGE and mass spectroscopy on a Bruker UltraFlex III MALDI-TOF instrument (Bruker, Massachusetts, USA).

The supernatant was filtered by a syringe filter (0.45 μm) and was loaded on a His Trap FF 5 mL affinity column (GE Healthcare, Chicago, IL, USA). Purification was carried out on the AKTA Prime Plus system (GE Healthcare). The column was equilibrated with 20 mM Tris-HCl, 10 mM Imidazole, 300 mM NaCl, with a pH of 8 and a flow of 1 mL/min y 0.5 MPa. Subsequently, the protein was eluted using an Imidazole gradient started with 50 mM. The Cr-cathepsin protein was eluted between 200–250 mM of imidazole. The elution fractions were dialyzed against PBS for complete refolding. The protein concentration in the final samples was determined using the BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were identified by 12% SDS-PAGE analysis under reducing conditions and the western blotting of anti-His-HRP (Thermo Fisher Scientific, Waltham, MA, USA) (Figure S1).