Recombinant Archease Protein Purification

Human Archease isoform 1 (UniProt A8K0B5; note the deviating translation start sites from UniProt Q8IWT0 by twelve additional residues) was expressed and purified as described previously^{10 (link)}. In short, the fusion protein with an N-terminal His₆ tag followed by a thrombin protease cleavage site was expressed in E. coli BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) cells. The clarified lysate was applied to a 5 mL Ni-NTA column (Cytiva). After tag removal (optional) with thrombin (Cytiva), the sample was re-applied onto the Ni-NTA column to remove the affinity tag. The flow-through fraction was concentrated using a centrifugal filter (Amicon Ultra, MWCO 10 kDa, Sigma) and purified by size-exclusion chromatography with buffer containing 25 mM HEPES, pH 7.5, 100 mM NaCl₂, 5% Glycerol, 1 mM TCEP on HiLoad16-600 Superdex 75 pg column (Cytiva). Archease containing peak fractions were concentrated, flash-frozen in liquid nitrogen and stored at −80 °C. We determined a 260/280 nm ratio of 0.56 for the final protein preparation indicating the absence of nucleic acid contaminants (see Source Data).