Production and Purification of Recombinant Proteases

Enzymatically active PCs were produced as described previously [3 (link),4 (link)]. Truncated forms of the enzymes containing the catalytic domain and the regulatory P-domain were produced in Sf9 insect cells using the baculovirus expression system (Invitrogen). The secreted enzymes were purified from culture supernatants by nickel-affinity chromatography, which was supported by a poly-histidine tail engineered at the carboxyl terminal end of the proteins, followed by size exclusion chromatography. Care was given to remove glycerol during filtration and concentration as it interferes with the activation of PsV and cell entry assays. The concentration of active proteases was determined by active-site titrations using the covalent-bound inhibitor dec-Arg-Val-Lys-Arg-chloromethyl ketone (CMK, Bachem, Torrance, CA, USA) and an activity assay that employs pyr-Arg-Thr-Lys-Arg-amido-methylcoumarin (Bachem, Torrance, CA, USA) as substrate.