The first 997 amino acids of human SKI-1/S1P lacking the Cterminal transmembrane domain but containing a C-terminal 8-His-tag (PGDDDDKHHHHHHHHSGS) were expressed in Sf9 insect cells as previously described [47] . Two liters of cell culture supernatant were used for purification. Two hundred milliliters of 200 mM Tris/HCl pH 8.0, 500 mM NaCl was added, and then the pH was adjusted to pH 8.0 by further addition of 2 M NaOH. The resulting precipitate was removed by centrifugation at 10000 x g for 30 minutes and subsequently filtered through a glass filter. The cleared supernatant was then applied to a small (0.9 ml column volume) IMAC column (Ni-Sepharose, GE Healthcare, Freiburg, Germany) by continuous flow (1.0 ml/minutes). The column had previously been equilibrated in 50 mM Tris/HCl pH 8.0, 500 mM NaCl (buffer A), and bound recombinant SKI-1/S1P was eluted with a continuous gradient over 30 column volumes to buffer A plus 300 mM imidazole. Collected fractions were assayed for SKI-1/S1P enzymatic activity as previously described [47] using the paranitroanilide (p-NA) acetylated (Ac) tetrapeptidyl substrate Ac-RRLL-pNA [custom synthesized by Peptides International (Louisville, Kentucky, USA)]. The most active fractions were pooled. Concentration and buffer exchange to buffer A was then done using spin concentrators (Millipore, Billerica, MA, USA) with a molecular weight cutoff of 30 kDa. The final preparation was, after addition of 30% v/v glycerol, stored at -80uC and had a specific activity of 0.018 U/mg (measured as above). Recombinant His-tagged furin (0.432 mg/ ml) was purchased from R & D Systems (Minneapolis, MN, USA), and reactions with adenovirus recombinant serpin were performed under the buffer conditions for furin assays as previously described [54, 58] .