Thrombin protease

GST-Tip110, GST-Tip110s, Tip110 deletion mutants ∆NLS and ∆RRM, and GST proteins were expressed in and purified from E. coli BL21, as previously described [20 (link)]. Briefly, expression plasmids were transformed into BL21 and induced with 1 mM isopropyl-d-thiogalactopyranoside for 2 h at 37°C. GST fusion proteins were purified using a Pierce GST fusion protein purification kit (Rockford, IL, USA). When necessary, GST was removed by treating the eluted protein with thrombin protease (10 units) (Invitrogen, Carlsbad, CA, USA) at room temperature for 18 h. The digested protein solution was dialyzed overnight in 4 l of phosphate-buffered saline and cleared of GST protein by additional incubations with fresh glutathione beads. The purified proteins were electrophoresed on a 8% SDS-polyacrylamide gel and stained with Gold–Blue (Pierce) to ensure the complete removal of GST protein as well as undigested fusion protein and other contaminated proteins.

Ammonia water, silver nitrate (AgNO₃), sodium citrate (Na₃C₆H₅O₇·2H₂O), hydrazine hydrate (N₂H₄·H₂O), glutathione (GSH), and phenylmethanesulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich (American). Isopropylthio-β-d-galactoside (IPTG), Tris-HCl (pH 8.0), thrombin protease, and GST tag were purchased from Invitrogen (American). All chemicals and reagents were used as received without further purification.