Precission protease

Primate transferrin orthologs were cloned into the pFASTBac vector (Invitrogen) containing an N-terminal Strep-Flag-Precission protease tag. Generation of baculoviral DNA from DH10Bac cells was performed using the Bac to Bac system (Invitrogen) per the manufacturer’s instructions. Sf9 insect cells (Expression Systems) were transfected with baculoviral DNA using Cellfectin II reagent (Invitrogen). After three passages to amplify viral titers, a one litre culture of Sf9 cells was infected, and cell pellets were collected 48 hours post-infection. Cell pellets were lysed in tris-buffered saline (TBS; 50 mM Tris pH 8.0, 150 mM NaCl) containing 1% Triton X100, protease inhibitors, iron-nitrilotriacetic acid (Fe-NTA), and 15 U avidin, then sonicated for three 30 second cycles with a needle sonicator (Branson). Lysates were centrifugred at >30,000xG for 90 minutes and passed through a 0.4 micron low protein binding filter. Clarified lysates were loaded onto a 5 mL StrepTrap HP column (GE Healthcare), washed with up to 1 M NaCl, and eluted in TBS containing 2.5 mM D-desthiobiotin (Sigma). Precission protease (Invitrogen) was added to samples and dialyzed against 1 L of TBS overnight.