Expression and Purification of Recombinant His-Tagged Human Furin

The expression plasmid was kindly provided by Sven Dahms and the expression procedure adapted from Dahms et al. (2014) and Aricescu et al. (2006). Briefly, the coding sequence of human pro-furin, covering the amino acids 23–574, was inserted into the plasmid pHLsec. Using the restriction site AgeI, the insert was placed downstream to a secretion signal sequence encoded by the expression vector. The native protein sequence was modified with a Thrombin cleavage site and a His-tag, resulting in the artificial C-terminus SGSLVPRGSHHHHHH that is expressed after Ala574 (Aricescu et al., 2006; Dahms et al., 2014). Transfection and expression were performed in HEK293 cells. HEK293 cells were maintained in a humidified atmosphere at 37°C, 5% CO₂ in Dulbecco’s Modified Eagle’s medium (DMEM high glucose, Gibco) supplemented with L-glutamine (Euroclone), non-essential amino-acids (Gibco), 10% fetal bovine serum (FBS;Euroclone) and 1% penicillin/streptomycin (Euroclone). Transfection was performed with Lipofectamine^® 2000 at the ratio (w/v) of plasmid-DNA to transfection reagent 1:5 (8.4 mg of plasmid-DNA and 42 ml of transfection reagent in 10-cm tissue culture dish). Transfection was carried out at approx. 80–90% confluence of the culture. The transfection medium was replaced after 4–5 h, lowering the serum concentration to 0.2%. Conditioned medium was harvested 72 and 120 h post transfection. 12 ml and 8 ml medium were used during the first and second culture period, respectively. The conditioned samples were centrifuged (20 min, 4500 g, 20°C), filtered using a PES membrane (0.22 mm pore size, Sarstedt) and stored at −20°C. Prior to purification the samples were thawed in ice, pooled and dialysed against a 20-fold excess of 25 mM Tris, pH 8,0, 250 mM NaCl, 5 mM CaCl₂ with Visking dialysis membrane (cut-off 12-14 kDa, Medicell Membranes Ltd.) for 16 h at 4°C. His-tagged human furin was purified by affinity chromatography, using an ÄKTApurifier 100 FPLC system (GE Healthcare) with a 1 ml HisTrap FF Crude (GE-Healthcare) affinity column. Before purification, imidazole was added to the resulting samples, up to a concentration of 10 mM. The column was then washed with 10 CV of buffer A (100 mM Tris, pH 8.0, 500 mM NaCl, 5 mM CaCl₂) supplemented with 10 mM imidazole. The protein was eluted with a linear gradient from 2 to 100% of buffer B (buffer A + 500 mM imidazole) at 1 ml/min flow in 20 CV and 2ml fractions collected. The ones corresponding to the UV (280 nm) FPLC peak were pooled and dialyzed against 10 mM Hepes, pH 7,5, 100 mM NaCl, 2 mM CaCl₂ and then concentrated to 1 mg/ml, using an Amicon ultrafiltration membrane with 10 kDa cut-off (Merck-Millipore). The enrichment and the purity of the protein were monitored by SDS-PAGE analysis.