Profinia protein purification system

The following antibodies were used for Western blotting: A mouse hybridoma clone expressing monoclonal antibody 15A6 recognizing an epitope in the CHR domain of PCSK9 was a generous gift from J. Horton (UTSouthwestern Medical Center, Dallas, TX). The IgG fraction was purified from hybridoma culture medium by Protein A chromatography on a Profinia™ protein purification system (Bio-Rad) as per manufacturer’s instructions. Rabbit anti-serum 3143 against the C-terminal 14 amino acids of LDLR was the kind gift of J. Herz (UTSouthwestern Medical Center). Anti-actin mouse monoclonal ascites fluid (clone AC-40) was from Sigma. Mouse anti-human transferrin receptor antibody was from Life Technologies. Secondary IRDye-labeled goat anti-mouse and anti-rabbit IgG antibodies were from LI-COR Biosciences. Rabbit anti-serum 1697 raised against full-length human PCSK9 was used for immunoprecipitation and was custom produced by Biomatik (Cambridge, Ontario, Canada)

All Thioredoxin (Trx)-6His-tagged and maltose binding protein (MBP)-tagged proteins were expressed and purified as described previously [3 (link), 60 (link)], with the exception of the Trx-6His-hsFIH and Trx-6His-tcFIH enzymes. Expression of these proteins was identical to those described above, but purification was carried out using the Profinia Protein Purification System (Bio-rad). Bacterial cell lysates were loaded onto a 1 mL Bio-scale Mini Profinity IMAC cartridge, washed with 6 column volumes (CV) of lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM imidazole, 1 mM PMSF (added fresh), 0.5 mM DTT (added fresh)), 6 CV wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM imidazole), and then eluted in 3.5 CV elution buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 250 mM imidazole). 2.5 mL of eluate was then loaded onto a PD-10 column (GE) and buffer exchanged into protein storage buffer (20 mM Tris-HCl pH 8, 150 mM NaCl) as per the manufacturer’s instructions. Due to a tendency of Trx-6His-tcFIH to precipitate after short term storage at 4°C, purified protein was immediately diluted to approximately 10 μM after buffer exchange. Recombinant polypeptide concentrations were calculated using their extinction coefficients and absorbance at 280 nm, and purity was assessed by densitometry of Coomassie-stained SDS PAGE gels.

Both proteins comprising C-terminal poly-histidine tags were purified by immobilized metal ion affinity chromatography (IMAC) using a Profinia protein purification system (Bio-Rad Laboratories, Munich, Germany). Before purification, supernatants were concentrated and dialyzed against 150 mM NaCl, 30 mM HEPES, 30 mM imidazole, 10% (v/v) glycerol, pH 8.0 using a hollow fibre module (cut-off 10 kDa, Spectrum Laboratories, CA). The affinity tag of IFNα, comprising His₈ and a Twin-Strep-sequence, was cleaved with an excess of TEV-protease (2 mg/ml) overnight at 4°C to prevent binding of αIFNα to the Ni-NTA tips during biolayer interferometry. The protease and uncleaved IFNα was separated from IFNα without tag. This was performed by IMAC using a 1 ml HisTrap FF column (GE Healthcare) and the ÄKTAprime Plus protein purification system.
αIFNα-ab and mouse IFNα4 without tag were concentrated with a Vivaspin concentrator (10,000 MWCO, Sartorius AG, Göttingen, Germany) and further purified by gel exclusion chromatography on a Superdex 75 16/60 column. Proteins were applied to the column after concentration with Vivaspin (Sartorius AG, Germany, MWCO 10,000). Mouse IFNα4 was purified using HEPES buffer (150 mM NaCl, 30 mM HEPES, 10% [v/v] glycerol) and αIFNα-ab was purified using Na-phosphate buffer (150 mM NaCl, 16 mM Na₂HPO₄, 84 mM NaH₂PO₄ and 10% [v/v] glycerol).