Heparin hp column

E. coli strain M15 [pREP4] containing the plasmid pQMSsoII or pQMNlaX was grown at 37°C in LB medium with 30 µg/ml kanamycin and 50 µg/ml ampicillin to an A₆₀₀ value of 0.6. Protein expression was induced with 0.7 mM isopropyl 1-thio-β-D-galactopyranoside, and the cell culture was kept for 20 h at 20°C. The cells were harvested by centrifugation. The cell pellets were resuspended in buffer A (50 mM Na-phosphate, 100 mM NaCl, 5 mM β-mercaptoethanol, 5% (w/v) glycerol, pH 7.0) and lysed by sonication. The lysate was clarified by centrifugation at 18,000 g and loaded onto a Heparin HP column (GE Healthcare) pre-equilibrated with buffer A. The target protein was eluted with a gradient from 0.1 to 1.0 M NaCl. In case of M.SsoII, the fractions containing this protein were loaded onto a HisTrap HP column (GE Healthcare) and eluted with a 20–400 mM imidazole gradient. The target proteins were concentrated and their purity was estimated using 12.5% SDS-PAGE. Because of cytotoxicity of M.SsoII, the yield of purified M.SsoII was 0.14 mg from 1 l of cell culture, two orders of magnitude lower than that of M.NlaX.

HerA and NurA cells were re-suspended in lysis buffer (100 mM NaCl, 50 mM Hepes–NaOH pH 8.0, 5 mM DTT and 5% v/v glycerol) supplemented with an EDTA-Free SigmaFast Protease Inhibitor Cocktail Tablet (Sigma-Aldrich). The cells were lysed by sonication and the lysate was clarified by centrifugation. The supernatant was heated in a 70°C water bath for 20 min and subsequently clarified by centrifugation to remove precipitated protein. The supernatant was then loaded onto a 5 ml Heparin HP column (GE Healthcare) equilibrated with lysis buffer. Bound protein was eluted with a linear gradient from 100 mM to 1 M NaCl. Fractions containing the protein of interest were pooled and dialysed against 100 mM NaCl, 50 mM Hepes–NaOH pH 8.0, 5 mM DTT and 5% (v/v) glycerol for 18 h. The dialyzed protein was loaded onto a 5 mL HiTrap Q HP column (GE Healthcare) equilibrated with lysis buffer. Bound protein was eluted with a linear gradient from 100 mM to 1 M NaCl. Fractions containing the protein of interest were pooled and dialysed against 300 mM NaCl, 20 mM Hepes–NaOH pH 8.0, 5 mM DTT and 5% (v/v) glycerol for 18 h. The protein was concentrated with Amicon Ultra-15 centrifugal filter units (EMD Millipore) and aliquots were flash-frozen in liquid nitrogen and stored at –80°C. Protein concentrations were estimated from absorbance at 280 nm using extinction coefficients.

The sequence of human PCSK9 used here is identical to sequence of GenBank acc. no. CAC38896.1. The coding region was ligated into the pCpGfree-vitroNmcs expression vector and transformed into the E. coli strain GT115 encoding the pir gene (Invivogen). CHO-K1 cells stably transfected with PCSK9 were grown as a suspension in Hybridoma-SFM medium (GIBCO) and then expanded to Celline CL 350 Bioreactor flasks (Integra) or in triple layer flasks (Thermo Fischer Scientific). The medium from PCSK9-expressing CHO cells was 1:1 in 25 mM Tris pH 7.4 and applied to two serial-connected 5 mL HiTrap QFF columns (GEhealthcare). The columns were washed with 25 mM Tris pH 7.4 and protein subsequently eluted with 25 mM Tris pH 7.4 and 1 M NaCl. The eluted sample was dialyzed against 25 mM Tris pH 7.4 and 5% (v/v) glycerol at 4°C. The dialyzed sample was applied to a 5 mL Heparin HP column (GEhealthcare) and protein was eluted in 1 mL fractions by a linear gradient over 40 mL from 0 to 1 M NaCl in 25 mM Tris pH 7.4. Fractions containing PCSK9 were concentrated and further purified by SEC on a Superdex200 increase 10/300 or 16/600 in either PBS or 25 mM Tris pH 7.4 and 150 mM NaCl. Samples were analyzed by SDS-PAGE and PCSK9 was concentrated to 5–8 mg/mL based on absorbance at 280 nm and flash-frozen in liquid nitrogen before storage at-80°C.

For the labeling of the PX, 1 mg of AF647 C2 Maleimide kit (Life Technologies, A20347) was dissolved in 100 μL DMSO (ThermoFischer, Catalog No. BP231-100) to final 7.7 mM. For the final labeling reaction 250 μM PX, 2.5 mM TCEP, 385 μM AF647 dye was mixed with labeling buffer (50 mM HEPES pH 7.0, 200 mM KCl) to a total volume of 1 mL, and the reaction was left for 2 hr at room temperature on a roller at 10 rpm. After the incubation, 1 mM of DTT was added and the protein was loaded on a 5 mL Heparin HP column (GE Healthcare 17040701). The column was first washed with HA buffer (50 mM HEPES PH 8.0, 100 mM KCl, 1 mM TCEP) and then eluted with HB buffer (50 mM HEPES PH 8.0, 1 M KCl, 1 mM TCEP). The peak fractions were pooled and concentrated to 6.7 mg/mL (384 μM) with ~ 20% labelling efficiency.