Triple layer flasks

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.

The peptide sequence of PCSK9 used in this study is identical to GenBank acc. no. CAC38896.1. The PCSK9 coding sequence was ligated into the pCpGfree-vitroNmcs expression vector and transformed into the E. coli strain GT115 encoding the pir gene (Invivogen). Mutants were created by successive overlapping PCR and inserted into the pCpG-vitroNmcs vector. Plasmids were sequenced by Eurofins. WT PCSK9 and derived mutants were expressed transiently in CHO-K1 cells. CHO-K1 cells stably transfected with PCSK9 were adapted to growth in suspension in Hybridoma-SFM medium (GIBCO) and then expanded in Celline CL 350 Bioreactor flasks (Integra) or in triple layer flasks (Thermo).