Recombinant BPI Protein Production

Templates for recombinant BPI were designed by flanking the corresponding BPI sequences without native signal peptides (huBPI: amino acids 32–487; muBPI: amino acids 28–483; scoBPI: amino acids 19–473; osBPI: amino acids 20–477) with an N-terminal HA-signal peptide and a C-terminal FLAG-tag. Constructs were ligated into a pcDNA 3 (huBPI, muBPI) or pcDNA 3.1(+) (osBPI, scoBPI; Cat# V79020; Thermo Fisher Scientific, Waltham, MA) vector backbone. BPI orthologues were generated as described previously with slight modifications (Ederer et al., 2022 (link); Bülow et al., 2018 (link)). Expi293F cells (Cat# A14527, RRID:CVCL_D615; Thermo Fisher Scientific) were transfected using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Recombinant huBPI, muBPI, and scoBPI were purified by cation-exchange chromatography on a HiTrap SP HP column (Cytiva, Marlborough, MA) followed by size-exclusion chromatography (Superdex 200 increase 10/300 GL column; Cytiva). Due to insufficient binding to the cation-exchange column, osBPI was purified by affinity chromatography on an anti-FLAG (Sigma-Aldrich, Taufkirchen, Germany) coupled NHS-activated HP column (Cytiva) followed by size-exclusion chromatography.