High capacity endotoxin removal columns

CLEC14A-ECD-Fc was expressed and purified as described.^{3 (link)} MMRN2^495–674 and MMRN2^495–603 with His tag or avitag were expressed in E. coli strain BL21-DE3 pLysS (Promega, Southampton, UK) by induction with 0.5 mM IPTG at OD₆₀₀ 0.6 and grown at 18 °C overnight. The cell pellets were homogenized by high pressure lysis (17 000 p.s.i.) in Emulsiflex-C3 (4 °C; Avestin, Winchester, UK) in buffer; 50 mM Na₂PO₄ pH 7.4 400 mM NaCl, 10% (v/v) glycerol, 50 mM imidazole, 0.5 mM TCEP and complete EDTA-free protease inhibitors (Roche, Burgess Hill, UK), then loaded onto Nickel-NTA columns. Fractions eluted using 500 mM imidazole were purified by size exclusion chromatography on S200 columns on an AKTA FPLC machine (GE Healthcare) in buffer; 20 mM Tris pH8.0, 50 mM NaCl. Proteins were buffer exchanged into PBS and endotoxin removed using high capacity endotoxin removal columns (Thermo Fisher Scientific) then filter sterilized.

The plasmids for the recombinant expression of human Trx1, Grx1, Grx2, and Prx2 in E. coli were described before (15 (link)–17 (link)). All proteins were expressed as His-Tag proteins in E. coli and were purified by immobilized metal affinity chromatography and FPLC as described previously (18 (link)) (see Supplementary Figure 2). Note that this expression system gives rise to redox-active enzymes, as shown for Trxs, Grxs, and Prxs before (18 (link)). For the cell culture experiments mouse Grx2 was also expressed as His-Tag protein in ClearColi® BL21(DE3) electrocompetent cells (Lucigen), BL21(DE3) derived cells with several key mutations that result in a lack of outer membrane agonists for hTLR4/MD-2 activation and therefore contain significantly reduced endotoxicity (19 (link)). Proteins were purified by immobilized metal affinity chromatography (GE Healthcare), endotoxins were removed using high capacity endotoxin removal columns according to manufacturer’s instructions (Thermo Scientific, USA) and were tested for their endotoxin levels using the HEK-BlueTM LPS Detection Kit2 (InvivoGen, San Diego, USA).