Immobilized metal affinity chromatography

HIV-1 IIIB C34 and T20 peptides were obtained through the NIH AIDS Reagent Program (Division of AIDS, NIAID). A 28-mer MPER peptide (EQELLELDKWASLWNWFNITNWLWYIKL) was ordered to ThermoFisher Scientific. The OLP#19 peptide covering the C-terminal part of MPER was kindly provided by C. Brander (IrsiCaixa, Spain). MIN sequence was cloned in a pET-21d(+) expression vector (Novagen) and produced by E. coli BL21 DE3 strain (Invitrogen). Inclusion bodies were prepared from 1 L of bacterial culture and solubilised using 8 M urea. Highly pure protein was obtained through niquel-based Immobilized Metal Affinity Chromatography (GE Healthcare) and gel filtration using a Sephacryl S-100 HR column (GE Healthcare).

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).

Processed RFP_Strep was purified from ND‐1 and SC‐1 cell extracts using Strep‐Tactin chromatography (Qiagen, Hilden, Germany) according to the procedure published previously (Zhang et al., 2015). Unbound flow‐through from the Strep‐Tactin column was subject to hydrophobic interaction chromatography, followed by immobilized metal affinity chromatography (GE Healthcare, Marlborough, MA) for purification of processed GFP₁₇₂ as described previously (Peckham et al., 2006; Zhang et al., 2015). Purified GFP₁₇₂ and RFP_Strep were further processed using SDS‐PAGE and blotted onto PVDF membrane. Target protein bands were excised for N‐terminal amino acid sequencing using Edman degradation approach (performed by the Protein Facility at Iowa State University). ESI‐TOFMS analysis of purified processed GFP₁₇₂ protein was carried out as described previously (Zhang et al., 2015).