Pivex2.4d

Recombinant HlgA, HlgB, HlgC, LukE, LukD, LukM, LukF′, LukA, and LukB proteins were generated in E. coli with a non-cleavable N-terminal 6xHIS tag as described previously10 (link)21 (link)44 (link). In short, coding sequences were cloned into either a slightly modified pRSETB vector (Invitrogen), pQE-30 (Qiagen, Courtaboeuf, France) or pIVEX2.4d (Roche) and expression plasmids were transformed in E. coli Rosetta Gami (DE3) plysS or BL21 (DE3). Protein expression was induced with 1 mM Isopropyl β-D-1-iogalactopyranoside (IPTG). Proteins were isolated from a HiTrap chelating HP column under native conditions and eluted using an imidazole gradient. Subsequently, proteins were stored in PBS and purity (>95%) was confirmed using SDS-PAGE electrophoresis. LukA and LukB were eluted from the purification column under denaturating conditions. To prevent precipitation of LukB, the protein was first dialysed to a Tris-arginine buffer, pH10, and subsequently pH was slowly increased towards a physiological pH. Finally, both toxins were dialyzed and stored in a Tris-NaCl buffer.

The Escherichia coli strains DH5α and BL21(DE3) pLysS, as well as the expression vector pET-32a containing the Trx tag, were purchased from Novagen (Darmstadt, Germany); pIVEX 2.4d was purchased from Roche (Basel, Switzerland); and pET-SUMO-28a was constructed and conserved in our lab. The RTS^TM 100 Escherichia coli Disulfate Kit was purchased from Biotechrabbit GmbH (Berlin, Germany). Restriction enzymes Kpn I, EcoR I, Not I, and Mlu I, T4 DNA ligase, protein markers, protein HisPur^TM Ni-NTA Purification Kit, HAM’S/F12 medium, Fetal Bovine serum (FBS), penicillin, and streptomycin were purchased from Thermo Scientific (Waltham, MA, USA). Enterokinase was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Vydac C18 218TP54 columns (5 μm, 4.6 mm × 250 mm, 10 μm, 22 mm× 250 mm) were purchased from Grace (Deerfield, IL, USA). Acetonitrile (ACN, gradient grade for HPLC), and other chemical reagents were all of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

The p7 protein sequence from HCV strain H77 genotype 1a was synthesized by DNA2.0. The gene was cloned directly into a pIVEX2.4d (Roche Applied Science) using NdeI/XhoI restriction sites. Cell-free expression was performed according to Soranzo et al.^{18 (link)}. Here, mixtures of either hydrogenated or deuterated amino acids (4.08 mg/mL) were used to express isotopically labelled proteins. Expressions reactions were performed at 30 °C for 9 hrs for NR and 6 hrs for electrophysiology measurements.