Superdex 75 hr 10 30

As described previously [17 (link)], a single-chain variable region fragment (scFv) of J591 in the VH-VL orientation was PCR-amplified from the SFG P28z vector and cloned into pSEC-tag2 (Invitrogen, Carlsbad, CA, USA) mammalian expression vector pMS-C with N-terminal Ig-kappa leader and a C-terminal (His)₆-tag followed by a Cys residue (J591c-scFv). HEK293T cells were transfected with the expression vector using Lipofectamine 2000 (Life Technologies, Paisley, UK), and transfected cells were selected with 100 μg/ml Zeocin (Life Technologies) before expanding to triple flasks for protein production. The J591c-scFv was purified from HEK293T culture supernatant by Ni-NTA chromatography (5 ml Ni-NTA Superflow Cartridge, Qiagen, Manchester, UK); the collected fractions were concentrated by centrifugal concentrations, then further purified by FPLC gel filtration using an AKTA system (Superdex 75 HR 10/30, GE Healthcare, Little Chalfont, UK). Purified protein in phosphate-buffered saline (PBS; pH 7.0) for subsequent maleimide conjugation was concentrated to > 1 mg/ml and stored in aliquots at − 80 °C. Purity was assessed by Coomassie staining after sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE; see Additional file 1) and analytical size-exclusion high-pressure liquid chromatography (HPLC) (BioSep SEC-s2000, Phenomenex, Cheshire, UK; see Additional file 1).

All ATPase₃ domain proteins and substrate Esx proteins were purified by a similar method. Briefly, cells were thawed and resuspended in buffer A (20 mmol/L Hepes pH 7.0, 150 mmol/L NaCl, 5% (w/v) glycerol, 1 mmol/L MgCl₂, 5 mmol/L ATP). The resuspended cells were then lysed by passing through a French Press at 800 bar after adding 1 mmol/L PMSF. Cell debris was then removed by centrifugation at 18,000 rpm for 30 min at 4 °C. The supernatant was applied to Ni-NTA agarose beads (GE Healthcare) for 2 h at 4 °C. The beads were rinsed with buffer A containing 30 mmol/L imidazole. For MtEccCb1-ATPase₃, MtEccC2-ATPase₃ and Esx proteins, the N-terminal tag was cleaved by 3C protease and then eluted. For MtEccC3-ATPase₃ and MtEccC5-ATPase₃, the recombinant protein was eluted from the beads with buffer A containing 300 mmol/L imidazole. Then the sample was concentrated and purified using a 5mL Hitrap Q HP (GE life science) column followed by size exclusion chromatography (SEC) using a Superdex 75 HR 10/30 (GE life science) column. The peak fractions were pooled and concentrated to approximately 10 mg/mL using a 10 kDa cut-off spin concentrator (Millipore). The separately purified MtEccCb1-ATPase₃ and MtEsxB were mixed in a 1:1 molar ratio, incubated and purified again by gel filtration. The fractions containing complex were pooled and concentrated for crystallization.

DR5 cDNA was purchased from Sino Biological INC (Beijing, China). The extra-cellular domain sequence of DR5 (residues 1–130, sDR5) was amplified by PCR and subcloned into pET21b with C-terminal 6× His-tags. The sDR5 protein was expressed in E. coli BL21(DE3) strain by addition of 0.2 mM IPTG. After induction at 20 °C for 16 h, the bacterial cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS) and disrupted by sonication. Following this, the sDR5 protein was purified by metal affinity chromatography on Ni-sepharose (GE Healthcare, Chicago, IL, USA), before Superdex 75 HR 10/30 (10 × 300 mm) size-exclusion chromatography was used (GE Healthcare).