Kta fplc system

Pro‐IL‐1α/β or pro‐caspase‐5 cDNAs were cloned into pGEX‐4T‐3 (GE) or pcDNA3 (Invitrogen), with mutations introduced by site‐directed mutagenesis. For bacterial expression, IPTG‐induced Rosetta cultures were lysed (PBS 1×; DTT 1 mM; EDTA 10 mM; benzonase 30 U/ml; lysozyme 55 kU/g (both Novagen); protease inhibitor cocktail) with sonication, and clarified by centrifugation (5,525 g, 1 hr, 4°C). Filtered supernatants were loaded onto a GSTrap column (GE), washed (PBS; DTT 1 mM; EDTA 1 mM) and eluted (Tris 50 mM; NaCl 100 mM; DTT 1 mM; reduced l‐glutathione 50 mM; adjusted to pH 8) using an ÅKTA FPLC system (GE). Eluted protein was dialysed overnight (Tris 10 mM, pH8; NaCl 50 mM) and stored in glycerol (10%) at −80°C. IL‐1 protein concentration was normalized by SDS‐PAGE, Coomassie staining (G‐250; Bio‐Rad) and quantitative imaging (Odyssey, Li‐Cor). Recombinant IL‐1 (4 µg/ml) was incubated (1 hr, 37°C) with active caspase (100 U or 10 U; all Enzo) in reaction buffer (human—HEPES 50 mM, pH 7.4; NaCl 100 mM; CHAPS 0.1%; EDTA 1 mM; glycerol 10%; DTT 10 mM) (mouse—MES 100 mM, pH 6.5; CHAPS 0.1%; PEG 10%; DTT 10 mM). Where indicated, LEVD‐FMK (330 µM; Enzo) was added. Recombinant IL‐1 (4 µg/ml) was incubated (1 hr, RT) with active calpain (100 U; Calbiochem) in reaction buffer (NaCl 100 mM; CaCl2 2 mM; DTT 1 mM).

Suspension HEK293 cells were transfected with sterile-filtered plasmid DNA using polyethylenimine in OptiPro serum-free medium (Thermo Fisher). TA99 was purified using rProtein A Sepharose Fast Flow resin (GE Healthcare) as previously described^{40 (link)}. His-tagged proteins were isolated from HEK293 supernatant using TALON Metal Affinity Resin (Takara Bio Inc.). Some cytokine-fusion proteins were then further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column on an ÄKTA FPLC system (GE Healthcare) that had been pretreated overnight with 1 M NaOH to remove endotoxin and subsequently equilibrated in sterile PBS (Corning). After purification, all proteins were buffer exchanged into sterile PBS (Corning), 0.2 μm sterile-filtered (Pall Corporation), and confirmed to contain minimal endotoxin (<0.1 EU per injection) using a chromogenic LAL assay (Lonza). To confirm their molecular weights, proteins were run alongside a Novex Prestained Sharp Protein Ladder on a 4–12% NuPAGE Bis-Tris protein gel (Life Technologies) with 1% MES running buffer. All proteins were stored at −80 °C, but before therapeutic injection or in vitro assessment, cytokine fusion proteins were thawed on ice.

Enrichment was carried out at room temperature in an anoxic chamber with an atmosphere of 98% N₂ and 2% H₂ (Coy Laboratory Products Inc., Grass Lake, MI, USA) using an ÄKTA-FPLC system (GE Healthcare Europe GmbH, Freiburg, Germany). Cells were grown in 2 L Schott bottles (aqueous to gas phase ratio 1:1) with pyruvate and fumarate. Harvesting, disruption and fractionation was done as described in the previous chapter, except that 1% digitonin was used as detergent. The filtered solubilized ME was fractionated via an anion exchange column (Q-Sepharose HP column 10/10, GE Healthcare Europe GmbH, Freiburg, Germany). The Q-Sepharose column was pre-equilibrated with 50 mM Tris-HCl (pH 8.0), 0.5 mM DTT and 0.1% (v/v) Triton X-100. Subsequently, the column was washed with 200 ml of the same buffer. Elution of proteins was achieved with a linear salt gradient from 0 to 0.5 M KCl at a flow rate of 2 ml/min. Fractions containing hydrogenase activity eluted at approximately 0.2 M KCl. H₂-oxidizing activity was checked photometrically and by activity stained blue native (BN) polyacrylamide gel electrophoresis (PAGE), while purity was checked by SDS PAGE and silver-stained BN PAGE.