Hiload 16 600 superdex 200

Expression and purification of Dg_GH4185a, Dg_GH4185b, Phage_GH4185, Myxo_GH4185, Dg_GH172a, Dg_GH172b, Dg_GH172c, and Myc_GH172.
Recombinant proteins were expressed in competent E. coli Tuner cells (Novagen) using pET28a vectors generated as above. Cells were grown in LB media at 37 °C with shaking, until turbidity reached an OD₆₀₀ of ~0.6, wherein expression was induced with 0.2 mM IPTG and cells were further cultured for 16 hours at 16 °C. Sonication was used to lyse cells in 20 mM Tris, pH 8.0, 200 mM NaCl.
Enzymes were purified using immobilized metal affinity chromatography on cobalt TALON resin. Proteins were dialyzed into 20 mM HEPES, pH 8.0, 150 mM NaCl buffer dialysis (Medicell). For crystallography proteins were purified further via size-exclusion chromatography (HiLoad 16/600 Superdex 200, GE Healthcare) in 20 mM HEPES, pH 8.0, 150 mM NaCl. Protein purity was ascertained by SDS-PAGE and protein concentrations were determined using Nanodrop spectroscopy (Thermofisher).

Sequences corresponding to regions of human SYCE2 (full-length, 1-218; core, 57-165; ΔS2C, 57-154) and TEX12 (full-length, 1-123; ΔCtip, 1-113; core, 49-123; core ΔCtip, 49-113) were cloned into pRSF-Duet1 (Novagen®) expression vectors for expression as TEV-cleavable N-terminal MBP- and His6-fusion proteins, respectively. Constructs were co-expressed in BL21 (DE3) cells (Novagen®), in 2xYT media, induced with 0.5 mM IPTG for 16 hours at 25°C. Cells were lysed by sonication in 20 mM Tris pH 8.0, 500 mM KCl, and fusion proteins were purified from clarified lysate through consecutive Ni-NTA (Qiagen), amylose (NEB) and HiTrap Q HP (GE Healthcare) ion exchange chromatography. Affinity tags were removed by incubation with TEV protease and cleaved samples were purified by HiTrap Q HP ion exchange chromatography and size exclusion chromatography (HiLoad™ 16/600 Superdex 200, GE Healthcare) in 20 mM Tris pH 8.0, 150 mM KCl, 2 mM DTT. Protein samples were concentrated using Pall Microsep™ Advance centrifugal devices, and were stored at -80°C following flash-freezing in liquid nitrogen. Protein samples were analysed by SDS-PAGE with Coomassie staining, and concentrations were determined by UV spectroscopy using a Cary 60 UV spectrophotometer (Agilent) with extinction coefficients and molecular weights calculated by ProtParam (http://web.expasy.org/protparam/).

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.