DNA sequences corresponding to human MST2 (residues 1–484) and MBP‐tagged λ‐phosphatase were cloned into separate opening readings frames in pRSF‐Duet (Novagen). Nucleotides corresponding to human SAV1‐FL (residues 1–383) or a truncated variant, SAV1ΔN, (residues 196–383) were cloned into a modified pGEX‐2TK (Sigma‐Aldrich) encoding an N‐terminal His8‐StrepTagII‐mYFP tag.
T7 Express cells (New England Biolabs) were co‐transformed with both plasmids. Cells were grown in Terrific Broth to an OD600 of 2.0, protein expression induced by 0.25 mM IPTG and cells were additionally grown overnight at 16°C. Cells were lysed in 50 mM Tris pH 8.5, 400 mM NaCl and 10%(v/v) Glycerol. MST2:SAV1 complexes were purified by a combination of immobilised metal affinity chromatograph (IMAC; BioRad), streptactin affinity chromatography (Cytiva) and size‐exclusion chromatography (Superose 200; GE Healthcare). Complex was concentrated to approximately 10 μM in 10 mM Tris pH 8.5, 400 mM NaCl, 5% Glycerol and 5 mM βME and used immediately.
T7 Express cells (New England Biolabs) were co‐transformed with both plasmids. Cells were grown in Terrific Broth to an OD600 of 2.0, protein expression induced by 0.25 mM IPTG and cells were additionally grown overnight at 16°C. Cells were lysed in 50 mM Tris pH 8.5, 400 mM NaCl and 10%(v/v) Glycerol. MST2:SAV1 complexes were purified by a combination of immobilised metal affinity chromatograph (IMAC; BioRad), streptactin affinity chromatography (Cytiva) and size‐exclusion chromatography (Superose 200; GE Healthcare). Complex was concentrated to approximately 10 μM in 10 mM Tris pH 8.5, 400 mM NaCl, 5% Glycerol and 5 mM βME and used immediately.