Actriia

The extracellular domains of human ActRIIA (residues 1–134) and rat ActRIIB (residues 1–120) were produced as previously described (Goebel et al., 2019a (link)). Specifically, both receptors were subcloned into the pVL1392 baculovirus vector with C-terminal Flag and His₁₀ tags (ActRIIA) or a C-terminal His₆ tag followed by a thrombin cleavage site (ActRIIB). Recombinant baculoviruses were generated through the Bac-to-Bac system (ActRIIA; Invitrogen - Waltham, MA) or the Baculogold system (ActRIIB; Pharmingen - San Diego,CA). Virus amplification and protein expression were carried out using standard protocols in SF + insect cells (Protein Sciences, Meriden, CT). ActRIIA and ActRIIB were purified from cell supernatants by using Ni Sepharose affinity resin (Cytiva, Marlborough, MA) with buffers containing 50 mM Na₂HPO₄, 500 mM NaCl, and 20 mM imidazole, pH 7.5 for loading/washing and 500 mM imidazole for elution. ActRIIB was digested with thrombin overnight to remove the His₆ tag. ActRIIA and ActRIIB were subjected to size exclusion chromatography (SEC) using a HiLoad Superdex S75 16/60 column (Cytiva) in 20 mM Hepes, and 500 mM NaCl, pH 7.5.