Ni sepharose affinity resin

AMHR2 ECD residues 18 to 124 was subcloned into the pFastBac baculovirus vector containing an N-terminal octahistidine tag, myc tag, and MBP fusion. Recombinant baculovirus was produced and amplified using the Bac-to-Bac expression system, per manufacture protocol (Invitrogen). Protein expression was conducted using standard manufacture protocols in SF+ insect cells (Protein Sciences). Cells were harvested 60 h postinfection, and cell debris was cleared through centrifugation at 3,200 rpm for 20 min at 4 °C. Conditioned media was applied to Ni Sepharose affinity resin (Cytiva) equilibrated with 20 mM Phosphate buffer pH 7.4, 500 mM NaCl, and 20 mM imidazole. Protein was eluted with equilibration buffer + 500 mM imidazole. The elutions were concentrated and applied to a HiLoad Superdex S200 16/60 column (Cytiva) in 20 mM Hepes pH 7.5 and 500 mM NaCl. Fractions containing pure AMHR2-MBP fusion were digested at 4 °C overnight with HRV-3C protease in 25 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA, and 1% ethylene glycol. After digestions, the protein was applied to a HiLoad Superdex S75 16/60 column (Cytiva) in 20 mM Hepes pH 7.5 and 500 mM NaCl. Fractions containing pure AMHR2 ECD were dialyzed into 10 mM HCl then concentrated and stored at −80 °C until used in experiments.

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.