Hiload superdex s75 16 60 column

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.

Both Fst-288 and Fstl3 were produced as previously described (Stamler et al., 2008 (link)). Fst-288 was expressed from a stably transfected CHO cell line and purified from CM by binding to a heparin-Sepharose column (abcam) in 100 mM NaBic pH 8 and 1.5 M NaCl, with a low salt gradient to elute followed by cation exchange over a Sepharose fast flow (Cytiva) in 25 mM HEPES pH 6.5, 150 mM NaCl with a high salt elution gradient. Finally, Fst-288 was then purified over an HPLC SCX column in 2.4 mM Tris, 1.5 mM imidazole, 11.6 mM piperazine pH 6 with a high salt, high pH (10.5) gradient elution. Fstl3 was cloned into the pcDNA3.1/myc-His expression vector and expressed transiently in HEK293F cells. CM was harvested after 6 days and applied to His-affinity resin (Cytiva), followed by washing with a buffer of 500 mM NaCl, 20 mM Tris pH 8 and elution with 500 mM imidazole. Fstl3 was then subjected to SEC using a HiLoad Superdex S75 16/60 column (Cytiva) in 20 mM HEPES pH 7.5 and 500 mM NaCl.