Recombinant SIRPα Protein Expression and Purification

All antibodies were expressed in Expi293 cells (Invitrogen) using standard manufacturer’s protocol. Expression cultures were typically grown for 5 days at 37 °C in 8% CO₂. Supernatants were harvested via centrifugation and sterile filtered. Proteins were affinity purified utilizing MabSelect Sure LX resin (GE Healthcare). For SPR screening, the IgV domains of human SIRPα v1 (NP_542970.1) and human SIRPα v2 (CAA71403.1) are expressed in Expi293 cells as described above as either a His-tagged or His-Avi-tagged fusions and purified using Ni-Sepharose 6 Fast Flow affinity purification and polished via gel filtration through a superdex hi-prep resin (GE Healthcare). The anti-SIRPα v1 specific antibody clone HEF-LB was generated as described in reference [23 ]. For crystallography, the IgV domain of SIRPα v1 and Fab fragments was generated as described [14 (link)].