Refolding of the recombinant IgV domain of CD47 from E. coli for NMR studies.

CD47 is a widely expressed integrin-associated transmembrane protein that regulates many macrophage functions, including phagocytosis. Since many cancer cells overexpress CD47 to evade the immune system, targeting CD47 has been proposed as a strategy to enhance macrophage-mediated destruction of cancer cells. Consequently, developing antagonists that block the CD47-SIRPα interaction has drawn attention. However, the exclusive use of eukaryotic cell culture to produce CD47-IgV precludes isotope enrichment of the domain. This prevents the use of solution NMR as a tool for identifying CD47 inhibitors. Here, we describe a two-step refolding protocol for the CD47-IgV domain from inclusion bodies produced in E. coli. The yield of CD47-IgV is ∼30 mg/L of culture. The refolded domain interacts with the anti-CD47 antibody B6H12 with an affinity similar to glycosylated CD47-IgV produced in mammalian cells and binds the IgV domain of SIRPα. The method allowed us to produce N and C enriched CD47-IgV for NMR. The chemical shift assignments of the CD47-IgV domain backbone atoms confirmed that the refolded protein has the same secondary structure as the crystal structure of CD47-IgV produced in mammalian cells. Furthermore, NMR data showed refolded CD47-IgV interacts with SIRPα-IgV similarly to glycosylated CD47-IgV. The application of this method can advance the progress of biochemical investigations of the interaction between CD47-IgV and SIRPα and will be useful for the discovery of antibodies, small molecules, and peptides targeting the CD47/SIRPα axis in vivo.