Expression, purification and characterization of C2 domain of milk fat globule-EGF-factor 8-L

Milk fat globule-EGF-factor 8-L (MFG-E8L) is secreted by activated macrophages and functions as a linker protein or opsonin between the dying cells and phagocytes. MFG-E8L recognizes the apoptotic or dying cells by specifically binding to Phosphatidylserine (PS) exposed on the outer cell surface and enhances the engulfment of the apoptotic cells by phagocytes, thereby preventing the inflammation and autoimmune response against intracellular antigens that can be released from the dying cells. MFG-E8L contains two EGF-like domains, P/T (proline/threonine) rich domain followed by two discoidin-like domains (C1 and C2). Recent studies have shown that the C2 domain of MFG-E8L is specifically involved in interaction with PS exposed on the apoptotic cells. Towards understanding this specific molecular interaction between the MFG-E8L C2 domain and PS, we expressed, purified the C2 domain of MFG-E8L and performed the binding studies with phospholipids by 31P NMR experiment. We demonstrated that our recombinant construct and expression system were effective and allowed us to obtain the C2 domain and also showed that the purified C2 domain was stable and properly folded, and our 31P NMR studies indicated that the C2 domain had specific binding with PS.