| Human MFG-E8 (milk fat globule-EGF factor 8, also lactadherin, breast epithelial antigen BA46, and medin) is a 46kD, secreted, monomeric glycoprotein that is associated with the human milk fat globule (HMFG) membrane (1-3). It is expressed by mammary epithelial cell surfaces, artery media, and in several carcinomas (1-4). Human MGF-E8 is synthesized as a 387 amino acid (aa) precursor that contains a 23 aa signal sequence and a 364 aa mature region (SwissProt Q08431). The mature molecule contains four N-linked glycosylation sites, an amino-terminal EGF-like domain, plus C1 and C2 Ig-like domains which are related to discoidin I and homologous to those of human coagulation factors V and VIII (2, 3). Within the EGF-like domain is an Arg-Gly-Asp (RGD) sequence, which is strategically placed in a hairpin loop between two antiparallel beta strands (2, 3). In this way, the EGF-like domain serves as a scaffold for the RGD sequence, which is proposed to promote cell adhesion by binding cell-surface integrin receptors, such as avb3 (2, 3). Human MFG-E8 shares 59%, 58%, 56%, and 52% aa sequence identity with porcine, bovine, rat, and mouse MFG-E8, respectively. Functionally, MFG-E8 in milk is reported to block rotavirus infection (5). MFG-E8 expression by macrophages promotes the phagocytosis of apoptotic thymocytes by forming a bridge between phosphatidylserine on apoptotic cells, and avb3 integrin on phagocytes (4, 6). The MFG-E8 expression by macrophages controls the accumulation of apoptotic membrane fragments in atherosclerotic plaques and substantially limits lesion development (4). Recombinant protein corresponding to Leu24-Cys387 of human Milk Fat Globule-EGF Factor 8 with a C-terminal 6-His tag, expressed in NSO cells. Calculated molecular mass of ~ 41.6kD. As a result of glycosylation, the recombinant protein migrates as an ~ 45-50kD protein in SDS-PAGE under reducing conditions. Biological Activity: Measured by the ability of the immobilized protein to support the adhesion of SVEC4-10 mouse vascular endothelial cells. When 5x10e4 cells/well are added to rhMFG-E8 coated plates (10ug/ml, 100ul/well), ~45-75% adhered after 60 minutes at 37C. Storage and Stability: Lyophilized and reconstituted products are stable for 6 months after receipt at -20C. Reconstitute with sterile PBS. Aliquot to avoid repeated freezing and thawing. Store at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
