Expression and characterization of recombinant human milk fat globule-EGF factor VIII

Abstract

Apoptosis plays an important role in the pathobiology of sepsis. The opsonizing protein milk fat globule-EGF factor VIII (MFG-E8) is involved in apoptotic cell clearance. Our previous studies have shown that administration of rat MFG-E8-containing exosomes or recombinant murine MFG-E8 (rmMFG-E8) is protective in a rat model of sepsis induced by cecal ligation of puncture (CLP). However, one obstacle hampering the development of MFG-E8 as a therapeutic agent for septic patients is the potential immunogenicity of animal proteins in humans. The purpose of this study, therefore, was to express recombinant human MFG-E8 (rhMFG-E8) and characterize its biological activity. Using an E. coli system, we successfully expressed and purified the mature molecule of human MFG-E8 (Leu24-Cys387). The purity of rhMFG-E8 was over 99% and it was immunoreactive for specific antihuman MFG-E8 antibodies. Amino acid sequence analysis by LC-MS/MS identified the purified protein as human MFG-E8. Using primary rat peritoneal macrophages, we showed that rhMFG-E8 markedly increased peritoneal macrophage phagocytosis of apoptotic thymocytes, which was as effective as commercial rmMFG-E8. To determine the biological activity of rhMFG-E8 in vivo, male adult rats were subjected to sepsis by CLP. rhMFG-E8 or rmMFG-E8 were administered intravenously at the time of CLP. Our results demonstrated that both rhMFG-E8 and rmMFG-E8 reduced thymocyte apoptosis and plasma levels of lactate and IL-6 at 20 h after CLP, and improved the 10-day survival rate. Thus, we have successfully expressed and purified biologically active rhMFG-E8. Our newly-expressed rhMFG-E8 is highly effective in the rat model of sepsis.