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

Abstract

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.

Recombinant Human MFG-E8 Attenuates Intestinal Injury and Mortality in Severe Whole Body Irradiation in Rats

“…The recombinant protein was greater than 99% pure, identified as human MFG-E8 with 95% confidence, and was rendered endotoxin free with Triton-X-114 treatment [15]. Rats were exposed to WBI as described above and randomly assigned to sham, treatment or vehicle groups.…”

Variation in milk fat globule size and composition: A source of bioactives for human health

“…Purification from bovine milk fat globules were stablished (Hvarregaard et al 1996) Purified recombinant protein of human milk fat globule-EGF factor 8 protein have been developed and is commercially available (Qiang et al 2011) Clusterin/ apolipoprotein J Human, caprine and ovine More abundant in human and ovine Clusterin has been linked to cell damage and apoptosis and has been shown to be overexpressed at damaged or stressed tissues and to provide a chaperone-like activity to protect other proteins against damage (Hochgrebe et al 2000) Purification method from milk has not been established.…”

Recombinant human milk fat globule-EGF factor VIII (rhMFG-E8) as a therapy for sepsis after acute exposure to alcohol

“…The rhMFG-E8 protein was produced by TheraSource (Manhasset, NY). The pET-28a(+) expression plasmid containing the His-tagged mature form of the human MFG-E8 (NM_005928.2; Leu24-Cys387) (GeneCopoeia, Inc., Germantown, MD) was transformed into E. coli BL21 (DE3) cells and high-yield colonies were selected, induced with IPTG, collected, and sonicated (Qiang et al 2011). The sonicate was clarified by centrifugation and rhMFG-E8 was purified using metal affinity chromatography.…”

“…However, immunogenicity precludes the use of animal proteins in humans. Therefore, we have expressed, purified and characterized recombinant human (rh) MFG-E8 protein (Qiang et al 2011). Unlike its mouse orthologue, human MFG-E8 is a 387-amino acid 45-KDa protein with only one N-terminal EGF-like domain and no proline/threonine rich domain (Fig.…”