title: Expression and characterization of recombinant human milk fat globule-EGF             factor VIII.
abstract: Apoptosis plays an important role in the patho-biology 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 anti-human 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.
authors.affiliation: The Feinstein Institute for Medical Research, Manhasset, NY 11030,                     USA.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Qiang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Li
authors.forename: Rongqian
authors.initials: R
authors.lastname: Wu
authors.forename: Youxin
authors.initials: Y
authors.lastname: Ji
authors.forename: Wayne
authors.initials: W
authors.lastname: Chaung
authors.forename: Weifeng
authors.initials: W
authors.lastname: Dong
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D004195:Disease Models, Animal; D004926:Escherichia coli; D015870:Gene Expression; D006017:Glycolipids / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007275:Injections, Intravenous; D015850:Interleukin-6 / Q000032:analysis / Q000096:biosynthesis; D066292:Lipid Droplets; D017737:Macrophages, Peritoneal / Q000187:drug effects*; D008297:Male; D051379:Mice; D008894:Milk Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D008895:Milk, Human / Q000737:chemistry; D010587:Phagocytosis / Q000187:drug effects*; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D018805:Sepsis / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D060168:Thymocytes / Q000166:cytology; D014169:Transformation, Bacterial
chemical_list: D000954:Antigens, Surface; D006017:Glycolipids; D006023:Glycoproteins; D015850:Interleukin-6; C098942:MFGE8 protein, human; D008894:Milk Proteins; D011994:Recombinant Proteins; C110367:milk fat globule