Open AccessExpression and characterization of recombinant human milk fat globule-EGF factor VIII
Author(s) -
Xiaoling Qiang,
Jianhua Li,
Rongqian Wu,
Youxin Ji,
Wayne Chaung,
Weifeng Dong,
Haichao Wang
Publication year - 2011
Publication title -
international journal of molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.048
H-Index - 90
eISSN - 1791-244X
pISSN - 1107-3756
DOI - 10.3892/ijmm.2011.782
Subject(s) - biology , recombinant dna , immunogenicity , apoptosis , antibody , phagocytosis , in vivo , sepsis , microbiology and biotechnology , pharmacology , immunology , biochemistry , gene
Apoptosis plays an important role in the patho-biology of sepsis. The opsonizingprotein milk fat globule-EGF factor VIII (MFG-E8) is involved in apoptotic cellclearance. Our previous studies have shown that administration of rat MFG-E8-containingexosomes or recombinant murine MFG-E8 (rmMFG-E8) is protective in a rat modelof sepsis induced by cecal ligation of puncture (CLP). However, one obstacle hamperingthe development of MFG-E8 as a therapeutic agent for septic patients is the potentialimmunogenicity of animal proteins in humans. The purpose of this study, therefore,was to express recombinant human MFG-E8 (rhMFG-E8) and characterize its biologicalactivity. Using an E. coli system, we successfully expressed and purified themature molecule of human MFG-E8 (Leu24-Cys387). The purity of rhMFG-E8 was over99% and it was immunoreactive for specific anti-human MFG-E8 antibodies. Aminoacid 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 increasedperitoneal macrophage phagocytosis of apoptotic thymocytes, which was as effectiveas 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 administeredintravenously at the time of CLP. Our results demonstrated that both rhMFG-E8and rmMFG-E8 reduced thymocyte apoptosis and plasma levels of lactate and IL-6at 20 h after CLP, and improved the 10-day survival rate. Thus, we have successfullyexpressed and purified biologically active rhMFG-E8. Our newly-expressed rhMFG-E8is highly effective in the rat model of sepsis.