[Expression of ATR-Fc fusion protein in CHO cells].
ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor (ATR) and a fragment (hinge, CH2, and CH3 domains) of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen (PA), a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor (LF) and edema factor (EF) into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the Hind III and Not I sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3.1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fc-1D5, whose expression level was about 10 - 15 microg/(10(6) cells x d), was established. The recombinant protein expressed by the ATR-Fc-1D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fc and PA assessed by ELISA.
Gao
Li-Hua
LH
Beijing Institute of Biotechnology, Beijing 100071, China.
Hu
Xian-Wen
XW
Chen
Wei
W
Xu
Jun-Jie
JJ
Zhao
Jian
J
Chen
Hui-Peng
HP
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics*; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics*; D008840:Microfilament Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
C473287:ANTXR1 protein, human; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins