Expression and characterization of recombinant C-terminal biotinylated extracellular domain of human receptor for advanced glycation end products (hsRAGE) in Escherichia coli.
The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K(d)) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.
Kumano-Kuramochi
Miyuki
M
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, Japan.
Xie
Qiuhong
Q
Sakakibara
Yoshikiyo
Y
Niimi
Setsuko
S
Sekizawa
Kyoko
K
Komba
Shiro
S
Machida
Sachiko
S
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D017127:Glycation End Products, Advanced; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011994:Recombinant Proteins