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),...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2008-02, Vol.143 (2), p.229-236
Main Authors: Kumano-Kuramochi, Miyuki, Xie, Qiuhong, Sakakibara, Yoshikiyo, Niimi, Setsuko, Sekizawa, Kyoko, Komba, Shiro, Machida, Sachiko
Format: Article
Language:English
Subjects:
advanced glycation end products (AGE)
AviTag
Base Sequence
DNA Primers
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Escherichia coli - genetics
Glycation End Products, Advanced - metabolism
in vivo biotinylation
Receptor for Advanced Glycation End Products
receptor for advanced glycation end products (RAGE)
Receptors, Immunologic - genetics
Receptors, Immunologic - metabolism
receptor–ligand interaction
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
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Summary: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 (Kd) 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.
ISSN:0021-924X
DOI:10.1093/jb/mvm213