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Journal of Biochemistry Advance Access originally published online on November 21, 2007
Journal of Biochemistry 2008 143(2):229-236; doi:10.1093/jb/mvm213
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© 2007 The Japanese Biochemical Society.

Expression and Characterization of Recombinant C-Terminal Biotinylated Extracellular Domain of Human Receptor for Advanced Glycation End Products (hsRAGE) in Escherichia coli

Miyuki Kumano-Kuramochi, Qiuhong Xie, Yoshikiyo Sakakibara, Setsuko Niimi, Kyoko Sekizawa, Shiro Komba and Sachiko Machida*

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

*To whom correspondence should be addressed. Tel: +81 29 838 8061, Fax: +81 29 838 7996, E-mail: lili{at}affrc.go.jp

Received October 23, 2007; Accepted October 29, 2007


  Abstract

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.

Key Words: advanced glycation end products (AGE), AviTag, in vivo biotinylation, receptor for advanced glycation end products (RAGE), receptor–ligand interaction

Abbreviations: BSA, bovine serum albumin; CD, circular dichroism; SPR, surface plasmon resonance


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