Skip Navigation


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
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
143/2/229    most recent
mvm213v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Kumano-Kuramochi, M.
Right arrow Articles by Machida, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumano-Kuramochi, M.
Right arrow Articles by Machida, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.