Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli.

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Research Abstract Details 

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Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Abstract Text:

Rosemarie Wilton,Mohammed A Yousef,Poonam Saxena,Mercedes Szpunar,Fred J Stevens,

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Publishing Authors By Initials

R Wilton,MA Yousef,P Saxena,M Szpunar,FJ Stevens,

For similar proteins: membrane proteins: receptors, cell surface: receptors, immunologic research abstracts see: proteins: membrane proteins: receptors, cell surface: receptors, immunologic research

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MEDLINE DATE:

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Protein expression and purification

VOLUME: 47

Page Numbers: 25-35

Journal Abbreviation: Protein Expr. Purif.

ISSN: 1046-5928

DAY: 8

MONTH: 02

YEAR: 2006

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 9101496

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Keywords Mesh Terms:

KEYWORDS: Receptors, Immunologic

MESH TERMS: isolation & purification

Chemical & Substance for Abstract: Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli. Information

Substance Name: amyloid fibril protein AL

Registry Number: 0

Grant and Affiliation Information for Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli.

AFFILIATION: Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, and Department of Biochemistry and Molecular Biology and Biophysics Core Facility, University of Chicago 60637, USA. rwilton@anl.gov

Country: United States

AGENCY: United States NIA

GRANT: AG22167

ACRONYM: AG

MEDLINETA: Protein Expr Purif

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