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

Protein Expression and Purification
Rosemarie WiltonFred J Stevens

Abstract

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...Continue Reading

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Citations

Mar 3, 2010·Annual Review of Immunology·Gary P SimsAnthony J Coyle
Jul 31, 2010·Cardiovascular Psychiatry and Neurology·Estelle LeclercStefan W Vetter
Mar 6, 2008·Molecular Nutrition & Food Research·Timo M BuetlerThierry Delatour
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Dec 13, 2007·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Mar 1, 2020·Medicinal Research Reviews·Selena Le BaggeJosephine M Forbes
Feb 6, 2007·The Journal of Immunology : Official Journal of the American Association of Immunologists·Ke ZenYuan Liu
Jan 1, 2020·Scientific Reports·Alexander MoysaMichal Dadlez
Sep 11, 2016·Current Diabetes Reports·Sherman S LeungDanielle J Borg

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