Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris.
RAGE is a multi-ligand receptor involved in various human diseases including diabetes, cancer or Alzheimer's disease. Engagement of RAGE by its ligands triggers activation of key cellular signalling pathways such as the MAP kinase and NF-kappaB pathways. Whereas the main isoform of RAGE is a transmembrane receptor with both extra- and intracellular domains, a secreted soluble isoform (sRAGE), corresponding to the extracellular part only, has the ability to block RAGE signalling and suppress cellular activation. Administration of sRAGE to animal models of cancer or multiple sclerosis blocked successfully tumour growth and the course of the autoimmune disease. These findings demonstrate that sRAGE may have a potential as therapeutic. We present here a fast and simple purification protocol of sRAGE from the yeast Pichia pastoris. The identity of the protein was confirmed by mass spectrometry and Western blot. The protein was N-glycosylated and 95-98% pure as judged by SDS-PAGE.
Ostendorp
Thorsten
T
Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Germany.
Weibel
Mirjam
M
Leclerc
Estelle
E
Kleinert
Peter
P
Kroneck
Peter M H
PM
Heizmann
Claus W
CW
Fritz
Günter
G
D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011994:Recombinant Proteins