PMID: 9636295Nov 1, 1995Paper

A generic method for expression and use of "tagged" soluble versions of cell surface receptors

Bio/technology
E A WhitehornR W Barrett

Abstract

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ...Continue Reading

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Citations

May 5, 1999·Journal of Chromatography. B, Biomedical Sciences and Applications·C P Woodbury, D L Venton
Aug 6, 1998·Current Opinion in Chemical Biology·W J Dower
Dec 1, 2005·Langmuir : the ACS Journal of Surfaces and Colloids·Tomas D PerezLance Kam
Mar 1, 1996·Nature Biotechnology·A CrameriW P Stemmer
Nov 14, 1997·Nature Biotechnology·N C WrightonW J Dower
May 24, 2000·Proceedings of the National Academy of Sciences of the United States of America·B P EnglandM McKinnon
Jul 9, 1996·Proceedings of the National Academy of Sciences of the United States of America·S D YanofskyR W Barrett
May 30, 1998·Proceedings of the National Academy of Sciences of the United States of America·C WülfingM M Davis
Dec 17, 2003·Journal of Nanobiotechnology·Ezio Ricca, Simon M Cutting
Oct 22, 2003·Proceedings of the National Academy of Sciences of the United States of America·Sylvie GratepancheLouis H Miller
Sep 30, 2000·Stem Cells·W J DowerR W Barrett

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