A GENERIC METHOD FOR EXPRESSION AND USE OF TAGGED SOLUBLE VERSIONS OFCELL-SURFACE RECEPTORS

A general method for expression, purification, immobilization, detecti on and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subu nit 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 ser ves to generically ''tag'' the recombinant ECD. The resulting fusion p rotein contains a substrate sequence for protein kinase-A (PKA) adjace nt to the signal sequence from human placental alkaline phosphatase (H PAP). The HPAP signal sequence directs the formation of the phosphatid ylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are de tected on the cell surface and can be released by treatment with phosp hatidylinositol-specific phospholipase-C (PI-PLC). Based on protein pr ocessing known to occur for native HPAP, twenty amino acids from the H PAP signal sequence remain at the C-terminus of the ECD. A high affini ty monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In ad dition, the ECDs can be radiolabeled with P-32 by treatment with PKA a nd maintain the ability to bind their natural ligands. This ''tagging' ' method has been successfully applied to many other type I proteins w hich serve as cell surface receptors.