A generic method for expression and use of "tagged" soluble versions of cell surface receptors.
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 ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.
Whitehorn
E A
EA
Affymax Research Institute, Palo Alto, CA 94304, USA.
Tate
E
E
Yanofsky
S D
SD
Kochersperger
L
L
Davis
A
A
Mortensen
R B
RB
Yonkovich
S
S
Bell
K
K
Dower
W J
WJ
Barrett
R W
RW
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019040:E-Selectin / Q000235:genetics*; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism; D006801:Humans; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010920:Placenta / Q000201:enzymology; D021382:Protein Sorting Signals / Q000235:genetics; D017472:Receptors, Interleukin-1 / Q000235:genetics*; D015375:Receptors, Interleukin-2 / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D019040:E-Selectin; D017261:Glycosylphosphatidylinositols; D021382:Protein Sorting Signals; D017472:Receptors, Interleukin-1; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase