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-lRtI), the α-subunit of interleukin-2 receptor (IL-2Rα) 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 phosphatidylinos-itol-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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rose-John, S. and Heinrich, P.C. 1994. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300: 281–290.
de Vos, A.M., Ultsch, M. and Kossiakoff, A.A. 1992. Human growth hormone and extracellular domain of its receptor crystal structure of the complex. Science 255: 306–312.
Stern, L.J., Brown, J.H., Jardetzky, T.S., Gorga, J.C., Urban, R.G., Strominger, J.L. and Wiley, D.C. 1994. Crystal structure of the human class IIMHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368: 215–221.
Driscoll, P.C., Altman, J.D., Boniface, J.J., Sakaguchi, K., Reay, P.A., Omichinski, J.G., Appella, E. and Davis, M.M. 1993. Two-dimensional nuclear magnetic resonance analysis of a labeled peptide bound to a class II major histocompatibility complex molecule. J. Mol. Biol. 232: 342–350.
Fountoulakis, M., Juranville, J.-F., Stuber, D., Weibel, E.K. and Garotta, G. 1990. Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli. J. Biol. Chem. 265: 13268–13275.
Fuh, G., Mulkerrin, M.G., Bass, S., McFarland, N., Brochier, M., Bourell, J.H., Light, D.R. and Wells, J.A. 1990. The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain. J. Biol. Chem. 265: 3111–3115.
Cunningham, B.C., Ultsch, M., de Vos, M., V. A., Mulkerrin, M.G., Clauser, K.R. and Wells, J.A 1991. Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254: 821–825.
Altman, J.D., Reay, P.A. and Davis, M.M. 1993. Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 90: 10330–10334.
Fountoulakis, M., Schlaeger, E.-J., Gentz, R., Juranville, J.P., Manneberg, M., Ozmen, L. and Garotta, G. 1991. Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells. Eur. J. Biochem. 198: 441–450.
Gibbs, V.C., Williams, S.R., Gray, P.W., Schreiber, R.D., Pennica, D., Rice, G. and Goeddel, D.V. 1991. The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer. Mol. Cell. Biol. 11: 5860–5866.
Low, M.G. 1987. Biochemistry of the glycosyl-phosphatidylinositol membrane protein anchors. Biochem. J. 244: 1–13.
Wettstein, D.A., Boniface, J.J., Reay, P.A., Schild, H. and Davis, M.M. 1991. Expression of a class II major histocompatibility complex (MHC) heterodimer in a lipid-linked form with enhanced peptide/soluble MHC complex formation at low pH. J. Exp. Med. 174: 219–228.
Li, B.-L., Langer, J.A., Schwartz, B. and Pestka, S. 1989. Creation of phos-phorylation sites in proteins: construction of a phosphorylatable human interferon alpha. Proc. Natl. Acad. Sci. USA 86: 558–562.
Micanovic, R., Gerber, L.D., Berger, J., Kodukula, K. and Udenfriend, S. 1990. Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase. Proc. Natl. Acad. Sci. USA 87: 157–161.
Barrett, R.W., Cwirla, S.E., Ackerman, M.S., Olson, A.M., Peters, E.A. and Dower, W.J. 1992. Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage. Anal. Biochem. 204: 357–364.
Cull, M.G., Miller, J.F. and Schatz, P.J. 1992. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc. Natl. Acad. Sci. USA 89: 1865–1869.
Cwirla, S.E., Peters, E.A., Barrett, R.W. and Dower, W.J. 1990. Pepddes on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87: 6378–6382.
Takebe, Y., Seiki, M., Fujisawa, J.-I., Hoy, P., Yokota, K., Arai, K.-I., Yoshida, M. and Arai, N. 1988. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8: 466–472.
Leonard, W.J., Depper, J.M., Crabtree, G.R., Rudikoff, S., Pumphrey, J., Robb, R.J., Kronke, M., Svetlik, P.B., Peffer, N.J., Waldmann, T.A. and Greene, W.C. 1984. Molecular cloning and expression of cDNAs for the human interleukin-2 receptor. Nature 311: 626–631.
Lin, A.Y., Devaux, B., Green, A., Sagerstrom, C., Elliott, J.F. and Davis, M.M. 1990. Expression of T cell antigen receptor heterodimers in a lipid-linked form. Science 249: 677–679.
Chua, A.O. and Gubler, U. 1989. Sequence of the cDNA for the human fibroblast type inttrieukin-1 receptor. Nucleic Acids Res. 17: 10114.
Kam, W., Clauser, E., Kim, Y.S., Kan, Y.W. and Rutter, W.J. 1985. Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA. Proc. Natl. Acad. Sci. USA 82: 8715–8719.
Seed, B. 1987. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329: 840–842.
Koke, J.A., Yang, M., Henner, D.J., Volwerk, J.J. and Griffith, O.H. 1991. High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringumsis. Protein Expr. Purif. 2: 51–58.
Eisenberg, S.P., Evans, R.J., Arend, W.P., Verderber, R., Brewer, M.T., Hannum, C.H. and Thompson, R.C. 1990. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 343: 341–346.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Whitehorn, E., Tate, E., Yanofsky, S. et al. A Generic Method for Expression and Use of “Tagged” Soluble Versions of Cell Surface Receptors. Nat Biotechnol 13, 1215–1219 (1995). https://doi.org/10.1038/nbt1195-1215
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt1195-1215