Phage and Escherichia coli expression of the human high affinity immunoglobulin E receptor alpha-subunit ectodomain. Domain localization of the IgE-binding site.
The high affinity IgE receptor is a multisubunit complex that participates in IgE-dependent activation of mast cells and basophils. The IgE-binding portion of the receptor resides exclusively in the alpha-subunit and specifically within the 180 residues of the mature extracytoplasmic portion. In this study the contiguous two-domain human alpha-subunit has been displayed on the surface of a filamentous bacteriophage in a form that specifically binds both human and murine IgE but not other isotypes. Phage display of the individual immunoglobulin-like domains reveals that the IgE-binding portion resides exclusively in the COOH-terminal domain and that this domain appears to bind IgE with lower affinity than the comparably displayed two-domain fragment. Phage display results using a chimeric rat-human alpha-subunit fragment suggest that structural elements within the NH2-terminal domain are necessary to impart high affinity IgE binding activity to the alpha-chain ectodomain. The two-domain human receptor fragment was also expressed in a soluble form from Escherichia coli and purified in one step by affinity chromatography. The solution binding of the purified receptor fragment to IgE was measured by fluorescence quench which afforded an approximate equilibrium affinity constant of 2 x 10(9) M-1 together with a stoichiometry of 1 receptor molecule/molecule of IgE. The complementary approach of phage display and E. coli expression used in this study represents a useful strategy for further analysis of discrete receptor epitope(s) that contribute to IgE binding activity.
Robertson
M W
MW
Medical Research Council Centre for Protein Engineering, Cambridge, United Kingdom.
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003090:Coliphages / Q000235:genetics*; D004247:DNA; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D051381:Rats; D017455:Receptors, IgE / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D004247:DNA