Heterodimeric, disulfide-linked alpha/beta T cell receptors in solution.
Structural and functional analysis of T cell receptor (TcR)-ligand binding would be greatly advanced by the availability of an intact, assembled TcR in soluble form. We have produced such a molecule, by splicing the extracellular domains of a TcR to the glycosyl phosphatidylinositol membrane anchor sequences of Thy-1. The molecule is expressed in the absence of CD3 on the cell surface, and can be cleaved from the membrane by treatment with phosphatidylinositol-specific phospholipase C. The alpha and beta chains of the soluble molecule are paired in the native conformation as judged by reactivity with the anti-V beta 8 monoclonal antibody F23.1, and with the anti-clonotypic monoclonal antibody 1B2; it is a disulfide-linked dimer with a mol. mass of 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, and 47 kDa after reduction. We conclude that we have generated an alpha/beta TcR in soluble form.
Slanetz
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Department of Biology, Yale University School of Medicine, New Haven, CT 06510.
Bothwell
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AL
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D004220:Disulfides; D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D051379:Mice; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D012995:Solubility; D018800:Thy-1 Antigens; D014162:Transfection
D000954:Antigens, Surface; D004220:Disulfides; D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D010716:Phosphatidylinositols; D011134:Polysaccharides; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase