Slanetz A E, Bothwell A L
Department of Biology, Yale University School of Medicine, New Haven, CT 06510.
Eur J Immunol. 1991 Jan;21(1):179-83. doi: 10.1002/eji.1830210127.
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.
如果能获得完整的、组装好的可溶性形式的T细胞受体（TcR），将极大地推动对TcR-配体结合的结构和功能分析。我们通过将TcR的细胞外结构域与Thy-1的糖基磷脂酰肌醇膜锚定序列拼接，制备出了这样一种分子。该分子在细胞表面无CD3的情况下表达，并且可以通过用磷脂酰肌醇特异性磷脂酶C处理从膜上切割下来。通过与抗Vβ8单克隆抗体F23.1以及抗克隆型单克隆抗体1B2的反应性判断，可溶性分子的α链和β链以天然构象配对；它是一种二硫键连接的二聚体，在非还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上分子量为95 kDa，还原后为47 kDa。我们得出结论，我们已经产生了可溶性形式的α/β TcR。
