Engineering and expression of a secreted murine TCR with reduced N-linked glycosylation
Structural studies of TCR-alpha beta heterodimers would be greatly aided by the ability to produce nonchimeric, secreted material with less carbohydrate heterogeneity. Here, we report the engineering and expression of variants of the murine TCR 2B4 in which many of the potential N-linked glycosylation sites were eliminated. Specific truncations proximal to the transmembrane region were also introduced that result in a secreted heterodimer. Although elimination of N-linked oligosaccharide on the beta-chain does not significantly affect the expression levels of 2B4 heterodimers, ablation of N-linked oligosaccharide on the alpha-chain results in a measurable reduction in expression levels of membrane-associated molecules. Secreted forms of 2B4 heterodimers in which the N-linked glycosylation of the beta-chain has been eliminated can be expressed. The secreted receptor is shown by a variety of Ab determinants to be indistinguishable from native material.
© 1994 American Association of Immunologists. Received for publication April 25, 1994. Accepted for publication July 30, 1994. We thank Rochelle Diamond (Caltech Cell Sorting Facility, Pasadena, CA) for assistance with the flow cytometric analyses, Ari Helenius for suggesting the growth of cells at redueed temperatures, and Christa Litzenberger for immunopurification of α^((0))_(truncated)/β^(-3)_(truncated) for BIAcore analysis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.