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.
Strong
R K
RK
Division of Biology 156-29, California Institute of Technology, Pasadena 91125.
Penny
D M
DM
Feldman
R M
RM
Weiner
L P
LP
Boniface
J J
JJ
Davis
M M
MM
Bjorkman
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013462:Sulfur Radioisotopes; D014162:Transfection / Q000502:physiology
D017261:Glycosylphosphatidylinositols; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes