Crystallization of a deglycosylated T cell receptor (TCR) complexed with an anti-TCR Fab fragment.

The Journal of Biological Chemistry
J LiuE L Reinherz

Abstract

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cbeta domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15DeltaH and N15(s)DeltaH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 alpha and beta subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cbeta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15DeltaH and N15(s)DeltaH. Both N15(s)DeltaH-Fab[H57] and iN15DeltaH-Fab[H57] complexes crystallize...Continue Reading

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