CRYSTALLIZATION OF A DEGLYCOSYLATED T-CELL RECEPTOR (TCR) COMPLEXED WITH AN ANTI-TCR FAB FRAGMENT

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the ''Velcro'' leucine zipper sequence to fac ilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8 -specific/H2-K-b-restricted N15 TCR could be readily immunopurified us ing the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached g lycans were GlcNAc(2)-Man(5). Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking t he unpaired cysteine at amino acid residue 183 in the C beta domain, w as thrombin-cleaved and endoglycosidase H-digested, and the two deriva tives were termed iN15 Delta(H) and N15(s) Delta(H), respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C- terminal microsequencing analysis showed the expected unique termini o f N15 alpha and beta subunits. Nevertheless, neither protein crystalli zed under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR C beta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15 Delta(H) and N15(s) Delt a(H). Both N15(s) Delta(H)-Fab[H57] and iN15 Delta(H)-Fab[H57] complex es crystallize, with the former diffracting to 2.8-Angstrom resolution . These findings show that neither intact glycans nor the conserved an d partially exposed Cys-183 is required for protein stability. Further more, our results suggest that the H57 Fab fragment aids in the crysta llization of TCRs by altering their molecular surface and/or stabilizi ng inherent conformational mobility.