Crystallization of a Deglycosylated T Cell Receptor (TCR) Complexed with an Anti-TCR Fab Fragment | Academic Article individual record
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, with the former diffracting to 2.8-A resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.

author list (cited authors)
Liu, J. u., Tse, A., Chang, H., Liu, J., Wang, J., Hussey, R. E., ... Reinherz, E. L.
publication date
1996
keywords
  • Isoelectric Focusing
  • Crystallography, X-Ray
  • Chromatography, Gel
  • Leucine Zippers
  • Hexosaminidases
  • Antibodies, Monoclonal
  • Cricetinae
  • Glycosylation
  • Animals
  • Crystallization
  • Immunoglobulin Fab Fragments
  • Chromatography, Affinity
  • Mass Spectrometry
  • Protein Conformation
  • Baculoviridae
  • Receptors, Antigen, T-Cell
altmetric score

6.0

citation count

28