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 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.
Liu
J
J
Laboratory of Immunobiology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Tse
A G
AG
Chang
H C
HC
Liu
J h
Jh
Wang
J
J
Hussey
R E
RE
Chishti
Y
Y
Rheinhold
B
B
Spoerl
R
R
Nathenson
S G
SG
Sacchettini
J C
JC
Reinherz
E L
EL
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006224:Cricetinae; D003460:Crystallization; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments; D007525:Isoelectric Focusing; D016350:Leucine Zippers; D013058:Mass Spectrometry; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011948:Receptors, Antigen, T-Cell; D006596:Hexosaminidases