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
References
Apr 30, 1992·Nature·S WeberK Karjalainen
Apr 20, 1992·Journal of Molecular Biology·E S Ward
May 25, 1992·Proceedings of the National Academy of Sciences of the United States of America·W F HooD M Kranz
May 29, 1992·Science·I EngelR D Klausner
Jan 1, 1991·European Journal of Immunology·A E Slanetz, A L Bothwell
Aug 10, 1990·Science·A Y LinM M Davis
Sep 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·C GrégoireB Malissen
Oct 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·J NovotnyE L Reinherz
Jan 1, 1990·Annual Review of Immunology·J D Ashwell, R D Klusner
Feb 1, 1990·Virology·K KurodaH D Klenk
Aug 1, 1989·Analytical Biochemistry·F MaleyT H Plummer
Nov 1, 1989·Analytical Biochemistry·S C Gill, P H von Hippel
Jan 1, 1989·Immunology Today·J M ClaverieL Bougueleret
Feb 1, 1989·Molecular and Cellular Biology·P Stanley
Mar 30, 1989·Nature·M Reth
Aug 4, 1988·Nature·M M Davis, P J Bjorkman
Feb 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·J NovotnýH N Eisen
Aug 15, 1970·Nature·U K Laemmli
Jun 30, 1983·Nature·S C MeuerE L Reinherz
Jan 1, 1984·Annual Review of Immunology·S C MeuerE L Reinherz
Apr 1, 1983·The Journal of Experimental Medicine·K HaskinsP Marrack
Mar 31, 1995·Science·G A BentleyR A Mariuzza
Nov 22, 1994·Proceedings of the National Academy of Sciences of the United States of America·H C ChangS G Nathenson
Jun 1, 1994·Analytical Biochemistry·K B LinsleyV N Reinhold
Dec 15, 1995·Science·B A FieldsR A Mariuzza
Dec 15, 1995·Structure·L C KovariM G Rossmann
Oct 1, 1993·Current Biology : CB·E K O'SheaP S Kim
Citations
Jan 7, 1998·Protein Science : a Publication of the Protein Society·N M McKernC W Ward
Aug 28, 1998·Current Opinion in Biotechnology·K C Garcia, L Teyton
Jan 22, 1998·Current Opinion in Structural Biology·I A Wilson, K C Garcia
Feb 19, 1999·Immunity·D N Garboczi, W E Biddison
Dec 17, 2009·Nature Immunology·Björn F LillemeierMark M Davis
Feb 28, 1998·The EMBO Journal·J WangE L Reinherz
Mar 26, 1999·Protein Science : a Publication of the Protein Society·E H RydbergS G Withers
Feb 14, 2004·Journal of Virology·Rob J CenterBernard Moss
Jun 8, 1999·Annual Review of Immunology·K C GarciaI A Wilson
Jan 22, 2015·Proceedings of the National Academy of Sciences of the United States of America·Dibyendu Kumar DasMatthew J Lang
May 16, 1998·Current Biology : CB·M K TengJ H Wang
Jun 15, 2006·International Immunology·Chhanda BiswasYair Argon
Nov 5, 2017·The Journal of Biological Chemistry·Robert J MallisGerhard Wagner
Aug 15, 2000·FEBS Letters·P A HoyneC W Ward
Nov 4, 1998·FEBS Letters·S CostagliolaG Vassart
Mar 17, 2018·Biochemistry. Biokhimii︠a︡·G K SelikhanovA G Gabdulkhakov
Sep 10, 1999·The Journal of Biological Chemistry·P KernH C Chang
Sep 7, 2001·The Journal of Biological Chemistry·M KimE L Reinherz
Dec 7, 2000·The Journal of Biological Chemistry·Y XiongE Reinherz
Aug 17, 2000·The Journal of Biological Chemistry·B ChenD C Wiley
Aug 22, 2001·The Journal of Biological Chemistry·C W ZhangE L Reinherz
Oct 28, 2011·The Journal of Immunology : Official Journal of the American Association of Immunologists·Yanping HuangJanis K Burkhardt
Oct 21, 2016·The Journal of Biological Chemistry·Dibyendu Kumar DasEllis L Reinherz
Aug 9, 2020·The Journal of Biological Chemistry·Ayodele AkintayoPamela Stanley
Jun 27, 2021·Proceedings of the National Academy of Sciences of the United States of America·Robert J MallisEllis L Reinherz
Dec 3, 1999·Science·E L ReinherzJ Wang