Abstract
Human CD3 is an essential multisubunit complex that plays a fundamental role in T-cell signalling, T-cell development and surface expression of the αβ T-cell receptor. The CD3 complex comprises the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer. Here, the expression of the human CD3εγ and CD3εδ heterodimers, both of which were expressed as single-chain polypeptides, is reported. Following refolding, functional heterodimers were immunoaffinity purified from improperly folded heterodimers using OKT3, a therapeutic monoclonal antibody specific for the CD3ε chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3εγ and CD3εδ heterodimers. Crystals of scCD3εγ-FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris-HCl pH 8.0. Crystals of scCD3εδ- FabOKT3 were grown using 20%(w/v) PEG 3350, 200 mM potassium formate, 100 mM Tris-HCl pH 8.0, 2%(v/v) MPD. Crystals of both complexes diffract to beyond 3 Å resolution. scCD3εγ-FabOKT3 crystals belonged to space group P21, with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 Å, β = 100.85° and one complex per asymmetric unit. scCD3εδ-FabOKT3 crystals belong to space group P21, with unit-cell parameters a = 101.67, b = 50.36, c = 138.7 Å, β = 108.84°, suggesting two complexes per asymmetric unit.
| Original language | English |
|---|---|
| Pages (from-to) | 1425-1428 |
| Number of pages | 4 |
| Journal | Acta Crystallographica Section D: Biological Crystallography |
| Volume | 60 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - 1 Aug 2004 |
Cite this
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The production and purification of the human T-cell receptors, the CD3εγ and CD3εδ heterodimers : Complex formation and crystallization with OKT3, a therapeutic monoclonal antibody. / Dunstone, Michelle A.; Kjer-Nielsen, Lars; Kostenko, Lyudmila; Purcell, Anthony W.; Brooks, Andrew G.; Rossjohn, Jamie; McCluskey, James.
In: Acta Crystallographica Section D: Biological Crystallography, Vol. 60, No. 8, 01.08.2004, p. 1425-1428.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - The production and purification of the human T-cell receptors, the CD3εγ and CD3εδ heterodimers
T2 - Complex formation and crystallization with OKT3, a therapeutic monoclonal antibody
AU - Dunstone, Michelle A.
AU - Kjer-Nielsen, Lars
AU - Kostenko, Lyudmila
AU - Purcell, Anthony W.
AU - Brooks, Andrew G.
AU - Rossjohn, Jamie
AU - McCluskey, James
PY - 2004/8/1
Y1 - 2004/8/1
N2 - Human CD3 is an essential multisubunit complex that plays a fundamental role in T-cell signalling, T-cell development and surface expression of the αβ T-cell receptor. The CD3 complex comprises the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer. Here, the expression of the human CD3εγ and CD3εδ heterodimers, both of which were expressed as single-chain polypeptides, is reported. Following refolding, functional heterodimers were immunoaffinity purified from improperly folded heterodimers using OKT3, a therapeutic monoclonal antibody specific for the CD3ε chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3εγ and CD3εδ heterodimers. Crystals of scCD3εγ-FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris-HCl pH 8.0. Crystals of scCD3εδ- FabOKT3 were grown using 20%(w/v) PEG 3350, 200 mM potassium formate, 100 mM Tris-HCl pH 8.0, 2%(v/v) MPD. Crystals of both complexes diffract to beyond 3 Å resolution. scCD3εγ-FabOKT3 crystals belonged to space group P21, with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 Å, β = 100.85° and one complex per asymmetric unit. scCD3εδ-FabOKT3 crystals belong to space group P21, with unit-cell parameters a = 101.67, b = 50.36, c = 138.7 Å, β = 108.84°, suggesting two complexes per asymmetric unit.
AB - Human CD3 is an essential multisubunit complex that plays a fundamental role in T-cell signalling, T-cell development and surface expression of the αβ T-cell receptor. The CD3 complex comprises the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer. Here, the expression of the human CD3εγ and CD3εδ heterodimers, both of which were expressed as single-chain polypeptides, is reported. Following refolding, functional heterodimers were immunoaffinity purified from improperly folded heterodimers using OKT3, a therapeutic monoclonal antibody specific for the CD3ε chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3εγ and CD3εδ heterodimers. Crystals of scCD3εγ-FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris-HCl pH 8.0. Crystals of scCD3εδ- FabOKT3 were grown using 20%(w/v) PEG 3350, 200 mM potassium formate, 100 mM Tris-HCl pH 8.0, 2%(v/v) MPD. Crystals of both complexes diffract to beyond 3 Å resolution. scCD3εγ-FabOKT3 crystals belonged to space group P21, with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 Å, β = 100.85° and one complex per asymmetric unit. scCD3εδ-FabOKT3 crystals belong to space group P21, with unit-cell parameters a = 101.67, b = 50.36, c = 138.7 Å, β = 108.84°, suggesting two complexes per asymmetric unit.
UR - http://www.scopus.com/inward/record.url?scp=16644376417&partnerID=8YFLogxK
U2 - 10.1107/S0907444904012041
DO - 10.1107/S0907444904012041
M3 - Article
VL - 60
SP - 1425
EP - 1428
JO - Acta Crystallographica Section D: Structural Biology
JF - Acta Crystallographica Section D: Structural Biology
SN - 2059-7983
IS - 8
ER -