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

Michelle A. Dunstone, Lars Kjer-Nielsen, Lyudmila Kostenko, Anthony W. Purcell, Andrew G. Brooks, Jamie Rossjohn, James McCluskey

Research output: Contribution to journalArticleResearchpeer-review

4 Citations (Scopus)

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 languageEnglish
Pages (from-to)1425-1428
Number of pages4
JournalActa Crystallographica Section D: Biological Crystallography
Volume60
Issue number8
DOIs
Publication statusPublished - 1 Aug 2004

Cite this

@article{3e9863626c2b4be28431ff0a6bce57f8,
title = "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",
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 {\AA} resolution. scCD3εγ-FabOKT3 crystals belonged to space group P21, with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 {\AA}, 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 {\AA}, β = 108.84°, suggesting two complexes per asymmetric unit.",
author = "Dunstone, {Michelle A.} and Lars Kjer-Nielsen and Lyudmila Kostenko and Purcell, {Anthony W.} and Brooks, {Andrew G.} and Jamie Rossjohn and James McCluskey",
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month = "8",
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journal = "Acta Crystallographica Section D: Structural Biology",
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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 journalArticleResearchpeer-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

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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.

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JO - Acta Crystallographica Section D: Structural Biology

JF - Acta Crystallographica Section D: Structural Biology

SN - 2059-7983

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