The production and purification of the human T-cell receptors, the CD3γ and CD3δ heterodimers: complex formation and crystallization with OKT3, a therapeutic monoclonal antibody

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 CD3γ and CD3δ heterodimers and the CD3ζζ homodimer. Here, the expression of the human CD3γ and CD3δ 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 CD3 chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3γ and CD3δ heterodimers. Crystals of scCD3γ–FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris–HCl pH 8.0. Crystals of scCD3δ–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. scCD3γ–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. scCD3δ–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.