The production and purification of the human T-cell receptors, the CD3epsilongamma and CD3epsilondelta 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 alphabeta T-cell receptor. The CD3 complex comprises the CD3epsilongamma and CD3epsilondelta heterodimers and the CD3zetazeta homodimer. Here, the expression of the human CD3epsilongamma and CD3epsilondelta 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 CD3epsilon chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3epsilongamma and CD3epsilondelta heterodimers. Crystals of scCD3epsilongamma-FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris-HCl pH 8.0. Crystals of scCD3epsilondelta-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 A resolution. scCD3epsilongamma-FabOKT3 crystals belonged to space group P2(1), with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 A, beta = 100.85 degrees and one complex per asymmetric unit. scCD3epsilondelta-FabOKT3 crystals belong to space group P2(1), with unit-cell parameters a = 101.67, b = 50.36, c = 138.7 A, beta = 108.84 degrees, suggesting two complexes per asymmetric unit.
The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biochemical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Michelle A
MA
Dunstone
Lars
L
Kjer-Nielsen
Lyudmila
L
Kostenko
Anthony W
AW
Purcell
Andrew G
AG
Brooks
Jamie
J
Rossjohn
James
J
McCluskey
D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D047468:Immunoprecipitation; D016853:Muromonab-CD3 / Q000737:chemistry* / Q000276:immunology* / Q000627:therapeutic use; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*
D016853:Muromonab-CD3; D017260:Receptor-CD3 Complex, Antigen, T-Cell