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.

Dunstone MA; Kjer Nielsen L; Kostenko LPurcell AWBrooks AGRossjohn JMcCluskey J

首页> 外文期刊>Acta crystallographica.Section D. Biological crystallography >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.

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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.

机译：人类的生产和净化CD3epsilongamma和t细胞受体CD3epsilondelta:形成复杂的形成和结晶OKT3治疗单克隆抗体。

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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.

机译：人类CD3必不可少multisubunit复杂在t细胞中起着基础性作用信号,t细胞发育和表面alphabeta t细胞受体的表达。包含CD3epsilongamma和CD3复杂CD3epsilondelta形成和CD3zetazeta为。这两个被表示为单链多肽,是报道。功能是形成immunoaffinity纯化折叠形成不当使用OKT3治疗性单克隆抗体特定的CD3epsilon链。OKT3用于复杂的Fab片段单独CD3epsilongamma和CD3epsilondelta形成。scCD3epsilongamma-FabOKT3种植使用3350年15% (w / v)挂钩,200毫米氟化钾,1008.0毫米Tris-HCl pH值。scCD3epsilondelta-FabOKT3种植使用3350年20% (w / v)挂钩,200毫米甲酸钾,100mM Tris-HCl pH值8.0,2% (v / v) MPD。复合物衍射超出3一项决议。scCD3epsilongamma-FabOKT3晶体属于空间群P2(1),晶胞参数=67.70, b = 55.77, c = 96.05,β= 100.85度,一个复杂的不对称单元。scCD3epsilondelta-FabOKT3晶体属于空间群P2(1),晶胞参数=101.67, b = 50.36, c = 138.7,β= 108.84度,这两个配合物/不对称单位。

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《Acta crystallographica.Section D. Biological crystallography》 |2004年第8期|1425-1428|共4页
作者
Dunstone MA; Kjer Nielsen L; Kostenko LPurcell AWBrooks AGRossjohn JMcCluskey J;
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The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biochemical Sciences, Monash University, Clayton, Victoria 3800, Australia.;

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正文语种英语
中图分类化学;
关键词
T lymphocyte receptor; CD3 Antigens; Immunoglobulin Fab Fragmentscrystal structureComplexationPurificationcrystalsT-LymphocytesMuromonab-CD3cell receptorMonoclonal AntibodiesCrystallization;

机译：工厂Fragmentscrystal;

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.

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