Cloning, expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

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TitleCloning, expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.
Publication TypeJournal Article
Year of Publication1996
AuthorsLebedeva, MI, Fields, BA, Spits, H, Panchamoorthy, G, Brenner, MB, Mariuzza, RA
JournalProtein Sci
Volume5
Issue12
Pagination2638-42
Date Published1996 Dec
ISSN0961-8368
KeywordsAmino Acid Sequence, Base Sequence, Cloning, Molecular, Crystallization, Crystallography, X-Ray, Escherichia coli, Gene Expression, Humans, Immunoglobulin Variable Region, Molecular Sequence Data, Receptors, Antigen, T-Cell, gamma-delta
Abstract

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.

DOI10.1002/pro.5560051226
Alternate JournalProtein Sci.
PubMed ID8976572
PubMed Central IDPMC2143324