Assembly and crystallization of the complex between the human T cell coreceptor CD8α homodimer and HLA-A2

被引:26
作者
Gao, GF
Gerth, UC
Wyer, JR
Willcox, BE
O'Callaghan, CA
Zhang, ZH
Jones, EY
Bell, JI
Jakobsen, BK [1 ]
机构
[1] Univ Oxford, John Radcliffe Hosp, Inst Mol Med, Mol Immunol Grp, Oxford OX3 9DS, England
[2] Dyson Perrins Lab, Oxford OX1 3QY, England
[3] Lab Mol Biophys, Oxford OX1 3QU, England
关键词
CD8; HLA-A2; protein crystallization; protein folding; receptor-ligand complex;
D O I
10.1002/pro.5560070520
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8 alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8 alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta 2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8 alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8 alpha alpha dimer to one HLA-A2-peptide unit.
引用
收藏
页码:1245 / 1249
页数:5
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