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Assembly and crystallization of the complex between the human T cell coreceptor CD8α homodimer and HLA‐A2
Author(s) -
Gao George F.,
Gerth Ulrich C.,
Wyer Jessica R.,
Willcox Benjamin E.,
O'callaghan Christopher A.,
Bell John I.,
Jakobsen Bent K.,
Zhang Zhihong,
Jones E. Yvonne
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070520
Subject(s) - dimer , epitope , cd8 , human leukocyte antigen , t cell receptor , escherichia coli , peptide , chemistry , biology , microbiology and biotechnology , crystallization , hla b , signal peptide , cytotoxic t cell , gene , peptide sequence , antigen , biochemistry , t cell , immune system , genetics , in vitro , organic chemistry
A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8α 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 CD8α, 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, β2‐microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV‐1 were also expressed in E. coli , refolded and purified. CD8α/HLA‐A2 complexes were formed in solution and by co‐crystallization with a stoichiometry of one CD8αα dimer to one HLA‐A2‐peptide unit.