人T细胞共受体CD8α同二聚体与HLA - A2复合物的组装及结晶
Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.
作者信息
Gao G F, Gerth U C, Wyer J R, Willcox B E, O'Callaghan C A, Zhang Z, Jones E Y, Bell J I, Jakobsen B K
机构信息
Molecular Immunology Group, Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, United Kingdom.
出版信息
Protein Sci. 1998 May;7(5):1245-9. doi: 10.1002/pro.5560070520.
Abstract
A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha 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 CD8alpha, 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, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.
摘要
利用基因5'区域的密码子使用改变设计了一种在大肠杆菌中过表达人CD8α细胞外免疫球蛋白结构域的策略，其设计目的是防止mRNA中形成二级结构。从细菌包涵体中回收了一段CD8α片段，该片段包含成熟蛋白的1-120位残基，不包括信号肽和膜近端柄区，并进行重折叠以产生单一物种的同型二聚体可溶性受体。HLA-A2重链、β2-微球蛋白和对应于HIV-1 pol表位的合成肽抗原也在大肠杆菌中表达、重折叠并纯化。CD8α/HLA-A2复合物在溶液中形成，并通过共结晶形成，化学计量比为一个CD8αα二聚体对一个HLA-A2-肽单元。
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