HLA-A2-PEPTIDE COMPLEXES - REFOLDING AND CRYSTALLIZATION OF MOLECULES EXPRESSED IN ESCHERICHIA-COLI AND COMPLEXED WITH SINGLE ANTIGENIC PEPTIDES
HLA-A2-PEPTIDE COMPLEXES - REFOLDING AND CRYSTALLIZATION OF MOLECULES EXPRESSED IN ESCHERICHIA-COLI AND COMPLEXED WITH SINGLE ANTIGENIC PEPTIDES
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DOI：
10.1073/pnas.89.8.3429
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发表时间：
1992-04-15
影响因子：
11.1
通讯作者：
WILEY, DC
中科院分区：
文献类型：
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作者：
GARBOCZI, DN;HUNG, DT;WILEY, DC
人类 I 类组织相容性抗原 (HLA)-A2 的两个亚基在细菌细胞中以不溶性聚集体的形式高水平（20-30 毫克/升）表达。将聚集物溶解在 8 M 尿素中，然后在抗原肽存在的情况下通过去除尿素重新折叠以形成 HLA-A2-肽复合物。已知来自流感病毒基质蛋白和核蛋白的两种肽可与 HLA-A2 结合，并且均支持重组 HLA-A2 分子的重折叠。另一种肽（1 型人类免疫缺陷病毒 gp120 包膜蛋白的九聚体）也支持重折叠。纯化的重组HLA-A2 的产率为10-15%。在没有 HLA-A2 限制性肽的情况下，不会形成稳定的 HLA-A2 复合物。已知与天然 HLA-A2 结合的单克隆抗体也与重组 HLA-A2-肽复合物结合。从细菌产生的蛋白质聚集体中重折叠的三种纯化的 HLA-A2-肽复合物在与从人淋巴母细胞中纯化的 HLA-A2 相同的条件下结晶。重组 HLA-A2 分子与流感基质九聚肽 Mp(58-66) 复合的晶体，衍射分辨率 > 1.5 埃。
The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to > 1.5-angstrom resolution.