Generation of peptide-MHC class I complexes through UV-mediated ligand exchange
Generation of peptide-MHC class I complexes through UV-mediated ligand exchange
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DOI：
10.1038/nprot.2006.121
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Ovaa, Huib
中科院分区：
文献类型：
--
作者：
Rodenko, Boris;Toebes, Mireille;Ovaa, Huib
主要组织相容性复合体（MHC）I类分子在细胞表面上呈递肽配体以供适当的细胞毒性T细胞识别。MHC结合的肽对于MHC复合物的稳定性是关键的，并且用于生产重组MHC复合物的标准策略是基于与特定肽的体外重折叠反应。这种策略不适合于大量MHC分子集合的高通量生产。我们已经开发了条件性MHC配体，其与MHC分子形成稳定的复合物，但可以在UV照射后裂解。在天然条件下，所得到的空的肽受体MHC分子可以装载有选择的表位。在这里，我们描述了深入的程序，高通量生产的肽-MHC（pMHC）复合物的MHC交换，通过ELISA和平行生产的MHC四聚体的T细胞检测的肽交换效率的分析。通过体外重折叠反应可以在2周内实现条件pMHC复合物的产生，并且实际的高通量MHC肽交换和随后的MHC四聚体形成需要不到一天。
Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.