Purification and Reconstitution of the Antigen Transport Complex TAP A PREREQUISITE FOR DETERMINATION OF PEPTIDE STOICHIOMETRY AND ATP HYDROLYSIS
Purification and Reconstitution of the Antigen Transport Complex TAP A PREREQUISITE FOR DETERMINATION OF PEPTIDE STOICHIOMETRY AND ATP HYDROLYSIS
复制标题
DOI：
10.1074/jbc.m109.047779
复制
发表时间：
2009-12-04
影响因子：
4.8
通讯作者：
Abele, Rupert
中科院分区：
文献类型：
--
作者：
Herget, Meike;Kreissig, Nina;Abele, Rupert
与抗原加工相关的转运蛋白（TAP）是适应性免疫系统的基本机器，其将抗原肽从胞质溶胶易位到内质网腔中以装载主要组织相容性I类分子。为了详细研究这种ABC转运复合物的机制，经过广泛的筛选和优化，我们已经建立了TAP的增溶、纯化和重建，以保留其在每个步骤中的功能。这使我们能够通过荧光互相关光谱法确定TAP复合物的底物结合化学计量。此外，TAP复合物显示肽结合和ATP水解之间的严格偶联，揭示在不存在肽的情况下没有基础ATP酶活性。这些结果代表了一个最佳的出发点，详细的机械研究的运输周期的TAP通过单分子实验来分析单个步骤的肽易位和肽运输和ATP水解之间的化学计量。
The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.