FUNCTIONAL EXPRESSION AND PURIFICATION OF THE ABC TRANSPORTER COMPLEX-ASSOCIATED WITH ANTIGEN-PROCESSING (TAP) IN INSECT CELLS
FUNCTIONAL EXPRESSION AND PURIFICATION OF THE ABC TRANSPORTER COMPLEX-ASSOCIATED WITH ANTIGEN-PROCESSING (TAP) IN INSECT CELLS
复制标题
DOI：
10.1016/0014-5793(94)00908-2
复制
发表时间：
1994-09-12
期刊：
影响因子：
3.5
通讯作者：
TAMPE, R
中科院分区：
文献类型：
--
作者：
MEYER, TH;VANENDERT, PM;TAMPE, R
利用杆状病毒表达系统，将人tap1和tap2基因产物以野生型和低聚组氨酸融合蛋白的形式在frugiperda （Spodoptera frugiperda, Sf9）昆虫细胞中过表达。这两种基因产物在同一细胞内共表达，并在微粒体膜中富集。免疫沉淀和固定化金属亲和层析揭示了TAP1和TAP2之间形成的复合物。通过肽转运到Sf9细胞的微粒体中，显示了表达的TAP复合物的功能。肽转运严格需要TAP1和TAP2以及ATP。人类TAP复合物在昆虫细胞中的功能表达首次得到证实，这表明高度发达的免疫系统的其他辅助因子对于肽在微粒体膜上的转运不是必需的。
Using the baculovirus expression system the gene products of human tap1 and tap2 were over-expressed as wild-type as well as oligohistidine fusion proteins in Spodoptera frugiperda (Sf9) insect cells. Both gene products were co-expressed within the same cells and were found enriched in microsomal membranes. Immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between TAP1 and TAP2. The expressed TAP complex was shown to be functional by peptide translocation into microsomes of Sf9 cells. Peptide transport strictly requires TAP1 and TAP2 as well as ATP. For the first time the functional expression of the human TAP complex in insect cells has been demonstrated, indicating that additional cofactors of a highly developed immune system are not essential for peptide transport across microsomal membranes.