A general approach to detect protein expression in vivo using fluorescent puromycin conjugates
A general approach to detect protein expression in vivo using fluorescent puromycin conjugates
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DOI：
10.1016/j.chembiol.2004.05.011
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发表时间：
2004-07-01
影响因子：
--
通讯作者：
Roberts, RW
中科院分区：
文献类型：
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作者：
Starck, SR;Green, HM;Roberts, RW
了解已知和未知基因产物的表达是后基因组世界的关键挑战之一。在这里，我们已经开发了一类新的试剂，以检查蛋白质表达在体内，不需要转染，放射性标记，或候选基因的事先选择。为此，我们构建了一系列带有各种荧光和生物素部分的嘌呤霉素缀合物。这些化合物容易在体外掺入细胞裂解物中表达的蛋白质产物中，并有效地穿过细胞膜以在体内蛋白质合成中起作用，如流式细胞术、选择性富集研究和Western分析所示。总体而言，这项工作表明，荧光嘌呤霉素共轭物提供了一种通用的手段，检查蛋白质的表达在体内。
Understanding the expression of known and unknown gene products represents one of the key challenges in the post-genomic world. Here, we have developed a new class of reagents to examine protein expression in vivo that does not require transfection, radiolabeling, or the prior choice of a candidate gene. To do this, we constructed a series of puromycin conjugates bearing various fluorescent and biotin moieties. These compounds are readily incorporated into expressed protein products in cell lysates in vitro and efficiently cross cell membranes to function in protein synthesis in vivo as indicated by flow cytometry, selective enrichment studies, and Western analysis. Overall, this work demonstrates that fluorescent-puromycin conjugates offer a general means to examine protein expression in vivo.