Cell-free protein expression and functional assay in nanowell chip format
Cell-free protein expression and functional assay in nanowell chip format
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DOI：
10.1021/ac035114i
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发表时间：
2004-04-01
影响因子：
7.4
通讯作者：
Lueking, A
中科院分区：
文献类型：
--
作者：
Angenendt, P;Nyarsik, L;Lueking, A
大型蛋白质文库的表达和表征需要高通量工具来快速且经济高效地表达和筛选。满足这些要求的一种有前途的工具是芯片格式的小型化高密度板，由一系列亚微升体积的孔阵列组成。在这里，我们展示了纳米孔芯片技术与蛋白质无细胞转录和翻译的结合。使用压电分配器，我们将蛋白质转移到体积为 100 nL 的纳米孔中，并使用共焦激光扫描成功检测到荧光。此外，我们使用市售的偶联转录和翻译系统展示了纳升规模的蛋白质无细胞表达。为了降低成本，我们证明了在表达前稀释体外转录和翻译混合物的可行性。此外，我们在纳米孔中提出了一种酶抑制测定法，以预测进一步的应用，例如候选药物的高通量筛选或生物技术中新型酶的鉴定。
The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.