Cell-Free Protein Expression and Functional Assay in Nanowell Chip Format

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• 作者：Philipp Angenendt ; Lajos Nyarsik ; Witold Szaflarski ; Jö ; rn Glö ; kler ; Knud H. Nierhaus ; Hans Lehrach ; Dolores J. Cahill ; and Angelika Lueking
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：April 1, 2004
• 年：2004
• 卷：76
• 期：7
• 页码：Angenendt, Lajos Nya
• 全文大小：316K
• 年卷期：v.76,no.7(April 1, 2004)
• ISSN：1520-6882

文摘

The expression and characterization of large proteinlibraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool tomeet these requirements is miniaturized high-densityplates in chip format, consisting of an array of wells withsubmicroliter volumes. Here, we show the combinationof nanowell chip technology and cell-free transcription andtranslation of proteins. Using piezoelectric dispensers, wetransferred proteins into nanowells down to volumes of100 nL and successfully detected fluorescence usingconfocal laser scanning. Moreover, we showed cell-freeexpression of proteins on a nanoliter scale using commercially available coupled transcription and translationsystems. To reduce costs, we demonstrated the feasibilityof diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present anenzymatic inhibition assay in nanowells to anticipatefurther applications, such as the high-throughput screening of drug candidates or the identification of novelenzymes for biotechnology.