Broadus Matthew R, Yew P Renee, Hann Stephen R, Lee Ethan
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, 465 21st Avenue South, U-4213A Learned Lab/MRBIII, Nashville, TN, 37232-8240, USA.
Methods Mol Biol. 2015;1263:63-73. doi: 10.1007/978-1-4939-2269-7_5.
Screens for small-molecule modulators of biological pathways typically utilize cultured cell lines, purified proteins, or, recently, model organisms (e.g., zebrafish, Drosophila, C. elegans). Herein, we describe a method for using Xenopus laevis egg extract, a biologically active and highly tractable cell-free system that recapitulates a legion of complex chemical reactions found in intact cells. Specifically, we focus on the use of a luciferase-based fusion system to identify small-molecule modulators that affect protein turnover.
生物途径小分子调节剂的筛选通常利用培养的细胞系、纯化的蛋白质，或者最近利用模式生物（如斑马鱼、果蝇、秀丽隐杆线虫）。在此，我们描述了一种使用非洲爪蟾卵提取物的方法，这是一种具有生物活性且易于操作的无细胞系统，它概括了完整细胞中发现的大量复杂化学反应。具体而言，我们专注于使用基于荧光素酶的融合系统来鉴定影响蛋白质周转的小分子调节剂。
