A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.
A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.
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DOI：
10.1038/90264
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发表时间：
2001-07
影响因子：
46.9
通讯作者：
Choo, Y
中科院分区：
文献类型：
--
作者：
Isalan, M;Klug, A;Choo, Y
通过使用噬菌体展示选择锌指模块来工程化具有预定序列特异性的DNA结合结构域，从而允许构建定制的转录因子。尽管在这一领域取得了显著的进展，但现有的蛋白质工程方法在许多方面存在不足，从而阻碍了该技术的适用性。在这里，我们提出了一个快速和方便的方法，可用于设计锌指蛋白对各种DNA结合位点。这是基于一对预制的锌指噬菌体展示文库，其被平行地用于选择两个DNA结合结构域，每个DNA结合结构域识别给定的5个碱基对序列，并且其产物被重组以产生识别预定序列的复合（9个碱基对）位点的单个蛋白质。使用该系统的工程可以在不到两周的时间内完成，并产生与DNA序列特异性结合的蛋白质，其Kd值在纳摩尔范围内。为了说明该技术，我们选择了七种不同的蛋白质结合人类免疫缺陷病毒1（HIV-1）启动子的各个区域。
DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.