In vitro selection of zinc finger DNA-binding proteins through ribosome display
In vitro selection of zinc finger DNA-binding proteins through ribosome display
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DOI：
10.1016/j.bbrc.2006.05.029
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发表时间：
2006-07-07
影响因子：
3.1
通讯作者：
Kobatake, Eiry
中科院分区：
文献类型：
--
作者：
Ihara, Hiroshi;Mie, Masayasu;Kobatake, Eiry
具有序列特异性的dna结合蛋白具有多种应用。为了创造新的功能dna结合蛋白，体内选择方法已经发展起来。然而，也有……这些方法的关键问题，如文库大小的限制和宿主细胞表达有毒蛋白的困难。为了克服这些问题，我们开发了一种利用体外核糖体展示技术来选择dna结合蛋白的新方法。以锌指随机序列Zif268为基础，制备三个锌指dna结合蛋白文库，并在体外转录为mRNA。利用生物素化的靶DNA片段与链霉亲和素磁珠结合，在兔网网细胞体外翻译系统中选择由mRNA、核糖体和翻译后的DNA结合蛋白在翻译过程中形成的三元核糖体复合物。从选择的复合物中提取的mrna用反转录PCR扩增，然后测序。这是涉及体外核糖体展示技术的dna结合蛋白选择的第一个报告。(c) 2006爱思唯尔公司版权所有。
DNA-binding proteins with sequence specificities have a variety of applications. To create novel functional DNA-binding proteins, in vivo selection methods have been developed. There are, however,. Crucial problems with such methods, e.g., limitation of library size and difficulty of expression of toxic proteins for the host cells. In order to overcome these problems, we developed a novel way to select DNA-binding proteins using an in vitro ribosome display technique. The three zinc finger DNA-binding protein libraries, based on a Zif268 containing randomized sequence in each finger, were prepared and transcribed to mRNA in vitro. The ternary ribosomal complexes, formed by mRNA, ribosome, and translated DNA-binding protein during translation in a rabbit reticulocyte in vitro translation system, were selected with biotinylated target DNA fragments bound to streptavidin magnetic beads. The extracted mRNAs from the selected complexes were amplified using reverse transcription PCR and then sequenced. This is the first report of the selection of DNA-binding proteins involving an in vitro ribosome display technique. (c) 2006 Elsevier Inc. All rights reserved.