Ihara Hiroshi, Mie Masayasu, Funabashi Hisakage, Takahashi Fumio, Sawasaki Tatsuya, Endo Yaeta, Kobatake Eiry
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Biochem Biophys Res Commun. 2006 Jul 7;345(3):1149-54. doi: 10.1016/j.bbrc.2006.05.029. Epub 2006 May 12.
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.
具有序列特异性的DNA结合蛋白有多种应用。为了创建新型功能性DNA结合蛋白，人们开发了体内筛选方法。然而，这些方法存在一些关键问题，例如文库大小的限制以及有毒蛋白对宿主细胞表达的困难。为了克服这些问题，我们开发了一种使用体外核糖体展示技术来筛选DNA结合蛋白的新方法。制备了三个基于每个指状结构中含有随机序列的Zif268的锌指DNA结合蛋白文库，并在体外转录为mRNA。在兔网织红细胞体外翻译系统中翻译过程中，由mRNA、核糖体和翻译后的DNA结合蛋白形成的三元核糖体复合物，用与链霉亲和素磁珠结合的生物素化靶DNA片段进行筛选。从选定的复合物中提取的mRNA使用逆转录PCR进行扩增，然后进行测序。这是关于使用体外核糖体展示技术筛选DNA结合蛋白的首次报道。
