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Kinetic screening of phage display libraries by capturing cassette technology.

通过捕获盒技术对噬菌体展示库进行动力学筛选。

基本信息

批准号：
09450304
负责人：
FUKUSAKI Eiichiro
金额：
$ 8.64万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

A library of proteins with about 140 amino acid residues containing random sequences was prepared to serve as a model of ancestral proteins, and it was shown that a primordial enzyme may not take a unique conformation but can exert a low catalytic activity. Hence, the known high and specific activity of natural enzymes and the folding ability of natural proteins into unique conformations could have been acquired through the course of evolution. Then, phage libraries displaying artificial proteins with random sequences were constructed for in vitro evolution of the random proteins. Ionic strength was found to be one of the important parameters for screening of phage display libraries. To establish a system in which catalytic proteins are evolutionally selected in vitro, we employed Staphylococcal nuclease (SNase)-DNA as a model of enzyme-substrate. M13 phage displaying active SNase was prepared and modified with N-succinimidy1-6-maleimiddohexanoate. A substrate DNA, of which the both ends are biotinylated and thiolated, respectively, was prepared by PCR. The SNase displaying phage immobilized on a streptavidin beads through the DNA was recovered by activating the displayed SNase with Ca2+. Additionally, we tried to obtain high-affinity DNA ligands, termed "DNA aptamer " using in vitro selection method. A random ssDNA library of 100 bases (59mer random sequence was included) was subjected to the chitin column to obtain the several clones that bound to chitin. Sequencing shown that each clone has the stem loop or the bulge loop structures.

我们建立了一个含有140个氨基酸残基的随机序列蛋白质文库，作为祖先蛋白质的模型，结果表明，原始酶可能不具有独特的构象，但可以发挥较低的催化活性。因此，已知的天然酶的高活性和特定活性以及天然蛋白质折叠成独特构象的能力可能是通过进化过程获得的。然后，构建了展示具有随机序列的人工蛋白的噬菌体文库，用于随机蛋白的体外进化。离子强度是筛选噬菌体展示文库的重要参数之一。为了建立体外进化选择催化蛋白的体系，我们使用葡萄球菌核酸酶(SNase)-DNA作为酶-底物的模型。制备了M13噬菌体展示活性SNase，并用N-琥珀酰亚胺-1-6-马来酰亚胺己酸酯进行修饰。通过聚合酶链式反应制备了底物DNA，其两端分别为生物素化和硫代化。通过DNA固定化链霉亲和素小球上的展示噬菌体酶，用钙离子激活展示的噬菌体酶可回收展示噬菌体。此外，我们还试图通过体外筛选的方法获得高亲和力的DNA配体，称为DNA适配子。将100个碱基的随机单链DNA文库(包括59个随机序列)经几丁质柱层析，得到几个与几丁质结合的克隆。测序表明，每个克隆都具有茎环或凸起环结构。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ei-ichiro Fukusaki, Takahisa Kato, Hiroshi Maeda, Naoki kawazoe, Yoshihiro Ito, Atsushi Okazawa, Shin-ichiro Kajiyama and Akio Kobayashi: "DNA aptamers that bind chitin."Bioorganic and Medicinal Chemistry Letters.. 10 (in press). (2000)

Ei-ichiro Fukusaki、Takahisa Kato、Hiroshi Maeda、Naoki kawazoe、Yoshihiro Ito、Atsushi Okazawa、Shin-ichiro Kajiyama 和 Akio Kobayashi：“结合几丁质的 DNA 适体”。《生物有机和药物化学快报》.. 10（印刷中）。