Kinetic screening of phage display libraries by capturing cassette technology.

制备了含有随机序列的约140个氨基酸残基的蛋白质文库作为祖先蛋白质的模型，并且表明原始酶可能不具有独特的构象，但可以发挥低催化活性。因此，已知的天然酶的高活性和特异性活性以及天然蛋白质折叠成独特构象的能力可以通过进化过程获得。然后，构建展示具有随机序列的人工蛋白的噬菌体文库，用于体外进化随机蛋白。发现离子强度是筛选噬菌体展示文库的重要参数之一。为了建立一个体外进化选择催化蛋白的系统，我们采用葡萄球菌核酸酶（SNase）-DNA作为酶-底物的模型。用N-琥珀酰亚胺基-6-马来酰亚胺基己酸酯修饰M13噬菌体，制备了具有SNase活性的噬菌体。通过PCR制备两端分别被生物素化和巯基化的底物DNA。通过用Ca 2+激活展示的SNase，回收通过DNA固定在链霉亲和素珠上的SNase展示噬菌体。此外，我们还尝试通过体外筛选的方法获得高亲和力的DNA配体，称之为“DNA适体”。将100个碱基的随机ssDNA文库（包括59聚体随机序列）置于几丁质柱上，以获得结合几丁质的几个克隆。测序结果表明，每个克隆都具有茎环或隆突环结构。

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.