利用噬菌体展示随机表位文库鉴定蛋白质上的功能相互作用位点

Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries.

作者信息

van Zonneveld A J, van den Berg B M, van Meijer M, Pannekoek H

机构信息

Department of Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands.

出版信息

Gene. 1995 Dec 29;167(1-2):49-52. doi: 10.1016/0378-1119(95)00614-1.

DOI:10.1016/0378-1119(95)00614-1

PMID:8566810

Abstract

We describe a phage-display-based method to identify epitopes or interaction sites on proteins. DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50-200 bp. These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library. Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning. To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein. By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166. The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries.

摘要

我们描述了一种基于噬菌体展示的方法，用于鉴定蛋白质上的表位或相互作用位点。用DNA酶I对编码目标蛋白质的DNA进行部分降解，以产生50 - 200 bp的随机片段。然后将这些片段克隆到一个经过修饰的噬菌粒载体中，该载体可用于表达随机片段并构建（细菌）噬菌体展示的随机表位文库。通过亲和选择或淘选，可以从这些文库中筛选出展示功能性表位的噬菌体。为了测试该方法，我们构建了人纤溶酶原激活物抑制剂1的随机表位文库，并使用该文库来定位针对该蛋白质的单克隆抗体（mAb）的表位。通过对所选的含重叠表位片段进行比对，我们能够将mAb的表位定位在从E128到V166的39个氨基酸序列上。该方法也可应用于比单蛋白基因更复杂的系统，如病毒基因组或完整的cDNA文库。