A versatile puromycin-linker using cnvK for high-throughput in vitro selection by cDNA display
A versatile puromycin-linker using cnvK for high-throughput in vitro selection by cDNA display
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DOI：
10.1016/j.jbiotec.2015.08.020
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发表时间：
2015-10-20
影响因子：
4.1
通讯作者：
Nemoto, Naoto
中科院分区：
文献类型：
--
作者：
Mochizuki, Yuki;Suzuki, Takeru;Nemoto, Naoto
cDNA展示技术是一种功能强大的体外展示技术，用于通过体外筛选从庞大的文库中发掘功能肽和蛋白质。除了通过使用cDNA展示加速体外选择周期之外，对所选择的候选克隆进行简单快速的功能分析对于功能肽和蛋白质的高通量筛选是至关重要的。在这份报告中，一个多功能的嘌呤霉素的接头采用超快的光交联剂，3-氰基乙烯基咔唑核苷，介绍。它的效用在体外选择使用cDNA展示和蛋白质-蛋白质相互作用分析使用表面等离子体共振（SPR）系统进行了描述。使用这种通用的嘌呤霉素-接头，我们证明了FLAG表位的体外选择模型和基于SPR的测定来测量蛋白A和免疫球蛋白G的B结构域之间的解离常数。如本文所述的嘌呤霉素-接头的改进应使cDNA展示方法更容易用于设计基于蛋白质或肽的亲和试剂。(C)2015作者由爱思唯尔公司出版
cDNA display is a powerful in vitro display technology used to explore functional peptides and proteins from a huge library by in vitro selection. In addition to expediting the in vitro selection cycle by using cDNA display, easy and rapid functional analysis of selected candidate clones is crucial for high-throughput screening of functional peptides and proteins. In this report, a versatile puromycin-linker employing an ultrafast photo-cross-linker, 3-cyanovinylcarbazole nucleoside, is introduced. Its utility for both in vitro selection using cDNA display and protein-protein interaction analysis using a surface plasmon resonance (SPR) system is described. Using this versatile puromycin-linker, we demonstrated the model in vitro selection of the FLAG epitope and a SPR-based assay to measure the dissociation constant between the B domain of protein A and immunoglobulin G. Improvement of the puromycin-linker as described herein should make the cDNA display method easier to utilize for design of protein or peptide based affinity reagents. (C) 2015 The Authors. Published by Elsevier B.V.