Edwards Bryan M, He Mingyue
Crescendo Biologics Ltd., Babraham Research Campus, Cambridge, UK.
Methods Mol Biol. 2012;907:281-92. doi: 10.1007/978-1-61779-974-7_16.
Ribosome display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-ribosome-mRNA (ARM) complexes as the selection particles, (3) selection of ligand-binding ARM complexes on an immobilized ligand and (4) recovery of the selected genetic information as DNA by RT-PCR. Since PCR-based random or/and site-directed mutagenesis can be easily used to introduce mutations into the selected DNA pool in each cycle, ribosome display offers an efficient "protein evolution" tool for antibody optimization. Both prokaryotic and eukaryotic cell-free systems have been explored for ribosome display of different proteins. In this chapter, we describe the use of the eukaryotic rabbit reticulocyte ribosome display method to isolate variants of V(H) antibody fragments with improved affinities.
核糖体展示是一种无细胞技术，可从非常大的多样化DNA文库中进行体外抗体筛选和进化。它通过以下关键步骤进行：（1）生成PCR文库；（2）形成稳定的抗体-核糖体-mRNA（ARM）复合物作为筛选颗粒；（3）在固定化配体上选择与配体结合的ARM复合物；（4）通过RT-PCR将所选遗传信息作为DNA回收。由于基于PCR的随机或/和定点诱变可轻松用于在每个循环中向所选DNA池中引入突变，核糖体展示为抗体优化提供了一种高效的“蛋白质进化”工具。原核和真核无细胞系统都已用于不同蛋白质的核糖体展示。在本章中，我们描述了使用真核兔网织红细胞核糖体展示方法来分离具有更高亲和力的V(H)抗体片段变体。
