Kastelic Damjana, He Mingyue
University of Ljubljana, Ljubljana, Slovenia.
Methods Mol Biol. 2012;899:61-72. doi: 10.1007/978-1-61779-921-1_4.
Ribosome display is a cell-free display technology which enables in vitro selection of antibodies from large recombinant DNA libraries. It also allows continuous introduction of mutations into the selected DNA pool by PCR-based mutagenesis in each cycle, enabling selection of antibody variants with improved affinity, specificity, and stability, thus providing a powerful "protein evolution" tool for optimizing antibody therapeutics. Ribosome display selects required molecules by linking individual proteins (phenotype) with their corresponding mRNAs (genotype) through the formation of stable Protein-Ribosome-mRNA (PRM) complexes. By affinity interaction with an immobilized ligand, the captured PRM complexes are recovered as cDNA using RT-PCR from the ribosome-attached mRNA. The DNA is then subjected to subsequent ribosome display cycles for further enrichment of rare species or cloning, expression, and sequencing to identify wanted candidates. Both prokaryotic and eukaryotic cell-free systems have been developed for ribosome display of different proteins. In this chapter, we describe ribosome display of antibodies using the eukaryotic rabbit reticulocyte system with an in situ single-primer DNA recovery method. A high-throughput Escherichia coli expression format is also described for screening of individual antibody binders from the ribosome-selected population.
核糖体展示是一种无细胞展示技术，可用于从大型重组DNA文库中体外筛选抗体。它还允许在每个循环中通过基于PCR的诱变将突变连续引入所选DNA库中，从而能够筛选出亲和力、特异性和稳定性更高的抗体变体，从而为优化抗体治疗提供了一个强大的“蛋白质进化”工具。核糖体展示通过形成稳定的蛋白质-核糖体-mRNA（PRM）复合物，将单个蛋白质（表型）与其相应的mRNA（基因型）连接起来，从而选择所需的分子。通过与固定化配体的亲和相互作用，利用RT-PCR从核糖体附着的mRNA中回收捕获的PRM复合物作为cDNA。然后将DNA进行后续的核糖体展示循环，以进一步富集稀有物种，或进行克隆、表达和测序，以鉴定所需的候选物。已开发出原核和真核无细胞系统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系统和原位单引物DNA回收方法进行抗体的核糖体展示。还描述了一种高通量大肠杆菌表达形式，用于从核糖体选择的群体中筛选单个抗体结合物。
