Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Methods. 2013 Mar 15;60(1):55-69. doi: 10.1016/j.ymeth.2012.11.004. Epub 2012 Nov 28.
mRNA-display is an amplification-based, iterative rounds of in vitro protein selection technique that circumvents a number of difficulties associated with yeast two-hybrid and phage display. Because of the covalent linkage between the genotype and the phenotype, mRNA-display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected from a library with very high diversity. The purpose of this article is to provide a summary of the field and practical framework of mRNA-display-based selections. We summarize the advantages and limitations of selections using mRNA-display as well as the recent applications, namely, the identification of novel affinity reagents, target-binding partners, and enzyme substrates from synthetic peptide or natural proteome libraries. Practically, we provide a detailed procedure for performing mRNA-display-based selections with the aim of identifying protease substrates and binding partners of a target protein. Furthermore, we describe how to confirm the function of the selected protein sequences by biochemical assays and bioinformatic tools.
mRNA 展示是一种基于扩增的、迭代的体外蛋白质选择技术，它规避了酵母双杂交和噬菌体展示相关的许多困难。由于基因型和表型之间的共价连接，mRNA 展示为从具有非常高多样性的文库中选择肽或蛋白质序列后读取和扩增提供了强大的手段。本文的目的是提供基于 mRNA 展示的选择领域和实用框架的总结。我们总结了使用 mRNA 展示进行选择的优点和局限性，以及最近的应用，即从合成肽或天然蛋白质组文库中鉴定新型亲和试剂、靶标结合伴侣和酶底物。实际上，我们提供了一种详细的执行基于 mRNA 展示的选择的程序，目的是鉴定靶标蛋白的蛋白酶底物和结合伴侣。此外，我们描述了如何通过生化分析和生物信息学工具来确认所选蛋白质序列的功能。
