Eshelman School of Pharmacy and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Nat Protoc. 2011 Jul 21;6(8):1163-82. doi: 10.1038/nprot.2011.354.
mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca(2+)-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display-based selection can be accomplished in ~7 d.
mRNA 展示是一种强大但具有挑战性的体外选择技术，可用于从天然蛋白质组和组合多肽文库中鉴定具有所需特性的蛋白质。在无 RNA 酶的环境中，在低纳摩尔尺度上操纵展示的蛋白质时，蛋白质与其自身 RNA 的物理偶联带来了独特的挑战。本方案概述了从天然生物中产生 cDNA 文库的方法，以及产生 mRNA-蛋白质融合分子、体外功能选择和所选 cDNA 文库再生所需的步骤。从天然 cDNA 文库中鉴定蛋白酶底物和 Ca(2+)依赖的钙调蛋白结合蛋白的选择程序作为示例呈现。该方法通常可用于从各种天然蛋白质组文库中鉴定具有所需特性的蛋白质序列。一轮基于 mRNA 展示的选择可以在大约 7 天内完成。
