Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins
Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins
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DOI：
10.1016/j.jbiotec.2007.07.947
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发表时间：
2007-09-15
影响因子：
4.1
通讯作者：
Yanagawa, Hiroshi
中科院分区：
文献类型：
--
作者：
Doi, Nobuhide;Takashima, Hideaki;Yanagawa, Hiroshi
体外展示技术，如mRNA展示和DNA展示，是从定向蛋白质进化和蛋白质组学领域的组合文库中筛选具有所需功能的肽和蛋白质的有力工具。当筛选多肽（表型）的组合文库时，其每一个都展示在其基因（基因型）上，问题仍然是如何最好地回收其表型部分已结合至所需靶标的基因型部分。在这里，我们描述了在我们的DNA和mRNA展示系统中基因型（DNA或mRNA）和表型（蛋白质）之间的光可裂解2-硝基苄基接头的使用。该技术允许快速和有效的回收选定的核酸通过简单的UV照射在4摄氏度15分钟。此外，我们证实，光可裂解的DNA展示和mRNA展示系统是有用的表位肽，重组抗体，和药物-受体相互作用的体外选择。因此，这些改进的方法应该可用于治疗和诊断，例如，用于从随机肽和抗体文库中筛选高亲和力结合剂，如酶抑制剂和重组抗体，以及用于从cDNA文库中筛选药物-蛋白质相互作用。(C)2007 Elsevier B. V.保留所有权利。
In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries. (C) 2007 Elsevier B.V. All rights reserved.