cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.
cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.
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DOI：
10.1093/nar/gkp514
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发表时间：
2009-09
影响因子：
14.9
通讯作者：
Nemoto N
中科院分区：
文献类型：
--
作者：
Yamaguchi J;Naimuddin M;Biyani M;Sasaki T;Machida M;Kubo T;Funatsu T;Husimi Y;Nemoto N
我们报告了一种基于 cDNA-蛋白质融合的强大的显示技术，通过开发一种新颖且多功能的嘌呤霉素-接头 DNA，用于筛选富含二硫键的肽。该接头由四个主要部分组成：T4 RNA 连接酶的“连接位点”、用于固相处理的“生物素位点”、用于从不稳定的 mRNA-蛋白质融合物（mRNA 展示）有效且快速地转化为稳定的 mRNA/cDNA-蛋白质融合物（cDNA 展示）（其 cDNA 与其编码的蛋白质共价连接）的“逆转录引物位点”，以及用于从固相支持物释放复合物的“限制性酶位点”。这不仅能够稳定 mRNA-蛋白质融合，还能促进蛋白质折叠和二硫键改组反应。我们评估了不同模型系统中 cDNA 展示的性能，并证明每轮选择的富集效率为 20 倍。针对白细胞介素 6 受体的 32 个残基随机文库的选择产生了含有多个二硫键的新型肽，其功能具有独特的连接。发现肽在低纳摩尔范围内与靶标结合。这些结果表明我们的方法适用于体外选择富含二硫键的蛋白质和其他潜在应用。
We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.
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影响因子：
8
作者：
LABEAN, TH;KAUFFMAN, SA
通讯作者：
KAUFFMAN, SA
影响因子：
2.2
作者：
Siegel, DL;Chang, TY;Bunya, VY
通讯作者：
Bunya, VY
影响因子：
14.9
作者：
Kurz, M;Gu, K;Lohse, P A
通讯作者：
Lohse, P A
影响因子：
14.9
作者：
Miyamoto-Sato, E;Takashima, H;Yanagawa, H
通讯作者：
Yanagawa, H
影响因子：
2.9
作者：
Ja, WW;Roberts, RW
通讯作者：
Roberts, RW