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cDNA展示：一种通过固相合成和mRNA-蛋白质融合体稳定化筛选富含功能性二硫键肽的新方法。

cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.

作者信息

Yamaguchi Junichi, Naimuddin Mohammed, Biyani Manish, Sasaki Toru, Machida Masayuki, Kubo Tai, Funatsu Takashi, Husimi Yuzuru, Nemoto Naoto

机构信息

Rational Evolutionary Design of Advanced Biomolecules, Saitama Small Enterprise Promotion Corporation, Saitama Industrial Technology Center, Kawaguchi, Saitama 333-0844, Japan.

出版信息

Nucleic Acids Res. 2009 Sep;37(16):e108. doi: 10.1093/nar/gkp514. Epub 2009 Jun 15.

DOI:10.1093/nar/gkp514
PMID:19528071
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2760808/
Abstract

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.

摘要

我们报告了一种基于cDNA-蛋白质融合的用于筛选富含二硫键肽的强大展示技术，通过开发一种新型通用的嘌呤霉素连接子DNA来实现。该连接子包含四个主要部分：用于T4 RNA连接酶的“连接位点”、用于固相处理的“生物素位点”、用于将不稳定的mRNA-蛋白质融合体（mRNA展示）高效快速转化为稳定的mRNA/cDNA-蛋白质融合体（cDNA展示）的“逆转录引物位点”，其cDNA与编码的蛋白质共价连接，以及用于从固相支持物上释放复合物的“限制性酶切位点”。这不仅能够稳定mRNA-蛋白质融合体，还能促进蛋白质折叠和二硫键重排反应。我们在不同模型系统中评估了cDNA展示的性能，并证明每轮筛选的富集效率为20倍。针对白细胞介素-6受体筛选一个32个残基的随机文库，产生了含有多个二硫键且具有独特连接方式以发挥其功能的新型肽。发现这些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筛选富含二硫键的蛋白质及其他潜在应用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/73743683b3f7/gkp514f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/a58795fb916d/gkp514f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/643c9a1630e4/gkp514f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/af669b3eb37c/gkp514f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/10318f61a928/gkp514f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/f0af0646a216/gkp514f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/a358141d5420/gkp514f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/73743683b3f7/gkp514f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/a58795fb916d/gkp514f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/643c9a1630e4/gkp514f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/af669b3eb37c/gkp514f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/10318f61a928/gkp514f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/f0af0646a216/gkp514f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/a358141d5420/gkp514f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/be16/2760808/73743683b3f7/gkp514f7.jpg

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Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation.从组合文库中筛选出的抗体可阻断一种在免疫调节中起关键作用的肿瘤抗原。
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