Click display: a rapid and efficient in vitro protein display method for directed evolution.
Click display: a rapid and efficient in vitro protein display method for directed evolution.
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DOI：
10.1093/nar/gkad643
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发表时间：
2023-09-08
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
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作者：
我们描述了一种新的体外蛋白质展示方法-点击展示-它不依赖于在生物淘选过程中保持RNA的完整性，并在2小时内从双链输入DNA产生共价连接的蛋白质-cDNA复合物。该显示以一锅格式实现，包括串联的转录、翻译和逆转录反应。蛋白质和编码cDNA之间的稳定连接是由修饰的DNA连接体-ML介导的，所述修饰的DNA连接体-ML通过含嘌呤霉素的寡核苷酸和cDNA合成引物之间的点击化学反应产生。与下一代测序分析偶联的点击展示模拟文库的生物淘选揭示了在单轮淘选内靶结合物的>600倍富集。在25 μl反应中使用点击展示产生具有1012个个体成员的设计锚蛋白重复蛋白（DARPins）的合成文库，并且针对模型蛋白进行六轮文库淘选产生一组纳摩尔结合物。这项研究建立了点击展示作为一个强大的工具，蛋白质结合剂发现/工程，并提供了一个方便的平台，即使在RNA酶丰富的环境，如在整个细胞的体外生物淘选选择。
We describe a novel method for in vitro protein display—click display—that does not depend on maintaining RNA integrity during biopanning and yields covalently linked protein–cDNA complexes from double-stranded input DNA within 2 h. The display is achieved in a one-pot format encompassing transcription, translation and reverse transcription reactions in series. Stable linkage between proteins and the encoding cDNA is mediated by a modified DNA linker—ML—generated via a click chemistry reaction between a puromycin-containing oligo and a cDNA synthesis primer. Biopanning of a click-displayed mock library coupled with next-generation sequencing analysis revealed >600-fold enrichment of target binders within a single round of panning. A synthetic library of Designed Ankyrin Repeat Proteins (DARPins) with ∼1012 individual members was generated using click display in a 25-μl reaction and six rounds of library panning against a model protein yielded a panel of nanomolar binders. This study establishes click display as a powerful tool for protein binder discovery/engineering and provides a convenient platform for in vitro biopanning selection even in RNase-rich environments such as on whole cells.
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影响因子：
64.8
作者：
Tunyasuvunakool K;Adler J;Wu Z;Green T;Zielinski M;Žídek A;Bridgland A;Cowie A;Meyer C;Laydon A;Velankar S;Kleywegt GJ;Bateman A;Evans R;Pritzel A;Figurnov M;Ronneberger O;Bates R;Kohl SAA;Potapenko A;Ballard AJ;Romera-Paredes B;Nikolov S;Jain R;Clancy E;Reiman D;Petersen S;Senior AW;Kavukcuoglu K;Birney E;Kohli P;Jumper J;Hassabis D
通讯作者：
Hassabis D
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
DOI：
10.3390/bioengineering9100511
发表时间：
2022-09-27
期刊：
Bioengineering (Basel, Switzerland)
影响因子：
--
作者：
通讯作者：
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影响因子：
14.8
作者：
Seelig, Burckhard
通讯作者：
Seelig, Burckhard
影响因子：
2.9
作者：
Jayathilake, Chathuni;Kumachi, Shigefumi;Nemoto, Naoto
通讯作者：
Nemoto, Naoto