Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies
Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies
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DOI：
10.21769/bioprotoc.4125
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发表时间：
2021-08-20
期刊：
影响因子：
0.8
通讯作者：
Murakami, Hiroshi
中科院分区：
文献类型：
--
作者：
Kondo, Taishi;Eguchi, Minori;Murakami, Hiroshi
最近，我们开发了结合嘌呤霉素连接子（TRAP）展示的转录/翻译技术，作为一种快速的体外筛选方法来获取类抗体蛋白。对于体外筛选而言，制备多样性高的mRNA文库非常重要。在此，我们描述了一种制备理论多样性大于10¹³的单域抗体mRNA文库的方法。首先，我们合成了两条长的单链DNA，它们对应于单域抗体DNA的片段，在BC环和FG环中具有随机密码子。在含有经修饰保护的3'端的引导寡核苷酸的支持下，这些寡核苷酸通过T4 DNA连接酶进行连接。通过PCR扩增产物DNA后，每个DNA片段的一端用II型限制酶BsaI进行酶切，然后使用T4 DNA连接酶连接所得的DNA片段。在扩增DNA产物之后，通过T7 RNA聚合酶合成mRNA。这种方法简单，可用于制备各种类抗体蛋白的mRNA文库。[图表]
Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody mRNA libraries with greater than 10(13) theoretical diversity. First, we synthesized two long single-stranded DNAs that corresponded to fragments of monobody DNA, with random codons in the BC and FG loops. These oligonucleotides were ligated by T4 DNA ligase with the support of guide oligonucleotides containing 3' ends that were protected by a modification. After amplifying the product DNAs by PCR, one end of each DNA fragment was digested with the type II restriction enzyme Bsal, and the resulting DNA fragments were ligated using T4 DNA ligase. After amplification of the DNA product, mRNAs were synthesized by T7 RNA polymerase. This method is simple and could be used for the preparation of mRNA libraries for various antibody-like proteins.[GRAPHICS].