An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution.
An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution.
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一种有效的结扎方法，用于生产体外病毒以用于体外蛋白的演化。
DOI：
10.1251/bpo33
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发表时间：
2002-10-28
影响因子：
6.4
通讯作者：
Husimi, Yuzuru
中科院分区：
文献类型：
--
作者：
Tabuchi, Ichiro;Soramoto, Sayaka;Suzuki, Miho;Nishigaki, Koichi;Nemoto, Naoto;Husimi, Yuzuru
“体外病毒”是一种执行进化蛋白质工程的分子构造。病毒粒子(=病毒颗粒)(信使核糖核酸-多肽融合)，是在试管中通过嘌呤霉素将新生蛋白质与其编码的信使核糖核酸结合在一起，进行体外翻译。在本工作中，使用Y-连接的方法将嘌呤霉素连接子连接到mRNA上，这是一种在双链茎的末端进行单链连接的方法，以形成茎环结构。该反应的产率约为95%。我们将Y-连接反应与另外两种连接反应进行了比较，结果表明，Y-连接反应的产率最高。用这种“病毒基因组”可以有效地扩增体外病毒。
The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated.