Ueno Shingo, Arai Hidenao, Suzuki Miho, Husimi Yuzuru
Department of Functional Materials Science, Saitama University, Shimo-okubo, Sakura-ku, Saitama, Japan.
Int J Biol Sci. 2007 Aug 29;3(6):365-74. doi: 10.7150/ijbs.3.365.
A novel method to link a nascent protein (phenotype) to its mRNA (genotype) covalently through the N-terminus was developed. The mRNA harboring amber stop codon at just downstream of initiation site was hybridized with hydrazide-modified ssDNA at upstream of coding region and was ligated to the DNA. This construct was then modified with 4-acetyl-phenylalanyl amber suppressor tRNA. This modified construct was fused with the nascent protein via the phenylalanine derivative when the mRNA uses the amber suppressor tRNA to decode the amber stop codon. The obtained fusion molecule was used successfully in selective enrichment experiments. It will be applicable for high-through-put screening in evolutionary protein engineering. In contrast to fusion molecules generated by other methods in which the protein is linked to genotype molecule through the C- terminus, our fusion molecule will serve to select a protein for which the C-terminus is essential to be active.
一种通过N端将新生蛋白质（表型）与其mRNA（基因型）共价连接的新方法被开发出来。在起始位点下游紧邻处含有琥珀色终止密码子的mRNA与编码区上游的酰肼修饰的单链DNA杂交，并与该DNA连接。然后用4-乙酰苯丙氨酰琥珀色抑制tRNA对该构建体进行修饰。当mRNA使用琥珀色抑制tRNA解码琥珀色终止密码子时，这种修饰后的构建体通过苯丙氨酸衍生物与新生蛋白质融合。所获得的融合分子成功用于选择性富集实验。它将适用于进化蛋白质工程中的高通量筛选。与其他方法产生的融合分子不同，在其他方法中蛋白质通过C端与基因型分子连接，我们的融合分子将用于选择一种蛋白质，其C端对于活性至关重要。
