非天然氨基酸蛋白文库构建的随机插入和删除突变。

H Murakami, T Hohsaka, M Sisido
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引用次数: 5

摘要

开发了一种新的方法来生成含有非天然氨基酸的突变蛋白库。“随机插入和删除(RID)诱变”方法是基于在随机位置上删除任意数量的碱基,同时在相同位置上插入任意序列。通过该方法,在绿色荧光蛋白(GFP)基因中随机选择三个连续的碱基替换为一个CGGT 4碱基密码子。当这个DNA文库在大肠杆菌中表达时,大约80%的菌落失去了荧光。采集非荧光菌落,对基因进行测序。在四个突变基因中,有两个基因被CGGT 4碱基密码子连续替换了三个碱基。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Random insertion and deletion mutagenesis for construction of protein library containing nonnatural amino acids.

A new method was developed for the generation of a library of mutant proteins that contained nonnatural amino acids. The method, "random insertion and deletion (RID) mutagenesis", is based on the deletion of an arbitrary number of bases at random positions and, at the same time, the insertion of an arbitrary sequence into the same position. By using this method, randomly selected three consecutive bases in the gene of green fluorescence protein (GFP) were replaced by a CGGT 4-base codon. When this DNA library was expressed in E. coli, about 80% of colonies lost the fluorescence. The non-fluorescent colonies were picked up and the genes were sequenced. Replacement of three consecutive bases by CGGT 4-base codon was found in two of the four mutated genes.

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