摘要

选择蛋白质编码序列的高度表达变体的策略通常基于尝试 - 错误方法，这是耗时且昂贵的。 我们使用翻译偶联抗生素抗性标记来解决这个问题。 该系统要求靶基因可以在3'末端与翻译偶联元件和抗生素抗性基因融合。 然后可以在存在高抗生素浓度的情况下使用快速且简单的全细胞存活测定来选择高表达的靶基因。 在这里，我们表明该系统可以用来从库中选择高度表达的克隆，取样翻译起始位点。

Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can be fused at the 3′-end with a translational coupling element and an antibiotic resistance gene. Highly expressed target genes can then be selected using a fast and simple whole cell survival assay in the presence of high antibiotic concentrations. Herein we show that the system can be used to select highly expressing clones from libraries sampling translation initiation sites.

https://link.springer.com/protocol/10.1007/978-1-4939-7295-1_16