利用反式翻译机制设计ssrA-X标签提高蛋白质定点非天然氨基酸引入质量

Design the ssrA-X Tag to Improve the Quality and Quantity of Proteins with Site-Incorporated Unnatural Amino Acids Based on the TransTranslation Mechanism

基于遗传密码扩增技术实现向蛋白质中终止密码子处定点引入非天然氨基酸会不可避免地造成截短型蛋白堆积以及产率降低。通过利用细菌天然存在反式翻译机制,即tmRNA介导的蛋白降解机制,作者设计了ssrA-X标签。将ssrA-X插入目标蛋白,利用截短型蛋白的C端暴露出ssrA标签能够被Ssp B识别,进而被clpX降解的特性,完全消除了截短型蛋白,同时增加了含非天然氨基酸的蛋白产量。通过荧光蛋白Cp Venus,融合蛋白GFP-Spycatcher,膜展示蛋白Omp A-Darpins分别验证了ssrA-X标签的可行性。

The site-incorporation of unnatural amino acids to the target protein at terminal conden based on the gentic code expanding usually have an inevitably issue.This issue is the low quality and quantity of protein caused by the accumulation of the truncated protein.Inspired by the natural trans-translation mechanism in bacteria,the authors designed the ssrA-X tag.This tag can be recognized by sspB,and the protein bearing this tag will be degraded once the tag is exposed at the C terminal of the truncated protein.This mechanism will eliminate the truncated protein and improve the quality and quantity of the full-length protein.To improve the quality and quantity of the proteins with site-incorprated unnatural amino acid,the authors constructed the ssrA-X tag and tested its function.In this study,the authors first verified the feasibility of the ssrA-X tag in the model fluorescent protein Cp Venus.The authors co-transformed several unnatural amino acids incorporation plasmids and the model protein Cp Venus plasmid into the host bacteria DH10B.The results show that this ssrA-tag fits these incorporation system well.Then the authors tested the function in the fusion protein GFP-Spycatcher.Due to the high molecular weight,this model fusion protein GFP-Spycatcher is hard to ferment efficiently.As mentioned above,the authors added this ssrA-tag to the model protein and co-transformed the two plasmids.The results of this part of experiment found clear support for the function of ssrA-tag to improve the quality and quantity in high molecular weight fusion protein fermentation.Finally,the authors applied this ssrA-tag to the bacteria surface display.The authors added the tag to the darpins selection plasmid,the Omp A-TAG-Darpins.The FCM data showed that no truncated or unladen OmpA was found on the membrane of the host bacteria.Overall,these experiments results suggest this ssrA-tag will provide a powerful tool for improving the quality and quantity of the unnatural amino acids site-incoporated protein.