文献类型：期刊文献

英文题名：A strategy for producing isotopically labeled peptides with antimicrobial activity or with short in vivo lifetime in Escherichia coli

作者：Ren, Qiongqiong[1];Fan, Zejun[1];Han, Rong[2,3];Sang, Meihui[1];Ma, Changxing[2,3];Zhao, Xiaoli[4];Wang, Shenlin[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Peking Univ, Coll Chem & Mol Engn, Beijing, Peoples R China;[3]Peking Univ, Beijing NMR Ctr, Beijing, Peoples R China;[4]Shandong Polytech, Jinan 250104, Peoples R China

年份：2024

卷号：116

期号：3

外文期刊名：PEPTIDE SCIENCE

收录：;EI(收录号：20240215374036);WOS:【SCI-EXPANDED(收录号:WOS:001140982700001)】；

基金：We thank the staff members of the Nuclear Magnetic Resonance System and the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for providing technical support and assistance in data collection and analysis. This work was supported by the National Natural Science Foundation of China (grant number 22274050), the Fundamental Research Funds for the Central Universities, and the Natural Science Foundation of Shandong Province (grant number ZR2022QC215). The authors thank Dr. Zhijun Liu from NFPS for assisting with the NMR data collection.

语种：英文

外文关键词：antimicrobial peptide; fusion expression system; fusion peptide; isotope labeling; NMR

摘要：Engineered Escherichia coli (E. coli) strains have been widely used to produce isotopically labeled peptides for NMR characterization on their structures and interactions. However, production of antimicrobial peptides (AMPs) by E. coli is still challenging, because AMPs are toxic to E. coli host and would lead to cell death after induction. On the other hand, expression of short peptides in E. coli host often encounter problems of the short in vivo lifetime of the peptides, which were rapidly degraded by endogenous enzymes during expression and purification steps. This report presents a practical method for overcoming these bottlenecks to enable E. coli to express AMPs and peptides that have short in vivo lifetime. This design uses the fusion of thioredoxin tags at both the N- and C-termini of the target peptides. The steric effect of the large soluble tags at both ends of the peptide reduces peptide accessibility, thereby enhancing their in vivo stability and eliminating the toxicity associated with AMPs. The approach was validated using an AMP A3K/L7K-LAH4 (K3K7) and a membrane fusion peptide (FP), which is a segment of the spike protein of SARS-CoV-2 and functions in fusing viral membranes and host cell membranes. Fusion expression of K3K7 with a thioredoxin tag only at the N-terminal resulted in high toxicity to the host cells, leading to impaired cell growth and a failure to obtain expressed fusion protein. In contrast, the fusion proteins from both termini were successfully expressed and purified. In the case of the FP, the fusion of thioredoxin at both termini significantly enhanced its stability, protecting it from enzymatic degradation during expression and purification steps. On the contrary, the FP with thioredoxin fused only at the N-terminal was found to be unstable in E. coli host strains. As stable isotope labeling on peptide is essentially important in NMR-based structure and interaction studies, we also demonstrated that the developed approach enables efficient 15N labeling for NMR studies. This strategy may also be extended to produce other challenging peptides.