High-yield and cost-effective biosynthesis process for producing antimicrobial peptide AA139

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-03-27 DOI:10.1016/j.pep.2024.106475

Ying Zhang , Yapeng Wang , Jianguang Lu , Zongqing Huang , Haoju Hua , Yanan Li , Jun Xu , Jun Feng

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引用次数: 0

Abstract

AA139, a variant of natural antimicrobial peptide (AMP) arenicin-3, displayed potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria. Nevertheless, there were currently few reports on the bioprocess of AA139, and the yields were less than 5 mg/L. Additionally, it was difficult and expensive to prepare AA139 through chemical synthesis due to its complex structure. These factors have impeded the further research and following clinical application of AA139. Here, we reported a bioprocess for the preparation of AA139, which was expressed in Escherichia coli (E. coli) BL21 (DE3) intracellularly in a soluble form via SUMO (small ubiquitin-related modifier) fusion technology. Then, recombinant AA139 (rAA139, refer to AA139 obtained by recombinant expression in this study) was obtained through the simplified downstream process, which was rationally designed in accordance with the physicochemical characteristics. Subsequently, the expression level of the interest protein was increased by 54% after optimization of high cell density fermentation (HCDF). Finally, we obtained a yield of 56 mg of rAA139 from 1 L culture with a purity of 98%, which represented the highest reported yield of AA139 to date. Furthermore, various characterizations were conducted to confirm the molecular mass, disulfide bonds, and antimicrobial activity of rAA139.

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生产抗菌肽 AA139 的高产、低成本生物合成工艺。

AA139 是天然抗菌肽 (AMP) arenicin-3 的变体，对耐多药（MDR）和广泛耐药（XDR）革兰氏阴性菌具有强效活性。然而，目前有关 AA139 生物工艺的报道很少，产量低于 5 毫克/升。此外，由于 AA139 的结构复杂，通过化学合成制备 AA139 既困难又昂贵。这些因素阻碍了 AA139 的进一步研究和后续临床应用。在此，我们报道了一种制备AA139的生物工艺，通过SUMO（小泛素相关修饰物）融合技术，AA139在大肠杆菌（E. coli）BL21（DE3）中以可溶性形式在细胞内表达。然后，根据理化特性，通过简化的下游工艺，合理设计得到重组 AA139（rAA139，指本研究中通过重组表达得到的 AA139）。随后，经过高细胞密度发酵（HCDF）优化后，相关蛋白的表达水平提高了 54%。最后，我们从 1 L 培养液中获得了 56 mg 的 rAA139，纯度为 98%，这是迄今为止报道的最高的 AA139 产率。此外，我们还对 rAA139 的分子质量、二硫键和抗菌活性进行了各种表征。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.