Lappöhn Carolin A, Oestreich Arne M, Stei Robin, Weber Linus G, Maerz Lea, Wolff Michael W
Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen (THM), Wiesenstr. 14, 35390 Giessen, Germany.
Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen (THM), Wiesenstr. 14, 35390 Giessen, Germany; Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Ohlebergsweg 12, 35392 Giessen, Germany.
J Biosci Bioeng. 2023 Nov;136(5):358-365. doi: 10.1016/j.jbiosc.2023.09.003. Epub 2023 Sep 26.
The production of antimicrobial peptides/proteins (AMPs) in sufficient quantities for clinical evaluation is challenging because complex peptides are unsuitable for chemical synthesis, natural sources have low yields, and heterologous systems often have low expression levels or require product-specific process adaptations. Here we describe the production of a complex AMP, the insect metalloproteinase inhibitor (IMPI), by adding a C-terminal C-tag to increase the yield compared to the unmodified peptide. We used a design of experiments approach for process intensification in Escherichia coli Rosetta-gami 2(DE3)pLysS cells and achieved a yield of 260 mg L, which is up to 30-fold higher than previously reported. The C-tag also enhanced product purity but had no effect on IMPI activity, making tag removal unnecessary and therefore simplifying process analytics and downstream processing. We have confirmed that the C-tag is compatible with the peptide and could form the basis of a platform technology for the expression, purification and detection of diverse AMPs produced in E. coli.
以足够数量生产抗菌肽/蛋白质（AMPs）用于临床评估具有挑战性，因为复杂肽不适合化学合成，天然来源产量低，而异源系统通常表达水平低或需要针对产品的工艺调整。在此，我们描述了一种复杂AMPs——昆虫金属蛋白酶抑制剂（IMPI）的生产方法，即通过添加C末端C标签来提高产量，与未修饰的肽相比产量有所增加。我们在大肠杆菌Rosetta-gami 2(DE3)pLysS细胞中采用实验设计方法进行工艺强化，产量达到260 mg/L，比之前报道的高出30倍。C标签还提高了产品纯度，但对IMPI活性没有影响，无需去除标签，从而简化了过程分析和下游加工。我们已证实C标签与该肽兼容，可为在大肠杆菌中表达、纯化和检测多种AMPs的平台技术奠定基础。
