内毒素或脂多糖 (LPS) 是细菌重组蛋白表达系统中备受关注的有效免疫刺激分子。革兰氏阴性细菌鲍曼不动杆菌表现出有趣且独特的表型，其特征是 LPS 完全丧失。在这项研究中，我们开发了一种新的系统，使用 LPS 缺陷的鲍曼不动杆菌生产完全没有内毒素污染的重组蛋白。我们纯化了无内毒素的功能性绿色荧光蛋白，将内毒素污染减少了大约三个数量级，并且还纯化了功能性细胞因子肿瘤坏死因子（TNF）-α。此外，利用鲍曼不动杆菌的Omp38信号肽能够在细胞外产生重链抗体的重链可变域(VHH)。凭借这些优势，从培养上清液中纯化出与严重急性呼吸综合征冠状病毒2型刺突蛋白特异性结合的多价VHH mNb6-tri-20aa，内毒素污染比传统方法减少约2×105倍。传统表达系统中的情况。病毒中和试验证明了纯化抗体在抑制病毒感染方面的功能。此外，我们应用我们的系统生产 ozoralizumab，这是一种多特异性 VHH，可与人 TNF-α 和白蛋白结合，并作为类风湿关节炎药物上市。我们成功地从内毒素污染中纯化出功能性抗体。该系统建立了一个新的、完全无内毒素的重组蛋白表达平台，这使其有别于其他细菌表达系统，并为未来的应用带来了希望。© 作者 2024。由牛津大学出版社代表美国国家科学院。

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.