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。革兰氏阴性菌鲍曼不动杆菌表现出有趣且独特的表型,其特征在于LPS的完全丧失。在这项研究中,我们开发了一种新的系统,用于使用缺乏LPS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,减少了大约三个数量级的内毒素污染,并纯化了功能性细胞因子肿瘤坏死因子(TNF)-α。此外,鲍曼不动杆菌的Omp38信号肽的利用使得能够在细胞外产生重链重链(VHH)抗体的可变结构域。有了这些优势,mNb6-tri-20aa,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的刺突蛋白特异性结合的多价VHH,与常规表达系统相比,内毒素污染减少了约2×105倍。病毒中和测定证明了纯化抗体在抑制病毒感染中的功能。此外,我们应用我们的系统来生产奥索利珠单抗,一种多特异性VHH,与人TNF-α和白蛋白结合,并作为类风湿性关节炎药物上市。我们成功地从内毒素污染中纯化了功能性抗体。这个系统建立了一个新的,用于表达重组蛋白的完全无内毒素的平台,这将其与其他细菌表达系统区分开来,并对未来的应用充满希望。
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.