Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium <i>Acinetobacter baumannii</i> 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 <i>A. baumannii</i>. 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 <i>A. baumannii</i> 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.