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

Author:

Kamoshida Go12ORCID,Yamaguchi Daiki2ORCID,Kaya Yuki2ORCID,Yamakado Toshiki2,Yamashita Kenta2,Aoyagi Moe2,Nagai Saaya2,Yamada Noriteru2,Kawagishi Yu2,Sugano Mizuki1,Sakairi Yoshiaki1,Ueno Mikako3,Takemoto Norihiko4,Morita Yuji1ORCID,Ishizaka Yukihito3ORCID,Yahiro Kinnosuke2ORCID

Affiliation:

1. Department of Infection Control Science, Meiji Pharmaceutical University , 2-522-1 Noshio, Kiyose, Tokyo 204-8588 , Japan

2. Laboratory of Microbiology and Infection Control, Kyoto Pharmaceutical University , 5 Misasagi-Nakauchi-cho, Yamashina-ku, Kyoto 607-8414 , Japan

3. Department of Intractable Diseases, National Center for Global Health and Medicine , 1-21-1 Toyama, Shinjyuku-ku, Tokyo 162-8655 , Japan

4. Pathogenic Microbe Laboratory, Research Institute, National Center for Global Health and Medicine , 1-21-1 Toyama, Shinjyuku-ku, Tokyo 162-8655 , Japan

Abstract

Abstract 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.

Funder

Ministry of Education, Culture, Sports, Science, and Technology of Japan

Science Research Promotion Fund

Kanehara Ichiro Memorial Foundation

Morinomiyako Medical Research Foundation

Kyoto Pharmaceutical University Fund

Promotion of Scientific Research

Agency for Medical Research and Development

AMED

Publisher

Oxford University Press (OUP)

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