Abstract

Microbial production of antibodies offers the promise of cheap, fast, and efficient production of antibodies at an industrial scale. Limiting this capacity in prokaryotes is the absence of the post-translational machinery, present in dedicated antibody producing eukaryotic cell lines, such as B cells. There has been few and limited success in producing full-length, correctly folded, and assembled IgG in the cytoplasm of prokaryotic cell lines. One such success was achieved by utilizing the genetically engineered Escherichia coli strain SHuffle with an oxidative cytoplasm. Due to the genetic disruption of reductive pathways, SHuffle cells are under constant oxidative stress, including increased levels of hydrogen peroxide (H₂O₂). The oxidizing capacity of H₂O₂ was linked to improved disulfide bond formation, by expressing a fusion of two endoplasmic reticulum-resident proteins, the thiol peroxidase GPx7 and the protein disulfide isomerase, PDI. In concert, these proteins mediate disulfide transfer from H₂O₂ to target proteins via PDI-Gpx7 fusions. The potential of this new strain was tested with Humira, a blockbuster antibody usually produced in eukaryotic cells. Expression results demonstrate that the new engineered SHuffle strain (SHuffle2) could produce Humira IgG four-fold better than the parental strain, both in shake-flask and in high-density fermentation. These preliminary studies guide the field in genetically engineering eukaryotic redox pathways in prokaryotes for the production of complex macromolecules.

Key points

• A eukaryotic redox pathway was engineered into the E. coli strain SHuffle in order to improve the yield of the blockbuster antibody Humira.

• The best peroxidase-PDI fusion was selected using bioinformatics and in vivo studies.

• Improved yields of Humira were demonstrated at shake-flask and high-density fermenters.

中文翻译：

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通过人类 PDI-GPx7 融合蛋白的共表达，改进了基因工程大肠杆菌 SHuffle 中 Humira 抗体的生产

摘要

抗体的微生物生产提供了以工业规模廉价、快速和高效生产抗体的前景。限制原核生物的这种能力是缺乏翻译后机制，存在于产生专用抗体的真核细胞系中，例如 B 细胞。在原核细胞系的细胞质中产生全长、正确折叠和组装的 IgG 的成功很少且有限。通过利用具有氧化细胞质的基因工程大肠杆菌菌株 SHuffle取得了这样的成功。由于还原途径的遗传破坏，SHuffle 细胞处于持续的氧化应激下，包括增加的过氧化氢 (H ₂ O ₂ ) 水平。H的氧化能力通过表达两种内质网驻留蛋白硫醇过氧化物酶 GPx7 和蛋白质二硫键异构酶 PDI 的融合，₂ O ₂与改善的二硫键形成有关。一致地，这些蛋白质介导 H ₂ O _{2 的}二硫化物转移通过 PDI-Gpx7 融合靶向蛋白质。这种新菌株的潜力用修美乐进行了测试，修美乐是一种通常在真核细胞中产生的重磅抗体。表达结果表明，无论是在摇瓶中还是在高密度发酵中，新的工程 SHuffle 菌株 (SHuffle2) 都能比亲本菌株产生四倍的 Humira IgG。这些初步研究指导了原核生物中真核生物氧化还原途径的基因工程领域，以产生复杂的大分子。

关键点

• 将真核氧化还原途径设计到大肠杆菌菌株 SHuffle 中，以提高重磅抗体 Humira 的产量。

• 使用生物信息学和体内研究选择了最好的过氧化物酶-PDI 融合体。

• 在摇瓶和高密度发酵罐中展示了 Humira 产量的提高。