Schilling Tobias, Biedendieck Rebekka, Moran-Torres Rafael, Saaranen Mirva J, Ruddock Lloyd W, Daniel Rolf, van Dijl Jan Maarten
University Medical Center Groningen, Department of Medical Microbiology, University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700RB Groningen, The Netherlands.
Braunschweig Centre of Systems Biology (BRICS) and Institute of Microbiology, Technische Universität Braunschweig, Rebenring 56, 38106 Braunschweig, Germany.
ACS Synth Biol. 2025 Mar 21;14(3):740-755. doi: 10.1021/acssynbio.4c00688. Epub 2025 Feb 27.
is a bacterial cell factory with outstanding protein secretion capabilities that has been deployed as a workhorse for the production of industrial enzymes for more than a century. Nevertheless, the production of other proteins with , such as antibody formats, has thus far been challenging due to specific requirements that relate to correct protein folding and disulfide bond formation upon export from the cytoplasm. In the present study, we explored the possibility of producing functional antibody formats, such as scFvs and scFabs, using the genome-reduced - and strain lineage. The applied workflow included selection of optimal chassis strains, appropriate expression vectors, signal peptides, growth media, and analytical methods to verify the functionality of the secreted antibody fragments. The production of scFv fragments was upscaled to the 1 L bioreactor level. As demonstrated for a human C-reactive protein-binding scFv antibody by mass spectrometry, biolayer interferometry, circular dichroism, free thiol cross-linking with -ethylmaleimide, and nano-differential scanning fluorimetry, strains can secrete fully functional, natively folded, disulfide-bonded, and thermostable antibody fragments. We therefore conclude that genome-reduced chassis strains are capable of secreting high-quality antibody fragments.
是一种具有出色蛋白质分泌能力的细菌细胞工厂，在一个多世纪以来一直被用作生产工业酶的主力。然而，由于从细胞质中输出时与正确的蛋白质折叠和二硫键形成相关的特定要求，迄今为止，生产其他蛋白质，如抗体形式，一直具有挑战性。在本研究中，我们探索了使用基因组简化的 菌株谱系生产功能性抗体形式，如单链抗体片段（scFvs）和单链抗体（scFabs）的可能性。应用的工作流程包括选择最佳底盘菌株、合适的表达载体、信号肽、生长培养基以及验证分泌抗体片段功能的分析方法。单链抗体片段（scFv）的生产规模扩大到了1 L生物反应器水平。通过质谱、生物层干涉术、圆二色性、与N - 乙基马来酰亚胺的游离巯基交联以及纳米差示扫描荧光法对人C反应蛋白结合单链抗体片段（scFv）抗体的验证表明， 菌株能够分泌功能完全正常、天然折叠、具有二硫键且热稳定的抗体片段。因此，我们得出结论，基因组简化的 底盘菌株能够分泌高质量的抗体片段。
