University of Oulu

Tungekar, A. A., & Ruddock, L. W. (2022). Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity. International Journal of Molecular Sciences, 23(23), 14740.

Efficient production of Fc fusion proteins in the cytoplasm of Escherichia coli : dissecting and mitigating redox heterogeneity

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Author: Tungekar, Aatir A.1; Ruddock, Lloyd W.1
Organizations: 1Protein and Structural Biology Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, Finland
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 2.7 MB)
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Language: English
Published: Multidisciplinary Digital Publishing Institute, 2022
Publish Date: 2023-06-06


Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG₁-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG₁ CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.

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Series: International journal of molecular sciences
ISSN: 1661-6596
ISSN-E: 1422-0067
ISSN-L: 1661-6596
Volume: 23
Issue: 23
Article number: 14740
DOI: 10.3390/ijms232314740
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: This work was funded by the People Programme (Marie Skłodowska-Curie Actions) of the European Union’s Horizon 2020 Programme under REA grant agreement no. 813979 (SECRETERS).
EU Grant Number: (813979) Secreters - A new generation of microbial expression hosts and tools for the production of biotherapeutics and high-value enzymes
Copyright information: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (