Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity
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 cat...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/23/14740 |
| _version_ | 1797463134946983936 |
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| author | Aatir A. Tungekar Lloyd W. Ruddock |
| author_facet | Aatir A. Tungekar Lloyd W. Ruddock |
| author_sort | Aatir A. Tungekar |
| collection | DOAJ |
| description | 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, <i>Escherichia coli</i>. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG<sub>1</sub>-based Fc fusion proteins in the <i>E. coli</i> 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 IgG<sub>1</sub> C<sub>H</sub>3 domain. Here, we communicate that mutating the cysteines in the C<sub>H</sub>3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of <i>E. coli</i> without compromising on yields and thermal stability. |
| first_indexed | 2024-03-09T17:46:22Z |
| format | Article |
| id | doaj.art-4a9392ed841c47d0ab5555b6b0db3df8 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T17:46:22Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-4a9392ed841c47d0ab5555b6b0db3df82023-11-24T11:07:08ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123231474010.3390/ijms232314740Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox HeterogeneityAatir A. Tungekar0Lloyd W. Ruddock1Protein and Structural Biology Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, FinlandProtein and Structural Biology Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, FinlandCost-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, <i>Escherichia coli</i>. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG<sub>1</sub>-based Fc fusion proteins in the <i>E. coli</i> 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 IgG<sub>1</sub> C<sub>H</sub>3 domain. Here, we communicate that mutating the cysteines in the C<sub>H</sub>3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of <i>E. coli</i> without compromising on yields and thermal stability.https://www.mdpi.com/1422-0067/23/23/14740<i>Escherichia coli</i>CyDisCo systemFc fusion proteinsredox heterogeneityoxidative folding |
| spellingShingle | Aatir A. Tungekar Lloyd W. Ruddock Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity International Journal of Molecular Sciences <i>Escherichia coli</i> CyDisCo system Fc fusion proteins redox heterogeneity oxidative folding |
| title | Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity |
| title_full | Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity |
| title_fullStr | Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity |
| title_full_unstemmed | Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity |
| title_short | Efficient Production of Fc Fusion Proteins in the Cytoplasm of <i>Escherichia coli</i>: Dissecting and Mitigating Redox Heterogeneity |
| title_sort | efficient production of fc fusion proteins in the cytoplasm of i escherichia coli i dissecting and mitigating redox heterogeneity |
| topic | <i>Escherichia coli</i> CyDisCo system Fc fusion proteins redox heterogeneity oxidative folding |
| url | https://www.mdpi.com/1422-0067/23/23/14740 |
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