University of Oulu

Tungekar, A.A., Ruddock, L.W. Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli. Sci Rep 13, 14188 (2023).

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli

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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, 1.8 MB)
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Language: English
Published: Springer Nature, 2023
Publish Date: 2023-10-09


With increased accessibility and tissue penetration, smaller antibody formats such as antibody fragments (Fab) and single chain variable fragments (scFv) show potential as effective and low-cost choices to full-length antibodies. These formats derived from the modular architecture of antibodies could prove to be game changers for certain therapeutic and diagnostic applications. Microbial hosts have shown tremendous promise as production hosts for antibody fragment formats. However, low target protein yields coupled with the complexity of protein folding result in production limitations. Here, we report an alternative antibody fragment format ‘FabH3’ designed to overcome some key bottlenecks associated with the folding and production of Fabs. The FabH3 molecule is based on the Fab format with the constant domains replaced by engineered immunoglobulin G1 (IgG₁) CH3 domains capable of heterodimerization based on the electrostatic steering approach. We show that this alternative antibody fragment format can be efficiently produced in the cytoplasm of E. coli using the catalyzed disulfide-bond formation system (CyDisCo) in a natively folded state with higher soluble yields than its Fab counterpart and a comparable binding affinity against the target antigen.

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Series: Scientific reports
ISSN: 2045-2322
ISSN-E: 2045-2322
ISSN-L: 2045-2322
Volume: 13
Issue: 1
Article number: 14188
DOI: 10.1038/s41598-023-41525-3
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
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