Production of modified antibody formats in the cytoplasm of Escherichia coli

Thesis event information

Date and time of the thesis defence

Place of the thesis defence

Leena Palotie Auditorium 101A, Aapistie 5, Kontinkangas Campus, University of Oulu

Topic of the dissertation

Production of modified antibody formats in the cytoplasm of Escherichia coli

Doctoral candidate

Master of Technology (Biotechnology) Aatir Tungekar

Faculty and unit

University of Oulu Graduate School, Faculty of Biochemistry and Molecular Medicine, Protein and Structural Biology

Subject of study

Biochemistry and Molecular Medicine


Professor Vesa Hytönen, University of Tampere


Professor Lloyd Ruddock, University of Oulu

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Efficient production of antibody formats using bacterial systems

The process of oxidative protein folding involves the formation of disulfide bridges to obtain a protein in its native and biologically active functional form. Disulfide bond formation, a key post-translational modification, is vital for the folding, stability, and function of many proteins. Many commercially and therapeutically relevant recombinant proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state.

The production of disulfide-bonded recombinant proteins using Escherichia coli is often challenging as the protein needs to be targeted to the oxidative periplasm for disulfide bond formation. Previously, a system called CyDisCo has been reported which involves the cytoplasmic pre-/co-expression of a sulfhydryl oxidase and a protein disulfide isomerase while leaving the natural reducing pathways intact. This system has been extensively proven to aid the oxidative
folding of recombinant proteins in the cytoplasm of wild-type E. coli. The main focus of this research project was to investigate and address the bottlenecks in the production of modified antibody formats using the CyDisCo system in the cytoplasm of E. coli.

We determined limitations in the soluble expression of a highly complex class of recombinant therapeutics called Fc fusion proteins using the CyDisCo system. The cause of redox heterogeneity was identified and addressed using a systematic approach leading to the production of the proteins of interest (POIs) in a single homogeneous redox state. In addition, we show that neutralizing antibody fragments including their variants with improved affinity, as well as the receptor binding domain of SARS-CoV-2 variants, can be solubly produced in a natively folded and functionally active form using the CyDisCo system.

Based on the combination of findings from the aforementioned investigations, we developed a novel and alternate antibody fragment format ‘FabH3’ to address certain key limitations in the folding and production of antibody fragments in E. coli. We show that this alternative antibody fragment format can be efficiently produced in the cytoplasm of E. coli using the CyDisCo system in a natively folded state with higher soluble yields than its Fab counterpart and has a comparable binding affinity against the target antigen.
Last updated: 6.2.2024