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Updated: Aug 19, 2026
Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development
Published on: March 16, 2022
Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer
Heather E Findlay1, Heather McClafferty, Richard H Ashley
1Division of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, UK. h.e.findlay@bristol.ac.uk
Background:
Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes.
Results:
C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) beta-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands.
Conclusions:
C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with beta-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins.
Insights
Chlamydia trachomatis Major Outer Membrane Protein (MOMP) was successfully expressed on E. coli surfaces, enabling functional studies and potential vaccine development. This breakthrough aids in understanding MOMP
Area of Science:
- Microbiology and immunology
- Protein biochemistry
- Structural biology
Background:
- Chlamydial Major Outer Membrane Protein (MOMP) is crucial for structure and immunity but difficult to express and purify.
- Recombinant MOMP expression in E. coli often results in misfolding and aggregation.
- Understanding MOMP's membrane topology and displaying its epitopes are key research goals.
Purpose of the Study:
- To optimize conditions for surface expression of correctly folded Chlamydia trachomatis MOMP in E. coli.
- To investigate the membrane topology of MOMP.
- To establish a system for displaying native and modified MOMP epitopes.
Main Methods:
- Optimized leader sequence, temperature, and medium for E. coli surface expression of C. trachomatis MOMP.
- Functional single-channel reconstitution to verify correct protein folding.
- Deletion mutagenesis of predicted surface-exposed loops and transmembrane strands to test topology models.
Main Results:
- MOMP was successfully expressed on the surface of E. coli, including in porin knockout strains.
- Functional reconstitution confirmed correct protein folding, with MOMP forming oligomers and disulfide bonds.
- Deletion of surface-exposed loops did not impede outer membrane incorporation, unlike transmembrane strand deletions.
Conclusions:
- Functional surface expression of C. trachomatis MOMP in E. coli was achieved under optimized conditions.
- Experimental data support a beta-barrel oligomer model with immunogenic regions on surface-exposed loops.
- This system facilitates immunological studies, vaccine development, and co-expression of chlamydial membrane proteins.
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