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Title: Expression and folding of recombinant chlamydial outer membrane proteins
Author: Findlay, Heather E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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The Chlamydiaceae are obligate intracellular pathogens of eukaryotic cells. These few species are responsible for a wide variety of human and animal diseases, including trachoma, sexually transmitted genital infections, pneumonia and abortion. Female infertility secondary to chronic pelvic inflammation is a particularly important and relatively common complication of C. trachomatis infection. The cysteine-rich Major Outer Membrane Protein (MOMP) is the immunodominant chlamydial protein and a primary vaccine target. It is predicted to function as a general diffusion porin that may also, through intra- and/or intermolecular disulphide bond formation, play an important structural role in maintaining stability of the organism in the absence of a peptideoglycan layer. However, vaccine development and examining the specific biochemistry of MOMP in situ is problematic due to difficulties involved in culturing the organisms and the presence of other chlamydial membrane proteins. A dual approach was taken to develop a recombinant system to express MOMP in E. coli. Full length MOMP expressed with the E. coli ompT signal sequence was shown to be successfully transported to the outer membrane of the cell. A proportion of the protein solubilised from the membrane eluted as trimers during size exclusion chromatography, and formed porin-like channels in planar lipid bilayers. Mature MOMP lacking a signal sequence accumulated in inclusion bodies when expressed in E. coli. These were denatured and refolded in vitro to produce higher order complexes of MOMP composed of SDS resistant trimers. Cysteine residues were found to play a critical role in the stabilisation of both secondary and tertiary structures. The ability to express properly folded recombinant MOMPs, and other chlamydial outer membrane proteins, will allow more detailed analysis of the structural and functional roles of MOMPS, and will contribute to the drive for vaccine development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available