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dc.contributor.advisorWall, J.Gerard
dc.contributor.authorWronska, Malgorzata Anna
dc.date.accessioned2015-06-24T14:57:16Z
dc.date.available2015-06-24T14:57:16Z
dc.date.issued2014-09-26
dc.identifier.urihttp://hdl.handle.net/10379/5035
dc.description.abstractA crystallographic approach was taken to investigate the structure and function of two proteins of interest previously isolated in our research group and identified as having potential application in improving the production of recombinant proteins in Escherichia coli. The first, CzrB from the thermophilic bacterium Thermus thermophilus, is a membrane-bound protein involved in the transport of metal ions to ensure cell homeostasis. The second target, trigger factor (TF) from the psychrophilic bacterium Psychrobacter frigidicola (TFPf), is a molecular chaperone that assists the folding of newly synthesised polypeptides in the cell. Here, we present a systematic study of the expression and characterisation of both proteins with a view towards obtaining diffraction-quality crystals for analysis by X-ray crystallography. Initial investigation based on previously established protocols led to successful expression of both targets in E. coli. CzrB was found to occur as a heterogeneous population of largely monomeric and dimeric molecules, however, which is unsuitable for crystallisation. The use of chromatographic size exclusion did not separate monomeric and dimeric CzrB, while the use of a range of maltoside (DDM, UDM, DM,)- and glucoside (OG, NG)-based detergents also failed to commit CzrB to an exclusively dimeric form. Well-shaped pentagonal crystals were obtained with CzrB purified in UDM exhibited a high detergent content, while CzrB purified in DDM formed triangular crystals that diffracted to 7 Å but were identified as the commonly IMAC-co-purified E. coli membrane protein AcrB. Construction and screening of a series of new CzrB constructs containing different fusion tags led to identification of two candidate constructs for large-scale production, while a non-dimerising CzrB construct design is also being investigated to reduce heterogeneity of protein preparations. The expression of C-terminal His-tagged TFPf resulted in a high yield of recombinant protein, however the extraction and purification of the protein was characterised by extensive degradation. Optimisation using ion exchange chromatography yielded protein of higher purity and quality, leading to needle-like crystals in a wide range of crystallisation conditions. The morphology did not improve upon pH screening or the inclusion of additives or seeding. Design and generation of a diverse panel of full-length TFPf constructs and component domains with a variety of fusion partners and recombinant tags identified differing degradation patterns during expression and extraction, indicating that constructs lacking the C-domain may be less susceptible to degradation. A full-length trigger factor construct that exhibited reduced degradation and is potentially suitable for crystallisation screening was also identified. Overall, significant progress was made in solubilisation and purification of the two proteins of interest expressed in E. coli. While extensive crystallisation investigations did not yield diffraction quality crystals of CzrB or TFPf, alternative approaches for future studies have been identified to overcome the bottlenecks of protein heterogeneity and degradation, respectively, in order to allow higher quality crystals to be obtained. The work has also generated important insights into the differences between working with membrane and soluble proteins, and illustrates the limitations of low-throughput versus high-throughput crystallisation approaches.en_US
dc.subjectEscherichia colien_US
dc.subjectThermus thermophilusen_US
dc.subjectPsychrobacter frigidicolaen_US
dc.subjectCrystallographyen_US
dc.subjectMicrobiologyen_US
dc.titleInvestigation of the structure and function of the metal transporter CzrB from Thermus thermophilus and the molecular chaperone trigger factor from Psychrobacter frigidicola by X-ray crystallographyen_US
dc.typeThesisen_US
dc.local.finalYesen_US
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