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Title: Investigations into the formation of pdu microcompartments in mammalian and bacterial cells as novel 'organelles' for protein folding and post-translational modifications
Author: Ozanne, Angelica
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2013
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In some bacteria proteinaceous polyhedral structures known as microcompartments (MCPs) are formed that encapsulate particular metabolic processes. One such bacterial Mep is the 1,2-propanediol utilisation (Pdu) MCP expressed in Citrobacter freundii and it is possible to synthesise functional pdu MCPs in Esherichia coli (Eeoli) by transferring the pdu operon from Citrobaeter freundii into Ecolf. Subsequent investigations have shown that if only the genes encoding for the pdu MCP shell proteins are transferred into E.coli empty pdu MCP structures are formed. The minimum number of genes thought to be necessary to form a recombinant MCP structure is five. In addition, if only one of these essential genes (PduA) is expressed in E-coli then large structures are observed which are either hexagonallattice like in appearance or long and thin spanning much of the E.coli cell. Importantly for this project, recent work has also demonstrated that proteins can be selectively targeted to within the pdu MCPs using specific N-terminal sequence tags. This IJ project set out to investigate the formation of MCPs in mammalian and bacterial cells as novel 'organelles' for protein folding and post-translational modifications. Initially, it was investigated whether the essential pdu shell proteins required to form the MCP can be expressed in CHOKI cells and whether upon expression these can subsequently form proteinaceous structures within such cells. Using western blotting and fluorescent immunostaining it was apparent that individual pdu shell proteins with a V5 tag could be transiently expressed in CHOKI cells but stably expressing cell lines could not be established. However, stable cell lines expressing OFP tagged pdu proteins could be generated although the level of expression was much less than OFP alone cell lines. Electron microscopy analysis revealed that the transient expression of OFP-pduA or all five pdu shell protein with a OFP tag generated in some cells structures that resembled those seen in E.coli when the ratio of subunits were manipulated. This data suggests that pdu protein based structures can be formed in mammalian cells, however a threshold of expression must be obtained before organised structure are obseIVed which only occurred upon transient expression. Stable cell lines did not presumably exceed this threshold and hence organised structures were not observed. Following on from these studies, MCPs were successfully generated in E.coli and specific proteins targeted to within these using N-terminal tags. To determine whether the targeting of proteins to the MCPs could enhance protein folding, the protein vtPA, which requires the correct formation of a number of disulphide bridges to be functionally active, was targeted to the MCP with or without PDL When vtPA alone was targeted to the MCP an increase in vtPA activity was observed over the control and although the additional targeting ofPDI also led to an increase in activity over the control, the activity was less than when vtPA alone was targeted. This is likely to be the result of dilution of expression as the bacteria was making two proteins (vtPA and PDI) as opposed to one alone and hence development of bacterial strains into which the pdu and PDI genes were integrated into the genome would in all likelihood be more beneficial for enhanced expression and folding of a single protein. In conclusion, the work presented here has demonstrated that pdu proteins and organised structures can be formed in mammalian cells and that the targeting of specific proteins to MCPs in bacterial systems offers the potential to develop a novel environment for improved protein folding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available