Browsing by Author "Lee, Jason Wong"

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    Molecular characterisation of PHA synthase and the in vivo synthesis of functionalised PHA beads with surface immobilised proteins : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand.
    (Massey University, 2011) Lee, Jason Wong; Lee, Jason Wong
    Polyhydroxyalkanoates (PHAs) are naturally occurring biopolyesters, synthesized by a large range of bacteria and deposited as small spherical water-insoluble cytoplasmic inclusion bodies containing hydrophobic polyester core surrounded by a phospholipid monolayer and associated embedded proteins. The most common form of PHA identified in bacteria is polyhydroxybutyrate (PHB). Formation of PHA beads requires three important enzymes with PHA synthase (PhaC) being the most important, catalysing the final stereo-selective conversion of (R)-3-hydroxyacyl-CoA thioesters into PHA. Increasingly beads are used as microbeads, which display surface immobilised proteins for a range of applications in biotechnology and medicine. However, functionalised PHA beads are largely produced in Gram-negative bacteria which contain endotoxins that are known to co-purify with the beads and are considered undesirable in medical applications. In addition, despite extensive research towards understanding PHA synthases, to date no structural data is currently available. Here it was shown that functionalised PHB beads can be produced in vivo for both the purification of antibodies and the display of medically relevant antigens (e.g. Hepatitis C) on the surface of PHB beads from the Gram-positive bacterium L.lactis. In addition, it was shown that PHA synthase from R.eutropha can be highly overproduced, remains largely soluble and can be purified to greater than 90 % purity. The results demonstrated and supported the use of PHB beads as a platform for the production of functionalised PHA beads suitable for a large range of biotechnological or medical applications. Although no structural data for PHA synthases are currently available, our results demonstrate progress towards obtaining a three-dimensional protein structure for PHA synthase (PhaC).
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    Novel particulate vaccine candidates recombinantly produced by pathogenic and nonpathogenic bacterial hosts : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand.
    (Massey University, 2017) Lee, Jason Wong
    Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized as small spherical cytoplasmic inclusion bodies by a range of bacteria. Recently, PHA beads have been investigated for use as a vaccine delivery platform by using engineered heterologous production hosts that allowed the efficient display of vaccine candidate antigens on the beads surface and were found to greatly improve immunogenicity of the displayed antigens. However, like other subunit vaccines, these antigen-displaying (vaccine) PHA beads only provide a limited repertoire of antigens. In this thesis we investigate the idea of directly utilizing the disease causative pathogen or model organism to produce vaccine PHA beads with a large antigenic repertoire. These beads are hypothesized to have the potential to induce greater protective immunity compared to production of the same PHA bead in a heterologous production host. This concept was exemplified with Pseudomonas aeruginosa and Mycobacterium tuberculosis as model human pathogens. For P. aeruginosa we describe the engineering of this bacterium to promote PHA and Psl (polysaccharide) production. This represents a new mode of functional display for the engineering, production, and validation of a novel OprI/F-AlgE fusion antigen-displayed on PHA beads. For the disease tuberculosis we investigated the use of nonpathogenic M. smegmatis as a model organism for M. tuberculosis. We described the bioengineering, production, and validation of Ag85AESAT- 6 displayed on PHA beads produced in M. smegmatis. Here we showed that both organisms were harnessed to produce custom-made PHA beads for use as particulate subunit vaccines that carried copurifying pathogen-derived proteins as a large antigenic repertoire and the ability of these vaccine PHA beads to generate a protective immune response. This novel bioengineering concept of particulate subunit vaccine production could be applied to a range of pathogens naturally producing PHA inclusions for developing efficacious subunit vaccines for infectious diseases.