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    Design of bacterial polyester beads for recombinant protein production, biomolecule separation and detection : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Du, Jinping
    Protein recovery and biomolecule detection are commonly required for scientific research as well as industrial activities. However, it is generally complicated and costly either to produce and purify recombinant proteins (especially therapeutic proteins) from engineered Escherichia coli cells, or to directly separate proteins or detect other biomolecules from natural sources. Here the PHA synthase (PhaC) mediated polyhydroxyalkanoate (PHA) bead display technology was explored as a solution to these problems by developing streamlined processes with less complex steps to achieve protein recovery and biomolecule detection. Firstly, by fusing a target protein to PhaC via a self-cleavable linker tag of either sortase (sortase A from Staphylococcus aureus) or intein (DnaB mini intein from Synechocystis sp. PCC 6803), new self-cleavable recombinant protein production and purification resins were developed. It was shown that the PhaC fusion could mediate in vivo production of PHA beads displaying the target protein. Functional target protein could be obtained at high purity from isolated PHA beads by incubation with CaCl2 and triglycine (in the case of the self-cleavable sortase tag) or by a pH shift to 6 (in the case of the self-cleavable intein tag). Six recombinant proteins were successfully produced and purified via the intein approach, including 3 model proteins (Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, and the synthetic immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus) and 3 therapeutic proteins (human tumour necrosis factor alpha (TNFα), human interferon alpha-2b (IFNα2b), and human granulocyte colony-stimulating factor (G-CSF)). Of these, TNFα and IFNα2b were also successfully produced and purified via the sortase approach. Secondly, in vivo one-step production of PHA affinity resins was achieved by fusing to PhaC differently customised OBody ligands. These ligands were previously engineered by other groups from the OB-fold domain of aspartyl-tRNA synthetase (aspRS) from Pyrobactulum aerophilum, by using phage display technology, to have specific binding affinities to biomolecules of interest. The resulting recombinant OBody beads were used for lysozyme sepration from a complex substrate, and for progesterone (P4) binding. Further optimisation of the P4 binding condition is necessary before the OBody bead system can be used for P4 detection in bovine milk. However, recombinant immobilisation of OBody ligands on the surface of PHA beads expands not only the attractiveness of these emerging OBody scaffolds, but also the utility scope of PHA beads as affinity resins.
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    The refolding of recombinant human liver methylmalonyl-CoA mutase from inclusion bodies produced in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1998) Hayes, Michelle Marie
    Human methylmalonyl-CoA mutase (hMCM) is an adenosylcobalamin-dependent enzyme that catalyses the structural rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA as pan of the catabolism of the branched chain amino acids valine, leucine and isoleucine, odd chain fatty acids and intermediates of cholesterol metabolism. Reactions that require adenosylcobalamin (AdoCbl) have been intensively studied, and the first step in the catalysis is widely agreed to involve homolytic cleavage of the unusual carbon-cobalt bond in the cofactor. A reliable source of recombinant hMCM would be useful in defining more fully the mechanistic pathway of AdoCbl-dependent enzymes. Recombinant hMCM overexpressed in E. coli forms insoluble aggregates of inactive protein known as inclusion bodies. hMCM inclusion bodies were purified, solubilised and then several different in vitro refolding techniques were tested in attempts to produce active recombinant hMCM from purified solubilised inclusion body material. These methods included refolding by rapid dilution, refolding by dialysis, detergent-assisted refolding, refolding by gel filtration chromatography and chaperonin-assisted refolding. Chaperonin-assisted refolding necessitated the purification of recombinant E. coli chaperonins GroES and GroEL from the E. coli strain DH1/pGroESL. Refolding by rapid dilution of the GdmHCl-solubilised inclusion bodies into a refolding buffer was judged to be the simplest and most effective method, however the refolding process was extremely inefficient. Refolding by rapid dilution was scaled up to 2 litres to produce as much active hMCM as possible. The refolded protein was concentrated by batch adsorption to and stepwise elution from hydroxyapatite, and further purified using a synthesised 5'adenosylcobalamin- agarose 'affinity' chromatography column. The final refolded hMCM preparation contained a single ~29 kDa contaminant protein, tentatively identified as E. coli branched-chain amino acid aminotransferase (EC 2.6.1.42), present in approximately equal amounts to the hMCM, and had a specific activity of ~3.11 units/mg.
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    Recombinant protein immobilisation and display by alginate : 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, 2016) Jameson, Andrew
    Biopolymers are a diverse group of organic materials with important applications in a number of industries. Their ability to adsorb and encapsulate compounds has been widely utilised in both biotechnologies and pharmaceuticals. In the last decade, biopolymers have been given new and enhanced functionality, including the separation and purification of compounds. This field is of increasing relevance as advances in the bacterial cell culture process have improved productivity in the biomanufacturing industry, with the establishment of several bacterial host cell lines and optimised protein production systems. This increase in upstream productivity is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. While previous studies have generated functionalised protein biopolymers using polyhydroxyalkanoate (PHA) biopolyester beads, very few studies have examined the commercially significant biopolymer alginate. Alginate is an exopolysaccharide produced by algae and some bacteria, and is widely utilised in food, pharmaceutical, and biomedical industries because of its stabilising, haemostatic, biocompatible properties and its modifiable structure. In this study, a partially functional alginate-binding recombinant protein was produced, which contained an α-amylase domain from Bacillus licheniformis (BLA) translationally fused to the alginate-binding domain of Pseudomonas aeruginosa AlgX – an alginate acetyltransferase. An Ssp DnaB mini-intein was included between BLA and AlgX to facilitate recovery of BLA, following immobilisation and display on the surface of alginate. However, aberrant activity of the intein caused total cleavage of the recombinant protein between its BLA and AlgX domains before it could be recovered from the protein production system. Additionally, the absence of a key cysteine residue in the alginate-binding domain prevented the formation of a disulfide bond, which is an essential structural element for the folding and functionality of this region. While this study was unable to overcome intein hyperactivity, functional analysis of the BLA domain showed consistent and significant levels of α-amylase activity, leading to a positive outlook for the functionality of a full-length recombinant protein if proper intein activity can be restored and the necessary cysteine included. In this way, alginate could be specifically functionalised with a desired protein, and in turn, alginate beads could be used for the separation and enrichment of target proteins.