Recombinant Escherichia coli producing an immobilised functional protein at the surface of bio-polyester beads : a novel application for a bio-bead : a thesis presented in partial fulfillment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
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Date
2008
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Massey University
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Abstract
Polyhydroxyalkanoates (PHAs) are polyesters, produced by many bacteria and some
archaea. The most commonly characterised is polyhydroxybutyrate (PHB). Produced
when nutrients are growth limiting and carbon available in excess, PHA serves as a
carbon-energy storage material and forms generally spherical insoluble inclusions
between 50-500nm in diameter in the cytoplasm. The key enzyme for PHA synthesis
is the PHA synthase and this enzyme catalyses the polymerisation of (R)-3-hydroxy
fatty acids into PHA. PHA synthase remains covalently attached to the growing
polyester chain and is displayed on the surface of the PHA granule. Other proteins
associated with PHA granules include depolymerases for mobilisation or degradation
of granules, regulatory proteins and phasins, proteins that aid in PHA granule
stability.
PHA bio-beads displaying an IgG binding protein were produced and used to purify
IgG from serum demonstrating that the PHA synthase can be engineered to display
functional synthase fusion proteins at the PHA granule surface. Correctly folded
eukaryotic proteins were also produced and displayed at the PHA granule surface as
phasin fusion proteins. Multiple-functionality was also achievable by co-expression
of various hybrid genes suggesting that this biotechnological bead production
strategy might represent a versatile platform technology.
The production of functional eukaryotic proteins at the PHA bead surface represents
a novel in vivo matrix-assisted protein folding system. Protein engineering of PHA
granule surface proteins provides a novel molecular tool for the display of antigens
for FACS based analysis and offers promising possibilities in the development of
future biotechnological production processes. Overall, the results obtained in this
study strongly enhance the applied potential of these polyester beads in
biotechnology and medicine.
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Keywords
Escherichia coli, Biopolyester beads, Bio-beads, Polyhydroxyalkanoates, Polyhydroxybutyrate, Protein engineering