Functional protein display on the surface of biobeads produced by recombinant Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

Polyhydroxyalkanoic acids (PHAs) are biopolyesters produced by various bacteria. They are deposited as spherical water-insoluble cytoplasmic inclusions (beads) containing an amorphous hydrophobic polyester core and surrounded by a phospholipid monolayer and embedded proteins, including PHA synthase (PhaC), the key enzyme required for PHA bead formation. Although inactive PhaC cannot produce PHA beads, fusing inactive PhaC to green fluorescent protein (GFP) leads to GFP protein bead formation. Both PHA and protein beads could serve as a versatile platform for display of desired proteins suitable for various biotechnological and medical applications. The tuberculin skin test (TST) for diagnosing bovine tuberculosis (TB) in cattle uses the purified protein derivative (PPD) that is prepared from Mycobacterium bovis. However, some antigens in the PPD are also present in environmental mycobacteria. Therefore, the TST lacks specificity if animals are exposed to non-pathogenic environmental mycobacteria. In this study, three specific TB antigens, CFP10, ESAT6, and Rv3615c — which are present in pathogenic but absent in most non-pathogenic mycobacteria — were displayed on the surface of PHA beads. The results demonstrated that these triple antigen-displaying PHA beads can differentiate TB-infected from non-infected cattle, making this an attractive alternative to current skin test diagnostic reagents. IgG binding domains displayed on GFP protein beads have a higher IgG binding ability when compared to their counterpart displayed on PHA beads. However, it is unclear whether an enhancement of IgG binding ability due to GFP protein beads could be achieved by immobilization on other fluorescent protein (FP) beads. The results showed that other FP (including yellow, red and cyan) beads displaying IgG binding domains have an approximately 1.5–2 fold greater IgG binding ability when compared to PHA beads displaying the same binding domains. To investigate whether protein beads displaying iron-binding peptides could be magnetized while maintaining IgG binding function, an iron binding peptide was displayed. The results demonstrated that protein beads displaying both IgG and iron binding peptides can be magnetised by iron oxide and retain a strong IgG binding ability. Finally, this study revealed that different cell disruption techniques could affect the morphology and functionality of FP protein bead.