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Engineering and characterisation of anti-progesterone OBodies

Molecular interactions are fundamental to communication between different parts of the cell or of an organism. These interactions can be weak and transient or strong and semi-permanent. In the case of the adaptive immune system, strong interactions between foreign antigens and specific antibodies lead to a cascade of events comprising the immune response. This phenomenon has been exploited by industry to produce high affinity binding molecules that have been used as therapeutics or diagnostics and engineered antibodies have been at the forefront of these industries. More recently, novel non-immunoglobulin binding proteins, have been similarly engineered to produce high affinity binding proteins that can potentially replace the binding function(s) of antibodies or even surpass them. The OB-fold is a high affinity binding protein domain which has previously been engineered to bind to Hen Egg-white Lysozyme (HEL) with nanomolar affinity as a proof-of-concept technology and has been given the name OBodies. This thesis explores this concept further with an OBody (D7) engineered to bind to the small molecule progesterone (P4) with potential applications to detect P4 in cow’s milk to evaluate pregnancy. Obody-P4 binding was characterised with an optimized ELISA system. This was followed by the engineering of improved versions of this OBody using phage display technology and structural characterisation of one such Obody-P4 complex using X-ray Crystallography. The three-dimensional structure provided surprising insights into the nature of the molecular interactions between P4 and the OBody. During phage display selection a new signal sequence was fortuitously discovered that provided an advantage during phage display. The new signal sequence was investigated and characterised using mutants of GFP and a DARPin sequence to uncover the nature of the selective advantage. The combination of this new signal sequence and thermostable OBody libraries further demonstrates the potential of this system to produce robust bio-sensors for diagnostic and therapeutic applications.
Type of thesis
Chonira, V. K. (2018). Engineering and characterisation of anti-progesterone OBodies (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/11701
The University of Waikato
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