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Molecular study of odorant binding proteins to better understand insect chemosensation

Agnihotri, Aniruddha Ravindra (2021) Molecular study of odorant binding proteins to better understand insect chemosensation. PhD thesis, Murdoch University.

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Like many other organisms, insects use chemical stimuli to regulate behaviours including feeding, egg-laying, and mating. Odorant binding proteins (OBPs) are one of the crucial components of insect chemosensory system, which play an essential role in transporting the hydrophobic volatile odorant molecules to the olfactory receptors. Most of insect OBPs were investigated by using recombinant technology in bacterial cells due to its fast, cost-effective, and high production mechanism. However, one of the major concerns of bacterial expression system is that the protein is frequently expressed in an unfolded state in inclusion bodies (IBs), which requires further in-vitro protein refolding step to make the protein biologically active. While doing this, there are always high chances of protein misfolding which results in soluble or insoluble protein aggregation. Thus, it is highly important to confirm the efficiency of each refolding method, used for OBP refolding, in terms of getting the correctly folded structure of the target protein. Unfortunately, it was neglected in many previous studies, resulting in significant doubts on various functional studies of insect OBPs. In this study, I used three Helicoverpa armigera OBPs, HarmOBP2, HarmOBP5, and HarmGOBP2, as model proteins to compare the different protein refolding strategies in producing correctly folded recombinant OBPs. Along with that, I have developed a novel pH-dependent method of protein refolding which demonstrated as a more efficient and productive approach for selected HarmOBPs’ refolding compared to other used methods. Further, I also developed a novel reverse chemical ecology method to isolate and identify the candidate natural ligands from host plants for HarmOBPs. This study points out a crucial but largely ignored step of insect OBP research, protein refolding and the loopholes associated with it in previous studies, which will improve our understanding of insect chemosensation and help develop more efficient and environmentally friendly insect control strategies.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Agricultural Sciences
Supervisor(s): Xu, Wei, Jones, Michael and Wang, Penghao
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