The cloning expression, purification, and reconstitution of Olfactory receptor co-receptor (Orco) in nanodisc from insects for structural and function studies

• Main Author: Tan, Jia Wei
• Other Authors: Sandip Basak
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/166548
• Institution: Nanyang Technological University

Insect vectors host-seeking behaviours are being mediated by the olfactory system, making it an attractive target for managing vector-borne disease transmission. Odorant receptors (ORs) allow insects to recognize a large variety of odorant molecules based on simple combinatorial signalling. The normal function of ORs in insects is regulated by the highly conserved Orco protein, and a diverse tuning subunit. We can study a simplified Orco homotetramer to better understand the structure and function of ORs. Here, we investigated Drosophila melanogaster and Anopheles gambiae Orco tetramers. We have introduced a thermostabilized fusion protein apocytochrome b562RIL (BRIL) domain fused in the intracellular loop of Orco proteins to enable binding of BRIL antibody to protein, which further aids in structure determination by changing properties of Orco receptors. All recombinant Orco-BRIL constructs were successfully cloned Bacmid vectors, for transfection into Sf9 cells (Spodoptera frugiperda) for baculovirus generation, which was confirmed by agarose gel electrophoresis, sequencing, and PCR. The recombinant Orco-BRIL protein was effectively overexpressed in Sf9 cells and purified and reconstituted into a lipid nanodisc, determined by SDS-PAGE, western blot, negative staining electron microscopy. These findings suggests that recombinant Orco-BRIL homotetramer can be efficiently expressed and purified for downstream cryo-EM structural studies.