Abstract
Insect odorant receptor (OR) genes are routinely expressed in Human Embryonic Kidney (HEK) 293 cells for functional characterization (“de-orphanization”) using transient or stable expression. However, progress in this research field has been hampered because some insect ORs are not functional in this system, which may be due to insufficient protein levels. We investigated whether codon optimization of insect OR sequences for expression in human cells could facilitate their functional characterization in HEK293 cells with stable and inducible expression. We tested the olfactory receptor co-receptor (Orco) proteins from the bark beetles Ips typographus (“Ityp”) and Dendroctonus ponderosae (“Dpon”), and six ItypORs previously characterized in Xenopus laevis oocytes and/or HEK cells. Western blot analysis indicated that codon optimization yielded increased cellular protein levels for seven of the eight receptors. Our experimental assays demonstrated that codon optimization enabled functional characterization of two ORs (ItypOR25 and ItypOR29) which are unresponsive when expressed from wildtype (non-codon optimized) genes. Similar to previous Xenopus oocyte recordings, ItypOR25 responded primarily to the host/conifer monoterpene (+)-3-carene. ItypOR29 responded primarily to (+)-isopinochamphone and similar ketones produced by fungal symbionts and trees. Codon optimization also resulted in significantly increased responses in ItypOR49 to its pheromone ligand (R)-(−)-ipsdienol, and improved responses to the Orco agonist VUAA1 in ItypOrco. However, codon optimization did not result in functional expression of DponOrco, ItypOR23, ItypOR27, and ItypOR28 despite higher protein levels as indicated by Western blots. We conclude that codon optimization may enable or improve the functional characterization of insect ORs in HEK cells, although this method is not sufficient for all ORs that are not functionally expressed from wildtype genes.
Highlights
Insect odorant receptors (ORs) are seven-transmembrane proteins expressed in olfactory sensory neurons (OSNs) within the chemosensory sensilla on the antennae (Clyne et al, 1999; Vosshall et al, 1999; Sato et al, 2008; Wicher et al, 2008)
In order to function in the detection of odor ligands, insect ORs must be present in the OSN membrane with the olfactory receptor co-receptor known as Orco (Krieger et al, 2003; Vosshall and Hansson, 2011)
Our results show that codon optimization led to increases in Human Embryonic Kidney (HEK) cell protein levels for seven of the eight receptors and increased ligand-induced responses in four of them, including two ORs that responded to ligands only when expressed from codon-optimized genes
Summary
Insect odorant receptors (ORs) are seven-transmembrane proteins expressed in olfactory sensory neurons (OSNs) within the chemosensory sensilla on the antennae (Clyne et al, 1999; Vosshall et al, 1999; Sato et al, 2008; Wicher et al, 2008). Several heterologous expression systems are commonly used to functionally characterize ORs, including Human Embryonic Kidney (HEK) 293 cells (Corcoran et al, 2014), Spodoptera frugiperda Sf9 cells (Kiely et al, 2007), Xenopus laevis oocytes (Wagner et al, 2000), and the Drosophila “empty neuron system” (Dobritsa et al, 2003). Each of these systems has its own advantages and disadvantages, in all systems certain insect ORs are not functionally expressed. There appears to be significant variation in the levels of OR proteins in this system as indicated by Western blot analysis, and several ORs have been unresponsive to the tested odorants (Andersson et al, 2016; Yuvaraj et al, 2017, 2021; Miazzi et al, 2019a; Hou et al, 2020)
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