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Front Cell Neurosci
2021 Jan 01;15:744401. doi: 10.3389/fncel.2021.744401.
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Codon Optimization of Insect Odorant Receptor Genes May Increase Their Stable Expression for Functional Characterization in HEK293 Cells.
Roberts RE
,
Yuvaraj JK
,
Andersson MN
.
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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.
FIGURE 1. Western blots of HEK293 cells transfected with bark beetle Myc-tagged Orco genes and V5-tagged odorant receptor (OR) genes. (A) Detection of Ips typographus (Ityp) OR proteins from the first set of cell lines co-transfected with the wildtype (WT) ItypOrco gene and wildtype genes for ItypOR23, ItypOR25, ItypOR27, ItypOR28, ItypOR29, ItypOR46, and ItypOR49 (two cell lines for this gene: WT1 and WT2). Data for ItypOR46 and ItypOR49 from Yuvaraj et al. (2021) (modified image under CC BY 4.0 license; https://creativecommons.org/licenses/by/4.0/). (B) Detection of ItypORs from the second set of cell lines co-transfected with the wildtype ItypOrco gene and wildtype genes for ItypOR23, ItypOR25, ItypOR27, ItypOR28, ItypOR29, and ItypOR46. (C) Detection of ItypOR49 from cells co-transfected with the wildtype ItypOrco gene and the wildtype (third cell line: WT3) or codon optimized (HsCO) gene for ItypOR49. (D) Detection of Orco proteins from cells expressing the wildtype ItypOrco gene or the codon optimized ItypOrco gene, and (E) the wildtype Dendroctonus ponderosae (Dpon) Orco gene or the codon optimized DponOrco gene. (F) Detection of ItypORs from cells co-transfected with the wildtype ItypOrco gene and codon optimized genes for ItypOR23, ItypOR25, ItypOR27, ItypOR28, and ItypOR29. OR and Orco proteins were only detected from cells induced (+) to express the exogenous genes, and not from non-induced (−) cells, indicating proper regulation by the repressor system (A–F). Note, the detection of some receptors as “double-bands” is likely due to different protein confirmations in the blotting.
FIGURE 2. Response to VUAA1 of cells expressing bark beetle Orco genes. (A) Cells expressing the codon optimized I. typographus Orco gene (ItypOrcoHsCO; n = 6 biological replicates; ntotal = 18) demonstrate increased maximal response and a somewhat lower EC50 value as compared to cells expressing the wildtype Orco gene (ItypOrcoWT; n = 5 biological replicates [replicates 1–3 from Yuvaraj et al. (2021)]; ntotal = 15). Asterisks (*) indicate significantly higher responses in ItypOrcoHsCO as compared to ItypOrcoWT (p = 0.002 to <0.001). (B) Cells transfected with either the wildtype or codon optimized Orco gene from D. ponderosae (DponOrcoWT and DponOrcoHsCO) failed to respond to VUAA1 (n = 3 biological replicates, ntotal = 9), with similar Δfluorescence values in induced and non-induced cells (not shown in graph for clarity; see Supplementary Data 1). Error bars show SEM.
FIGURE 3. Response of cells co-expressing wildtype (WT) I. typographus Orco (ItypOrcoWT) and wildtype or codon optimized (HsCO) ItypOR49. (A) Dose-dependent responses of ItypOR49WT and ItypOR49HsCO to main ligand (R)-(−)-ipsdienol with higher responses of cells expressing the codon optimized gene (n = 4 biological replicates; ntotal = 12). Asterisks (*) indicate significantly higher responses in ItypOR49HsCO as compared to ItypOR49WT (p = 0.042 to <0.001). The EC50 value for ItypOR49WT was not calculated due to non-sigmoid dose-response curve. (B) Screening results (30 μM compound concentration) from cells co-expressing ItypOrcoWT and ItypOR49HsCO showing the same specificity as previously reported for ItypOR49WT (Yuvaraj et al., 2021) (n = 4 biological replicates; ntotal = 12). Asterisks (***) indicate significantly higher response in induced (+) versus non-induced (−) cells at p < 0.001. Error bars show SEM.
FIGURE 4. Response of cells co-expressing wildtype I. typographus Orco (ItypOrcoWT) and wildtype or codon optimized (HsCO) ItypOR25 to select compounds. (A) ItypOR25WT did not respond to any compound in the screening experiments (30 μM concentration; n = 2 biological replicates; ntotal = 6). (B) ItypOR25HsCO responded primarily to (+)-3-carene and secondarily to myrcene in the screening experiment (n = 3 biological replicates, ntotal = 9). Asterisks (***) indicate significantly higher response in induced (+) versus non-induced (−) cells at p < 0.001, and different lowercase letters indicate significant differences between compounds at p < 0.001. (C) Dose-dependent response of ItypOR25HsCO to (+)-3-carene (n = 3 biological replicates; ntotal = 9). Error bars show SEM. Responses of ItypOR25WT and ItypOR25HsCO to the full odor panel are presented in Supplementary Figure 1.
FIGURE 5. Response of cells co-expressing wildtype I. typographus Orco (ItypOrcoWT) and wildtype or codon optimized (HsCO) ItypOR29 to select compounds. (A) ItypOR29WT did not respond to any compound in the screening experiments (30 μM concentration; n = 2 biological replicates; ntotal = 6). (B) ItypOR29HsCO responded primarily to (+)-isopinocamphone followed by (+)-pinocamphone, (−)-pinocamphone, and (−)-isopinocamphone in the screening experiment (n = 3 biological replicates, ntotal = 9). Asterisks (***) indicate significantly higher response in induced (+) versus non-induced (−) cells at p < 0.001. Responses to the four active compounds were not significantly different (p = 0.059). (C) Dose-dependent response of ItypOR29HsCO to the four active ligands (n = 3 biological replicates; ntotal = 9). Error bars show SEM. EC50 values were not calculated due to non-sigmoid dose-response curves. Responses of ItypOR29WT and ItypOR29HsCO to the full odor panel are presented in Supplementary Figure 2.
Supplementary Figure 1. Response of cells co-expressing wildtype I. typographus Orco (ItypOrcoWT) and wildtype or codon optimized (HsCO) ItypOR25 to the full test odor panel. (A) ItypOR25WT did not respond to any compound in the screening experiments (30 μM concentration; n = 2 biological replicates; ntotal = 6). (B) ItypOR25HsCO responded primarily to (+)-3-carene and secondarily to myrcene in the screening experiment (n = 3 biological replicates; ntotal = 9). Asterisks (***) indicate significantly higher response in induced versus non-induced cells at p < 0.001, and different lowercase letters indicate significant differences between compounds at p < 0.001. Error bars show SEM. Abbreviation: (5S,7S)-tC = (5S,7S)- trans-conophthorin.
Supplementary Figure 2. Response of cells co-expressing wildtype I. typographus Orco (ItypOrcoWT) and wildtype or codon optimized (HsCO) ItypOR29 to the full test odor panel. (A) ItypOR29WT did not respond to any compound in the screening experiments (30 μM concentration; n = 2 biological replicates; ntotal = 6). (B) ItypOR29HsCO responded primarily to (+)-isopinocamphone followed by (+)-pinocamphone, (-)-pinocamphone, and (-)- isopinocamphone in the screening experiment (n = 3 biological replicates; ntotal = 9). Asterisks (***) indicate significantly higher response in induced versus non-induced cells at p < 0.001. Responses to the four active compounds were not significantly different (p = 0.059). Error bars show SEM. Abbreviation: (5S,7S)-tC = (5S,7S)-trans-conophthorin.
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