Low Ca2+ levels in the culture media support the heterologous expression of insect odorant receptor proteins in HEK cells

Abstract

Background: Heterologous expression of insect odorant receptors (ORs) in mammalian or insect cells is challenging due to the insufficient intracellular trafficking of ORs and their ability to form leak ion channels.

New method: We tested whether reducing the Ca²⁺ levels in the cell culture medium after electroporation by means of a Dulbecco's Modified Eagle Medium (DMEM) without calcium, in a 1:1 ratio with Ham's F12 nutrient mixture, together with 10% fetal calf serum, can improve the success rate of insect OR expression in HEK293 cells.

Results: We show that a reduced extracellular Ca²⁺ level supports functional expression of insect ORs by increasing the fraction of cells responding to the co-receptor agonist VUAA1 and by reducing the intracellular Ca²⁺ base level of transfected cells.

Comparison with existing method(s): A DMEM formula without calcium outperforms standard DMEM in a 1:1 ratio with Ham's F12 mix and 10% serum, when culturing HEK293 cells transiently expressing insect OR proteins.

Conclusions: Reducing the extracellular Ca²⁺ level of HEK293 cell culture media after transfection increases the success of functional insect OR expression.

Keywords: Calcium; Locusta migratoria; Odorant receptor; Orco.