Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems

Calcium imaging is an important functional tool for analysing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from the endoplasmic reticulum to incorporate the synthesized membrane proteins-like ion channels. Carboxylesterase is required to cleave the acetoxymethyl ester moiety of the chemical calcium indicators in order to ensure its immobility across the endoplasmic reticulum membrane. Absence or an inadequate amount of carboxylesterase in the endoplasmic reticulum of different eukaryotic cells poses a hindrance to perform calcium imaging in microsomes. In this work, we try to overcome this drawback and adapt the cell-based calcium imaging principle to a cell-free protein synthesis platform. Carboxylesterase synthesized in a Spodoptera frugiperda Sf21 lysate translation system is established as a viable calcium imaging tool in microsomes. Cell-free synthesized carboxylesterase inside microsomes is validated with esterase and dye loading assays. Native proteins from the endoplasmic reticulum, such as ryanodine channels and calcium ATPase, are analysed. Cell-free synthesized transient receptor potential channels are used as model proteins to demonstrate the realization of this concept.