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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....

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Autores principales: Dhandapani, Priyavathi, Dondapati, Srujan Kumar, Zemella, Anne, Bräuer, Dennis, Wüstenhagen, Doreen Anja, Mergler, Stefan, Kubick, Stefan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9030739/
https://www.ncbi.nlm.nih.gov/pubmed/35479141
http://dx.doi.org/10.1039/d0ra08397f
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author Dhandapani, Priyavathi
Dondapati, Srujan Kumar
Zemella, Anne
Bräuer, Dennis
Wüstenhagen, Doreen Anja
Mergler, Stefan
Kubick, Stefan
author_facet Dhandapani, Priyavathi
Dondapati, Srujan Kumar
Zemella, Anne
Bräuer, Dennis
Wüstenhagen, Doreen Anja
Mergler, Stefan
Kubick, Stefan
author_sort Dhandapani, Priyavathi
collection PubMed
description 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.
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spelling pubmed-90307392022-04-26 Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems Dhandapani, Priyavathi Dondapati, Srujan Kumar Zemella, Anne Bräuer, Dennis Wüstenhagen, Doreen Anja Mergler, Stefan Kubick, Stefan RSC Adv Chemistry 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. The Royal Society of Chemistry 2021-05-04 /pmc/articles/PMC9030739/ /pubmed/35479141 http://dx.doi.org/10.1039/d0ra08397f Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Dhandapani, Priyavathi
Dondapati, Srujan Kumar
Zemella, Anne
Bräuer, Dennis
Wüstenhagen, Doreen Anja
Mergler, Stefan
Kubick, Stefan
Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems
title Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems
title_full Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems
title_fullStr Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems
title_full_unstemmed Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems
title_short Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems
title_sort targeted esterase-induced dye (ted) loading supports direct calcium imaging in eukaryotic cell-free systems
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9030739/
https://www.ncbi.nlm.nih.gov/pubmed/35479141
http://dx.doi.org/10.1039/d0ra08397f
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