Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay

The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation. Therefore, PKA has been viewed increasingly as potential target for variety of drugs and environmental endocrine disr...

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Autores principales:	Kun Qiao, Yao Jiang, Tiantian Hu, Shuying Li, Wenjun Gui
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Protein kinase A alpha catalytic subunit Prokaryotic expression in vitro kinase activity detection Molecular docking Environmental pollution TD172-193.5 Environmental sciences GE1-350
Acceso en línea:	https://doaj.org/article/33abf30b4d97410b93271f6567bd5bc0
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spelling	oai:doaj.org-article:33abf30b4d97410b93271f6567bd5bc02021-11-28T04:27:26ZProkaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay0147-651310.1016/j.ecoenv.2021.113023https://doaj.org/article/33abf30b4d97410b93271f6567bd5bc02021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S0147651321011350https://doaj.org/toc/0147-6513The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation. Therefore, PKA has been viewed increasingly as potential target for variety of drugs and environmental endocrine disruptors. Consequentially, the preparation of PKA protein became an important initial step for the subsequent exploration of PKA’s character in endocrine disrupting effects of pesticides. To investigate PKA protein, which is potential to be the environmental endocrine toxicity target of triazole fungicides, a strategy to heterologously express protein kinase A catalytic alpha subunit of human (hPKAcα) and zebrafish (zPKAcα) in Escherichia coli (E. coli) BL21(DE3) host cells was demonstrated. After optimizing conditions and protein purification, we successfully obtained enzymatically active hPKAcα and zPKAcα. Western blot analysis indicated that the recombinant hPKAcα and zPKAcα still retained their characteristic antigenicity and binding activity, while in vitro kinase activity assays revealed that the recombinant hPKAcα and zPKAcα maintained enzyme activity. By in silico methods including homology modelling and molecular docking, the affinity of ligands and the models of hPKAcα and zPKAcα were further tested. The present study offered a valuable method to achieve the prokaryotic expression of a eukaryotic protein kinase and laid a foundation to facilitate further investigation of toxicological target of triazole pesticides.Kun QiaoYao JiangTiantian HuShuying LiWenjun GuiElsevierarticleProtein kinase A alpha catalytic subunitProkaryotic expressionin vitro kinase activity detectionMolecular dockingEnvironmental pollutionTD172-193.5Environmental sciencesGE1-350ENEcotoxicology and Environmental Safety, Vol 228, Iss , Pp 113023- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Protein kinase A alpha catalytic subunit Prokaryotic expression in vitro kinase activity detection Molecular docking Environmental pollution TD172-193.5 Environmental sciences GE1-350
spellingShingle	Protein kinase A alpha catalytic subunit Prokaryotic expression in vitro kinase activity detection Molecular docking Environmental pollution TD172-193.5 Environmental sciences GE1-350 Kun Qiao Yao Jiang Tiantian Hu Shuying Li Wenjun Gui Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay
description	The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation. Therefore, PKA has been viewed increasingly as potential target for variety of drugs and environmental endocrine disruptors. Consequentially, the preparation of PKA protein became an important initial step for the subsequent exploration of PKA’s character in endocrine disrupting effects of pesticides. To investigate PKA protein, which is potential to be the environmental endocrine toxicity target of triazole fungicides, a strategy to heterologously express protein kinase A catalytic alpha subunit of human (hPKAcα) and zebrafish (zPKAcα) in Escherichia coli (E. coli) BL21(DE3) host cells was demonstrated. After optimizing conditions and protein purification, we successfully obtained enzymatically active hPKAcα and zPKAcα. Western blot analysis indicated that the recombinant hPKAcα and zPKAcα still retained their characteristic antigenicity and binding activity, while in vitro kinase activity assays revealed that the recombinant hPKAcα and zPKAcα maintained enzyme activity. By in silico methods including homology modelling and molecular docking, the affinity of ligands and the models of hPKAcα and zPKAcα were further tested. The present study offered a valuable method to achieve the prokaryotic expression of a eukaryotic protein kinase and laid a foundation to facilitate further investigation of toxicological target of triazole pesticides.
format	article
author	Kun Qiao Yao Jiang Tiantian Hu Shuying Li Wenjun Gui
author_facet	Kun Qiao Yao Jiang Tiantian Hu Shuying Li Wenjun Gui
author_sort	Kun Qiao
title	Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay
title_short	Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay
title_full	Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay
title_fullStr	Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay
title_full_unstemmed	Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay
title_sort	prokaryotic expression of human-sourced and zebrafish-sourced protein kinase a alpha catalytic subunits combined with in vitro and in silico assay
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/33abf30b4d97410b93271f6567bd5bc0
work_keys_str_mv	AT kunqiao prokaryoticexpressionofhumansourcedandzebrafishsourcedproteinkinaseaalphacatalyticsubunitscombinedwithinvitroandinsilicoassay AT yaojiang prokaryoticexpressionofhumansourcedandzebrafishsourcedproteinkinaseaalphacatalyticsubunitscombinedwithinvitroandinsilicoassay AT tiantianhu prokaryoticexpressionofhumansourcedandzebrafishsourcedproteinkinaseaalphacatalyticsubunitscombinedwithinvitroandinsilicoassay AT shuyingli prokaryoticexpressionofhumansourcedandzebrafishsourcedproteinkinaseaalphacatalyticsubunitscombinedwithinvitroandinsilicoassay AT wenjungui prokaryoticexpressionofhumansourcedandzebrafishsourcedproteinkinaseaalphacatalyticsubunitscombinedwithinvitroandinsilicoassay
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Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assay

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