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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| Format: | Article |
| Language: | English |
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Elsevier
2021-12-01
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| Series: | Ecotoxicology and Environmental Safety |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0147651321011350 |
| _version_ | 1818459864411668480 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T23:21:08Z |
| format | Article |
| id | doaj.art-33abf30b4d97410b93271f6567bd5bc0 |
| institution | Directory Open Access Journal |
| issn | 0147-6513 |
| language | English |
| last_indexed | 2024-12-14T23:21:08Z |
| publishDate | 2021-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Ecotoxicology and Environmental Safety |
| spelling | doaj.art-33abf30b4d97410b93271f6567bd5bc02022-12-21T22:43:57ZengElsevierEcotoxicology and Environmental Safety0147-65132021-12-01228113023Prokaryotic expression of human-sourced and zebrafish-sourced protein kinase A alpha catalytic subunits combined with in vitro and in silico assayKun Qiao0Yao Jiang1Tiantian Hu2Shuying Li3Wenjun Gui4Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China; Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310058, PR ChinaInstitute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR ChinaInstitute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR ChinaInstitute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China; Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, PR China; Key Laboratory of Biology of Crop Pathogens and Insect Pests of Zhejiang Province, Zhejiang, Hangzhou 310058, PR China; Corresponding author at: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China.Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China; Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, PR China; Key Laboratory of Biology of Crop Pathogens and Insect Pests of Zhejiang Province, Zhejiang, Hangzhou 310058, PR ChinaThe 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.http://www.sciencedirect.com/science/article/pii/S0147651321011350Protein kinase A alpha catalytic subunitProkaryotic expressionin vitro kinase activity detectionMolecular docking |
| spellingShingle | 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 Ecotoxicology and Environmental Safety Protein kinase A alpha catalytic subunit Prokaryotic expression in vitro kinase activity detection Molecular docking |
| title | 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_short | 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 |
| topic | Protein kinase A alpha catalytic subunit Prokaryotic expression in vitro kinase activity detection Molecular docking |
| url | http://www.sciencedirect.com/science/article/pii/S0147651321011350 |
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