A dual prokaryotic (E. coli) expression system (pdMAX).

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogala...

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Autores principales:	Manabu Murakami, Agnieszka M Murakami, Shirou Itagaki
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/898cb8e561ae4d2ca8da9687fe58dcca
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spelling	oai:doaj.org-article:898cb8e561ae4d2ca8da9687fe58dcca2021-12-02T20:16:41ZA dual prokaryotic (E. coli) expression system (pdMAX).1932-620310.1371/journal.pone.0258553https://doaj.org/article/898cb8e561ae4d2ca8da9687fe58dcca2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0258553https://doaj.org/toc/1932-6203In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.Manabu MurakamiAgnieszka M MurakamiShirou ItagakiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10, p e0258553 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manabu Murakami Agnieszka M Murakami Shirou Itagaki A dual prokaryotic (E. coli) expression system (pdMAX).
description	In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.
format	article
author	Manabu Murakami Agnieszka M Murakami Shirou Itagaki
author_facet	Manabu Murakami Agnieszka M Murakami Shirou Itagaki
author_sort	Manabu Murakami
title	A dual prokaryotic (E. coli) expression system (pdMAX).
title_short	A dual prokaryotic (E. coli) expression system (pdMAX).
title_full	A dual prokaryotic (E. coli) expression system (pdMAX).
title_fullStr	A dual prokaryotic (E. coli) expression system (pdMAX).
title_full_unstemmed	A dual prokaryotic (E. coli) expression system (pdMAX).
title_sort	dual prokaryotic (e. coli) expression system (pdmax).
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/898cb8e561ae4d2ca8da9687fe58dcca
work_keys_str_mv	AT manabumurakami adualprokaryoticecoliexpressionsystempdmax AT agnieszkammurakami adualprokaryoticecoliexpressionsystempdmax AT shirouitagaki adualprokaryoticecoliexpressionsystempdmax AT manabumurakami dualprokaryoticecoliexpressionsystempdmax AT agnieszkammurakami dualprokaryoticecoliexpressionsystempdmax AT shirouitagaki dualprokaryoticecoliexpressionsystempdmax
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A dual prokaryotic (E. coli) expression system (pdMAX).

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