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

Manabu Murakami; Agnieszka M Murakami; Shirou Itagaki

doi:10.1371/journal.pone.0258553

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

PLoS One. 2021 Oct 21;16(10):e0258553. doi: 10.1371/journal.pone.0258553. eCollection 2021.

Authors

Manabu Murakami¹, Agnieszka M Murakami¹, Shirou Itagaki²

Affiliations

¹ Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
² Collaboration Center for Community and Industry, Sapporo Medical University, Sapporo, Japan.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli*
Isopropyl Thiogalactoside

Substances

Isopropyl Thiogalactoside

Grants and funding

This research was sponsored in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI nos. 17K08527 [MM], 17H04319 [MM], and 20K07255 [MM]). No additional external funding was received for this study.