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

Manabu Murakami,Shirou Itagaki,Agnieszka M Murakami,Dharam Singh

doi:10.1371/journal.pone.0258553

Manabu Murakami, Shirou Itagaki + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0258553

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Journal: PloS one	Publication Date: Oct 21, 2021
Citations: 3	License type: CC BY 4.0

Affiliation: Hirosaki University, Sapporo Medical University

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.

Highlights

A number of bacterial expression plasmids are used for prokaryotic expression in Escherichia coli (E. coli)
The arabinose expression unit featured araC, the araBAD promoter, the FLAG tag sequence, the EcoRV site for blunt-end cloning, and iUnit (Fig 1A); whereas the IPTG expression unit was composed of the lac promoter, the lac operator, the myc tag sequence, a SmaI site for blunt-end cloning, and an iUnit
The polymerase chain reaction (PCR)-amplified α-peptide sequence of the lacZ (β -galactosidase) gene was inserted between the blunt-end sites of EcoRV or SmaI of pdMAX

Summary

Introduction

A number of bacterial expression plasmids are used for prokaryotic expression in Escherichia coli (E. coli). The polymerase chain reaction (PCR)-amplified α-peptide sequence of the lacZ (β -galactosidase) gene was inserted between the blunt-end sites of EcoRV (in the arabinose expression unit) or SmaI (in the IPTG expression unit) of pdMAX. Secondary spot culture revealed decreased inducible gene expression in pdMAX, whereas the single-promoter pgMAX construct showed strong basal β-galactosidase activity, indicated by blue regions (S1 Fig, amp/Xgal, lane 3).

Results

Conclusion