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

Manabu Murakami1, Agnieszka M Murakami1, Shirou Itagaki2

  • 1Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

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|October 21, 2021
PubMed
Summary
This summary is machine-generated.

We developed a novel dual-promoter plasmid system (pdMAX) for Escherichia coli, enabling efficient subcloning and inducible expression of two distinct genes with FLAG and Myc tags. This system facilitates prokaryotic gene expression analysis.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Microbiology

Background:

  • Developing efficient systems for simultaneous gene expression in prokaryotes is crucial for complex biological studies.
  • Existing single-promoter systems limit the ability to analyze multiple genes concurrently.
  • Inducible promoters offer temporal control over gene expression, essential for studying gene function and interactions.

Purpose of the Study:

  • To introduce an efficient subcloning and dual-inducible expression system in Escherichia coli.
  • To enable the expression of two distinct genes with specific tags (FLAG and Myc) from a single plasmid.
  • To facilitate the analysis of dual gene expression in prokaryotic systems.

Main Methods:

  • Construction of a dual-promoter plasmid (pdMAX) in Escherichia coli, featuring arabinose and isopropyl-β-D-thiogalactoside (IPTG) inducible promoters.
  • Incorporation of an efficient subcloning (iUnit) system based on a DNA topoisomerase toxin for selection.
  • Co-expression of FLAG-tagged and Myc-tagged proteins, and fluorescent proteins (DsRed and EGFP) under dual induction.

Main Results:

  • The pdMAX system demonstrated efficient subcloning and inducible expression of two genes.
  • A decrease in lacZ (β-galactosidase) activity was observed compared to solo expression systems.
  • Successful expression of DsRed and EGFP fluorescent proteins was achieved under dual induction and low-temperature incubation.

Conclusions:

  • The novel pdMAX system allows for efficient subcloning and analysis of two distinct genes in prokaryotes.
  • This dual-inducible system provides a valuable tool for various prokaryotic gene expression studies.
  • The system's ability to express tagged proteins and fluorescent markers enhances its utility in molecular biology research.