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Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
Published on: June 27, 2017
A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium
Martin Gamer1, David Fröde, Rebekka Biedendieck
1Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Applied Microbiology and Biotechnology
|March 25, 2009
Summary
Researchers developed a T7 RNA polymerase expression system in Bacillus megaterium for high-level recombinant protein production. This system efficiently produced both intracellular green fluorescent protein (GFP) and secreted Lactobacillus reuteri levansucrase.
Area of Science:
- Biotechnology
- Molecular Biology
- Microbial Engineering
Background:
- Bacteriophage T7 RNA polymerase systems are favored for high-level recombinant protein production.
- Bacillus megaterium is a Gram-positive bacterium recognized for its utility in heterologous protein expression.
Purpose of the Study:
- To develop and evaluate a T7 RNA polymerase-dependent gene expression system in Bacillus megaterium.
- To assess the system's efficacy for both intracellular and extracellular protein production.
Main Methods:
- Construction of a T7 RNA polymerase-based expression vector for B. megaterium.
- Utilized green fluorescent protein (GFP) as an intracellular model protein.
- Employed Lactobacillus reuteri levansucrase as a secretory model protein.
- Investigated the effect of rifampicin on protein stability.
Main Results:
- Achieved high intracellular accumulation of GFP (up to 50 mg/l) after T7 RNA polymerase induction.
- Observed enhanced GFP stability with rifampicin treatment, inhibiting native B. megaterium RNA polymerase.
- Successfully secreted L. reuteri levansucrase into the supernatant (up to 20 U/l).
- Identified limitations in the Sec-dependent translocation process due to intracellular levansucrase accumulation.
Conclusions:
- The developed T7 RNA polymerase system is effective for high-yield intracellular protein production in B. megaterium.
- The system enables extracellular secretion of proteins, though translocation efficiency may require optimization.
- B. megaterium serves as a robust host for diverse recombinant protein production strategies.
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