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Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Published on: November 12, 2012
Expressivity tag: a novel tool for increased expression in Escherichia coli
Jon Gade Hansted1, Laura Pietikäinen, Friederike Hög
1Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
Journal of Biotechnology
|August 2, 2011
Summary
Researchers developed a novel expressivity tag, InfB(1-21), to enhance protein expression in Escherichia coli. This tag significantly boosts the production of recombinant proteins like streptavidin and scFv, addressing common expression challenges.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Expression
Background:
- Heterologous protein expression in Escherichia coli often faces challenges like low yield and insolubility.
- Existing fusion tags primarily address solubility and affinity, not expression levels directly.
Purpose of the Study:
- To define and characterize a novel fusion tag, termed an expressivity tag, to enhance protein expression levels.
- To investigate the efficacy of a specific InfB gene fragment as an expressivity tag.
Main Methods:
- Constructed progressively shortened fragments of the InfB(1-471) gene.
- Fused gene fragments to a green fluorescent protein (GFP) reporter gene.
- Tested the InfB(1-21) tag with streptavidin and a single-chain variable fragment (scFv) for expression enhancement.
Main Results:
- A 5-fold increase in GFP expression was observed using an optimal 21-nucleotide InfB(1-21) sequence.
- Expression of streptavidin and scFv was significantly improved when fused with the InfB(1-21) expressivity tag, as confirmed by SDS-PAGE.
- Thermodynamic analysis indicated higher free energies in the translation initiation region for proteins fused to InfB(1-21).
Conclusions:
- The InfB(1-21) sequence functions as an effective expressivity tag, enhancing recombinant protein yields in E. coli.
- This expressivity tag offers a new tool for optimizing heterologous protein production.
- The tag improves expression without altering codon usage or codon adaptation index.
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