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Engineered fluorescent proteins illuminate the bacterial periplasm.
Thorben Dammeyer1, Philip Tinnefeld1
1Institut für Physikalische und Theoretische Chemie, NanoBioSciences, Technische Universität Braunschweig, Hans Sommer Str. 10, 38106 Braunschweig, Germany.
Computational and Structural Biotechnology Journal
|April 2, 2014
Summary
Engineered Green Fluorescent Protein (GFP) variants can now fold and function within the oxidizing bacterial periplasm. This breakthrough enables enhanced protein production and new biotechnological applications using fluorescent reporters.
Area of Science:
- Microbiology and Biotechnology
- Protein Engineering
- Cellular Biology
Background:
- The bacterial periplasm offers advantages for recombinant protein production, including correct N-termini and disulfide bond formation.
- The oxidizing periplasmic environment hinders the proper folding and activity of standard Green Fluorescent Protein (GFP) due to interchain disulfide bonds.
- Protein engineering has yielded improved GFP variants with enhanced folding and stability properties.
Purpose of the Study:
- To review advancements in engineering Green Fluorescent Protein (GFP) for functional expression in the bacterial periplasm.
- To highlight progress in understanding bacterial protein export pathways and periplasmic protein organization.
- To explore new biotechnological applications leveraging periplasmic production and fluorescent reporters.
Main Methods:
- Review of recent studies on protein engineering of Green Fluorescent Protein (GFP) variants.
- Analysis of protein translocation pathways, including the twin-arginine translocation (Tat) pathway, general secretory pathway (Sec), and signal recognition particle (SRP) mediated secretion.
- Examination of the impact of the oxidizing periplasmic environment on protein folding and disulfide bond formation.
Main Results:
- Engineered GFP variants demonstrate improved folding and stability, enabling activity in the periplasm.
- Recent GFP variants can actively fold within the periplasm after secretion via Sec and SRP pathways.
- Some variants are active when translocated in a pre-folded state via the Tat pathway.
Conclusions:
- Protein engineering has overcome the challenges of expressing active Green Fluorescent Protein (GFP) in the bacterial periplasm.
- These advancements provide new tools for studying bacterial export and protein organization.
- The development opens avenues for novel biotechnological applications combining periplasmic production advantages with fluorescent reporter capabilities.
