An intein-based biosensor to measure protein stability in vivo

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Protein stability is crucial for cellular function and disease.
• Existing biosensors for in vivo protein stability often use tripartite designs.
• Inteins are protein-splicing elements that can be engineered for novel applications.

Purpose of the Study:

• To develop a novel and sensitive biosensor for measuring protein stability in vivo.
• To demonstrate the utility of intein-mediated protein splicing in biosensor design.
• To create a biosensor with dual readouts for protein folding and stability.

Main Methods:

• Engineered a novel biosensor by inserting a protein of interest (POI) within an intein.
• Intein was further inserted into an antibiotic resistance reporter gene.
• Utilized a slow-maturing fluorescent protein as the POI for a second readout.
• Assessed biosensor activity with stable and unstable POI variants and in the presence of GroEL.

Main Results:

• Protein splicing was essential for antibiotic resistance, linking POI stability to reporter activity.
• A stable POI within the intein resulted in a 100,000-fold increase in survival compared to an unstable variant.
• A dual-readout reporter system simultaneously monitored protein folding and stability.
• Co-expression of GroEL enhanced biosensor activity, confirming the influence of protein folding factors.

Conclusions:

• The developed intein-based biosensor offers a sensitive and versatile tool for assessing protein stability in vivo.
• This novel design provides a significant advancement over traditional tripartite biosensors.
• The biosensor system has broad implications for understanding protein folding, stability, and disease mechanisms.