Rapid and easy development of versatile tools to study protein/ligand interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Producing difficult-to-express protein domains is a significant challenge in biotechnology.
• Existing methods often lack efficiency in screening and production of functional proteins.

Purpose of the Study:

• To develop a novel system for expressing protein fragments within a carrier protein.
• To facilitate the production and screening of functional, difficult-to-express protein domains.

Main Methods:

• Utilized beta-lactamase (BlaP) from Escherichia coli as a carrier protein.
• Engineered protease-specific recognition sites for fragment cleavage.
• Employed constitutive expression vectors with positive selection via antibiotic resistance.

Main Results:

• Successfully expressed protein fragments in a solvent-exposed loop of BlaP.
• Demonstrated that BlaP carrier does not impede the activity of the inserted chitin-binding domain.
• Showcased the utility of hybrid protein beta-lactamase activity for monitoring molecular interactions and screening inhibitors.

Conclusions:

• The BlaP carrier system is a viable alternative for producing challenging protein domains.
• This approach simplifies the screening and purification of correctly folded and secreted hybrid proteins.
• The system enables functional assays and inhibitor screening based on the carrier's enzymatic activity.