Modular protein switches derived from antibody mimetic proteins

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Protein switches are crucial for biosensors and therapeutics but are typically developed using a trial-and-error approach.
• A modular platform allowing easy adaptation of existing switches to new ligands is highly desirable.

Purpose of the Study:

• To investigate the feasibility of a modular protein switch platform.
• To create adaptable protein switches using fusions of TEM-1 beta-lactamase (BLA) with designed ankyrin repeat proteins (DARPins) and monobodies.

Main Methods:

• Constructed gene libraries by randomly inserting the BLA gene into DARPin and monobody genes targeting maltose-binding protein (MBP).
• Employed a genetic selection system based on beta-lactamase activity to identify functional switch genes in Escherichia coli.
• Introduced mutations into antibody mimetic domains to alter ligand-binding specificity.

Main Results:

• Identified switch proteins exhibiting up to a 14-fold increase in enzymatic activity in the presence of MBP.
• Demonstrated that mutations in the antibody mimetic domain can confer desired ligand specificities to the protein switches.
• Confirmed the potential modularity of the developed protein switch platform.

Conclusions:

• The developed modular platform shows promise for creating customizable protein switches.
• This approach facilitates the engineering of protein switches with tunable responses to various ligands.
• The study highlights a significant advancement in the design of protein-based biosensors and therapeutics.