Cell surface display yields evolvable, clickable antibody fragments.

Area of Science:

• Biochemistry
• Protein Engineering
• Molecular Biology

Background:

• Non-canonical amino acids (ncAAs) offer versatile tools for modifying protein properties.
• Introducing ncAAs can lead to unpredictable effects on protein function, requiring robust screening methods.
• Directed evolution is a powerful technique for optimizing protein characteristics.

Purpose of the Study:

• To develop and validate an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments.
• To investigate the impact of ncAAs on antibody fragment binding affinity and kinetics.
• To assess the utility of azide-functionalized antibody fragments for chemical modification.

Main Methods:

• Utilized an Escherichia coli cell surface display system for high-throughput screening.
• Employed directed evolution to select for antibody fragments with improved digoxigenin binding.
• Incorporated non-canonical amino acids, including azide-functionalized variants, into antibody fragments.
• Characterized binding kinetics and affinity of evolved antibody fragments.
• Demonstrated chemical modification via azide-alkyne cycloaddition.

Main Results:

• Successfully isolated antibody fragments with enhanced digoxigenin binding and moderate affinity maturation in various ncAA contexts.
• Azide-functionalized antibody fragments demonstrated superior binding kinetics compared to methionine-containing counterparts.
• Confirmed facile chemical modification of azide-functionalized fragments through click chemistry without compromising binding properties.
• Showcased the retention of protein binding capabilities after chemical modification.

Conclusions:

• The developed cell surface display platform facilitates the directed evolution of clickable antibody fragments.
• Non-canonical amino acids can modulate molecular recognition events and protein properties.
• Chemically modified proteins can be directly screened, opening new avenues in protein engineering.
• This approach enables the engineering of proteins with tailored chemical functionalities and improved binding characteristics.