Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Area of Science:

• Bioconjugation Chemistry
• Protein Engineering
• Molecular Biology

Background:

• Click chemistry offers bio-orthogonal, selective, and high-yield protein functionalization under mild conditions.
• Designed Ankyrin Repeat Proteins (DARPins) are stable binding proteins suitable for incorporating clickable amino acids.
• Methionine-free DARPin scaffolds with mutations like M34L maintain biophysical properties while allowing clickable modifications.

Purpose of the Study:

• To develop a method for site-specific dual functionalization of DARPins using click chemistry.
• To evaluate the impact of polyethylene glycol (PEG)ylation on DARPin binding kinetics in a tumor targeting context.
• To demonstrate the utility of modified DARPins for biomedical applications.

Main Methods:

• Incorporation of N-terminal azidohomoalanine (Aha) for click chemistry.
• C-terminal cysteine introduction for secondary modification.
• Site-specific conjugation of PEG and Alexa488 to DARPins.
• Selection of an EpCAM-targeting DARPin for functionalization.
• Measurement of binding kinetics on tumor cells.

Main Results:

• Clickable DARPins were produced in high yield (30 mg/L E. coli culture) with retained stability, specificity, and affinity.
• Site-specific dual modification with PEG (N-terminus) and Alexa488 (C-terminus) was achieved.
• PEGylation slightly decreased association rate and maximal cell binding but did not affect dissociation rate.
• Functionalized DARPins showed effective binding kinetics on EpCAM-expressing tumor cells.

Conclusions:

• Click chemistry provides a versatile platform for site-specific dual functionalization of DARPins.
• Modified DARPins retain key binding properties, demonstrating their potential for targeted therapies.
• This approach facilitates the simultaneous addition of multiple functional moieties for diverse biomedical applications.