Enzyme-Mediated, Site-Specific Protein Coupling Strategies for Surface-Based Binding Assays

Area of Science:

• Biochemistry
• Surface Chemistry
• Assay Development

Background:

• Traditional protein immobilization for binding assays relies on non-specific lysine residue attachment.
• This non-specific method presents challenges for receptors with lysine residues near binding pockets or those disfavored by surface electrostatics.
• Limitations include difficulties in probing certain receptors and achieving homogeneous surface functionalization.

Purpose of the Study:

• To adapt and optimize enzymatic coupling strategies for site-specific protein immobilization on biolayer interferometry surfaces.
• To overcome limitations associated with non-specific lysine-based immobilization.
• To improve the homogeneity and functionality of surface-immobilized receptors for binding assays.

Main Methods:

• Adaptation and optimization of three enzymatic coupling strategies: 4'-phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase.
• Site-specific and covalent ligation of proteins of interest via short recognition sequences using these enzymes.
• Application of these enzymatic methods for biolayer interferometry surface modification.

Main Results:

• Successful adaptation and optimization of enzymatic coupling strategies for protein immobilization.
• Demonstrated site-specific and covalent ligation of proteins under mild conditions, preserving receptor functionality.
• Effective study of binding kinetics for two different receptor-ligand pairs using the optimized enzymatic surface functionalization.

Conclusions:

• Enzymatic coupling strategies offer a robust alternative to non-specific immobilization for protein binding assays.
• These methods enable site-specific functionalization, improving assay homogeneity and allowing the study of challenging receptors.
• The optimized enzymatic approach enhances the reliability and scope of surface-based binding kinetic studies.