A general strategy for protein affinity-ligand oriented-immobilization and screening for bioactive compounds

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Natural products are vital drug sources, but screening for active compounds is inefficient.
• Existing methods for immobilizing proteins for screening are often time-consuming and complex.

Purpose of the Study:

• To develop a facile and efficient protein affinity-ligand oriented-immobilization strategy for bioactive compound screening.
• To validate the strategy using model proteins and identify a specific enzyme inhibitor.

Main Methods:

• Utilized SpyTag/SpyCatcher (ST/SC) chemistry for one-step protein immobilization.
• Employed ST-fused green fluorescent protein (GFP) and PqsA for method verification.
• Characterized affinity carriers using infrared spectroscopy, fluorography, electrophoresis, and fluorescence analyses.
• Validated the strategy by immobilizing PqsA and screening for inhibitors.

Main Results:

• Successfully immobilized ST-labeled GFP and PqsA onto SC-coupled agarose with specific orientation.
• Confirmed the spontaneity and site-specificity of the ST/SC ligation reaction.
• Demonstrated the isolation of a PqsA inhibitor (2-amino-6-fluorobenzoic acid) from a fermentation broth.
• Established acceptable pH stability for the affinity carriers (pH < 9).

Conclusions:

• The ST/SC-based strategy provides a rapid and efficient method for oriented protein immobilization.
• This approach facilitates the screening of compounds that specifically interact with immobilized protein ligands.
• The validated method holds promise for accelerating the discovery of bioactive compounds from natural products.