Engineering caspase 7 as an affinity reagent to capture proteolytic products

Area of Science:

• Biochemistry
• Molecular Biology
• Protease Engineering

Background:

• Proteases exhibit high substrate specificity, suggesting their binding sites can be engineered into affinity reagents.
• Differentiating between protease substrates and products is crucial for understanding enzymatic activity and developing targeted therapies.

Purpose of the Study:

• To engineer a caspase 7-based scaffold capable of specifically trapping protease products.
• To develop a versatile affinity reagent for recognizing apoptotic cells and other targets.

Main Methods:

• Engineered a catalytically inactive caspase 7 mutant that adopts a substrate-binding conformation.
• Introduced three specific mutations to create a 'product-only trap' by compensating for charge changes upon product formation.
• Fused the engineered product trap with a fluorescent protein to create a cellular imaging agent.

Main Results:

• Successfully generated a caspase 7 variant that binds peptides with affinity comparable to the wild-type enzyme.
• Developed a 'product-only trap' that specifically recognizes protease cleavage products.
• Demonstrated that the hybrid fluorescent protein reagent specifically identifies apoptotic cells.

Conclusions:

• Protease scaffolds can be engineered into highly specific affinity reagents for trapping reaction products.
• This approach offers a versatile platform for developing novel affinity and imaging agents for various proteases.
• The developed reagent shows promise for specific detection of apoptotic cells and potentially other biological targets.