Macrocyclic phage display for identification of selective protease substrates.

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Traditional protease substrate identification relies on limited linear peptides.
• Phage display offers a powerful platform for screening diverse molecular libraries.

Purpose of the Study:

• To develop a novel phage display method for identifying highly selective protease substrates.
• To overcome limitations of traditional methods using chemically modified cyclic peptides.

Main Methods:

• Utilized phage display to screen chemically modified cyclic peptide libraries.
• Incorporated reactive linkers, affinity tags, and fluorescent reporters for selection and quantification.
• Demonstrated utility with Fibroblast Activation Protein alpha (FAPα) and dipeptidyl peptidase-4 (DPP4).

Main Results:

• Identified highly selective substrates for FAPα and DPP4.
• Demonstrated the efficiency of the turn-on fluorescent reporter for cleavage quantification.
• Validated substrate identification and optimization using recombinant enzymes and cells.

Conclusions:

• The developed phage display strategy provides a rapid, unbiased platform for discovering non-natural protease substrates.
• Identified substrates show potential for basic research and fluorescence image-guided surgery (FIGS).
• This method overcomes key limitations of existing protease substrate identification techniques.