Library-on-Library Intercellular Labeling for Selection of Biotin Ligase and Acceptor Peptides

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Library-on-library (LOL) selection is a powerful technique for screening combinatorial protein libraries.
• Existing LOL methods are primarily limited to stable protein-protein interactions.
• Transient enzyme-substrate interactions present unique challenges for LOL screening.

Purpose of the Study:

• To develop a generalizable sequential LOL platform for transient enzyme-substrate pairs.
• To demonstrate the platform's efficacy using the Escherichia coli biotin ligase (BirA) and its acceptor peptide (AP).
• To engineer novel enzyme-peptide substrate pairs with tailored activities.

Main Methods:

• Development of a sequential trans- and cis-labeling platform for LOL selection.
• Utilizing yeast surface display for screening mutant libraries.
• Employing SpyTag-SpyCatcher-mediated cis-labeling for enrichment of matched enzyme and substrate variants.

Main Results:

• Successful proof-of-concept selection of acceptor peptide (AP) variants trans-biotinylated by specific biotin ligase (BirA) mutants.
• Enrichment of matched BirA mutants using the cis-labeling platform.
• Demonstration of the first enzyme-peptide substrate LOL selection.

Conclusions:

• The developed platform is the first to enable LOL selection for transient enzyme-peptide substrates.
• This versatile framework facilitates the engineering of new enzyme-peptide substrate pairs.
• The method offers potential for creating enzymes with varied substrate specificities and activities.