Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

Abstract

Abstract. We present the construction and screening of yeast display libraries of cyclic peptides wherein site-selective enzymatic cyclization of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve cyclization in the endoplasmic reticulum prior to yeast surface display. The cyclization yield was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-cyclized peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified cyclic peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.67 μM for YAP and 0.84 μM for WW as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of yeast surface display for screening transglutaminase-cyclized peptide libraries, and efficient identification of cyclic peptide ligands.

A novel approach for selecting potent peptide inhibitors of the SARS-CoV2-Mpro protease

“…Biosynthetic libraries can easily contain as many as 10 9 peptides, while chemical synthesis is limited to approximately10 4 peptides. The most commonly used biosynthetic selection methods are phage display 11 , yeast display 12 and RNA display 13,14 . All of these methods select peptides that bind to the target protein most tightly.…”