Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.

John Bowen,Collin Labar,Balaji M Rao,Kaitlyn Bacon,John Schneible,Stefano Menegatti

doi:10.3390/ijms22041634

John Bowen, Collin Labar + Show 4 more

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https://doi.org/10.3390/ijms22041634

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Abstract

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment 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 peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated 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 peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.

Highlights

The use of cyclic peptides in basic research, as well as diagnostic and therapeutic applications, has grown significantly owing to their superior bioactivity and biochemical stability compared to their linear counterparts [1,2,3]
A notable difference in residual c-myc level was observed between untreated cells, which respectively showed a significant decrease in fluorescence (Figure 2B,D), and cells treated with transglutaminase, which showed only a slight decrease (Figure 2C,D), after tobacco etch virus (TEV) treatment
In this study we explored the use of transglutaminase to cyclize peptides displayed on the surface of yeast cells, given the versatility of this enzyme in modifying peptides with varying length and composition provided that the appropriate flanking residues are present

Summary

Introduction

The use of cyclic peptides in basic research, as well as diagnostic and therapeutic applications, has grown significantly owing to their superior bioactivity and biochemical stability compared to their linear counterparts [1,2,3]. A wide range of enzymes have been employed to achieve peptide or protein cyclization such as sortase [26,27], butelase 1 [28], the S-adenosylmethionine enzyme AlbA [29], subtiligase [30], the thioesterase domain of tyrocidine synthetase [31], inteins [32,33,34,35], and transglutaminase [24] Despite their proven potential, only a few examples are reported where enzymes were used to engineer display libraries of cyclic peptides for de novo discovery of bioactive peptides [36,37,38]

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