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

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.

Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface

“…The values obtained with assays conducted in scFv display format were consistent with the values of assays performed in solution in scFv-Fc format. While previous reports have described titrations of yeast to evaluate inhibition 30 , we are not aware of any work in which IC 50 values were determined on yeast. Yeastbased assays for IC 50 value determination are expected to support multiple key evaluations during inhibitor discovery and characterization.…”

Structural principles of insulin formulation and analog design: A century of innovation

“…Receptor-targeted phage display peptides, for example, bind at or near insulin's native receptor-binding sites but stimulate a distinct pattern of signaling outputs [ [228] , [229] , [230] ]. Yeast display technology may enable this approach to generalize to libraries of insulin variants [ [231] , [232] , [233] ] as demonstrated by Chou and colleagues [ 234 ]. The concept of biased agonism may also be exploited to reduce the baseline mitogenicity of insulin, of interest in relation to epidemiological relationships between T2D and several common cancers [ [235] , [236] , [237] ].…”

Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research

“…Recent computational docking methods for protein and peptide interactions, and progress in peptide library-use concerning synthetic and/or structurally modified peptides, enable comparative studies and engineering of both continuous and discontinuous peptides and selection of potential motifs/lead compounds [20,[28][29][30][31]. Novel protein-peptide docking procedures are based on different aspects of interaction studies, including inhibitor screening, model prediction, experimental data interpretation, specificity of prediction, and design of interfering peptides [28].…”