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.

中文翻译：

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筛选酶环化肽的酵母展示文库以发现大环肽配体

摘要。我们介绍了环肽的酵母展示文库的构建和筛选，其中使用细菌转谷氨酰胺酶实现了线性肽的位点选择性酶环化。为此，我们开发了两种替代方法，即（i）酵母展示线性肽，然后在溶液中用重组转谷氨酰胺酶处理；（ii）在酵母表面展示之前，线性肽和转谷氨酰胺酶在细胞内的共表达以在内质网中实现环化。通过流式细胞术通过正交检测整合在酵母展示肽中的抗原决定簇标签，以及通过烟草蚀刻病毒（TEV）蛋白酶比较环肽与线性肽的裂解，来评估环化产率。后来，使用磁性选择和荧光激活细胞分选（FACS）筛选了转谷氨酰胺酶环化的肽的酵母展示文库，以分离与Yes-Associated蛋白（YAP）及其WW域的N端区域结合的结合物。鉴定出的环肽环[E-LYLAYPAH-K]的KD对YAP为1.67μM，对WW为0.84μM，并且对白蛋白和溶菌酶具有高结合选择性。这些结果证明了酵母表面展示用于筛选转谷氨酰胺酶环化的肽文库和有效鉴定环肽配体的有用性。WW的浓度为84μM，对白蛋白和溶菌酶的结合选择性高。这些结果证明了酵母表面展示用于筛选转谷氨酰胺酶环化的肽文库和有效鉴定环肽配体的有用性。WW的浓度为84μM，对白蛋白和溶菌酶的结合选择性高。这些结果证明了酵母表面展示用于筛选转谷氨酰胺酶环化的肽文库和有效鉴定环肽配体的有用性。