Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USA.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
Int J Mol Sci. 2021 Feb 5;22(4):1634. doi: 10.3390/ijms22041634.
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[LYLAYPAH] featured a K 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.
我们构建并筛选了经翻译后修饰肽的酵母展示文库，其中采用细菌转谷氨酰胺酶实现线性肽的位点选择性酶处理。为此，我们开发了两种替代途径，即 (i) 线性肽的酵母展示，然后在溶液中用重组转谷氨酰胺酶处理；或 (ii) 线性肽和转谷氨酰胺酶的细胞内共表达，以在酵母表面展示之前在内质网中实现肽修饰。通过流式细胞术正交检测整合在酵母展示肽中的表位标签，以及通过烟草蚀纹病毒 (TEV) 蛋白酶对假定的环状与线性肽的比较切割，评估了肽修饰的效率。随后，筛选经转谷氨酰胺酶处理的肽的酵母展示文库，以使用磁选和荧光激活细胞分选 (FACS) 分离与 Yes 相关蛋白 (YAP) 的 N 端区域及其 WW 结构域结合的配体。鉴定出的肽 cyclo[LYLAYPAH]对 YAP 的 Kd 为 1.75 μM，对 YAP 的 WW 结构域的 Kd 为 0.68 μM，并且对白蛋白和溶菌酶具有高结合选择性。这些结果表明酶介导的环化在筛选组合文库以鉴定环状肽结合物方面的有用性。
