Palei Shubhendu, Jose Joachim, Mootz Henning D
International Graduate School of Chemistry (GSC-MS), Institute of Biochemistry, University of Muenster, Münster, Germany.
Methods Mol Biol. 2022;2371:193-213. doi: 10.1007/978-1-0716-1689-5_11.
Semisynthetic cyclic peptides bearing both non-proteinogenic and genetically encoded amino acids are excellent ligands for peptide-based drug discovery. While semisynthesis expands the chemical space, genetic encoding allows access to a large library via randomization at the nucleic acid level. Selection of novel binders of such macrocyclic ligands requires linking their genotype to phenotype. In this chapter, we report a bacterial cell-surface display system to present cyclic peptides composed of synthetic and genetically encoded fragments. The synthetic fragment along with the split intein partner and an aminooxy moiety is ligated and cyclized with the recombinant backbone containing an unnatural amino acid by protein trans-splicing and intramolecular oxime ligation, respectively. A pH-shift protocol was applied to accelerate on surface cyclization. This method will enable generation of semisynthetic cyclic peptide libraries and their selection by fluorescence-activated cell sorting.
同时含有非蛋白原性氨基酸和基因编码氨基酸的半合成环肽是基于肽的药物发现的优秀配体。虽然半合成扩展了化学空间，但基因编码允许通过核酸水平的随机化获得大量文库。选择此类大环配体的新型结合物需要将其基因型与表型联系起来。在本章中，我们报告了一种细菌细胞表面展示系统，用于展示由合成片段和基因编码片段组成的环肽。合成片段与分裂内含肽伴侣和氨氧基部分分别通过蛋白质反式剪接和分子内肟连接与含有非天然氨基酸的重组骨架连接并环化。应用pH值转换方案加速表面环化。该方法将能够生成半合成环肽文库并通过荧光激活细胞分选对其进行筛选。
