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Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display
bioRxiv - Synthetic Biology Pub Date : 2020-04-18 , DOI: 10.1101/2020.04.16.044438
Kaitlyn Bacon , Abigail Blain , Matthew Burroughs , Nikki McArthur , Balaji M. Rao , Stefano Menegatti

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 µM). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative estimates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

中文翻译:

使用酵母表面展示分离化学环化的肽结合剂

具有工程蛋白结合活性的环肽在治疗和生物技术应用中越来越受到关注。我们描述了使用酵母表面展示从遗传编码的组合库中有效分离和表征环肽结合剂。在这里,肽的环化是通过戊二酸二琥珀酰亚胺基介导的线性肽序列中的胺基团交联来实现的,线性肽序列表现为酵母细胞表面融合。使用这种方法,我们首先通过磁选择和荧光激活细胞分选(FACS)筛选了环状七肽文库,以分离具有低微摩尔结合亲和力(K D)的模型目标(溶菌酶)的结合剂〜1.2-3.7 µM)。分离的肽选择性地且仅在环化时结合溶菌酶。重要的是,我们表明酵母表面展示的环状肽可用于有效获得结合亲和力的定量估计,而无需化学合成选定的肽。随后,为了证明我们方法的广泛适用性,我们使用酵母菌展示的IL-17作为磁性选择的目标,分离了结合人白介素17(IL-17)的环状七肽,然后使用重组IL-17进行了FACS。分子对接模拟和后续实验分析确定了候选环状肽,该环状肽以中等亲和力与受体结合区中的IL-17结合(K D〜300 nM)。两者合计,我们的结果表明,酵母表面展示可用于有效地分离和表征由组合文库中化学修饰产生的环状肽。
更新日期:2020-04-18
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