Optimized Construction of a Yeast SICLOPPS Library for Unbiased In Vivo Selection of Cyclic Peptides.

IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Biochemistry Biochemistry Pub Date : 2024-12-17 Epub Date: 2024-12-06 DOI:10.1021/acs.biochem.4c00013
Nanna Birkmose, Emilie U Frydendahl, Charlotte R Knudsen
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引用次数: 0

Abstract

DNA-encoded libraries hold great potential for discovering small, cyclized peptides with drug potential. Split-intein circular ligation of peptides and proteins (SICLOPPS) is a well-established method for in vivo selection of cyclic peptides targeting specific intracellular components. However, the method has mainly been used in prokaryotic cells. In contrast, selection studies performed directly in eukaryotic cells allow for the identification of cyclic peptides promoting a functional outcome, without the need to define a specific cellular target. Here, we report the construction of a Saccharomyces cerevisiae-specific SICLOPPS library of 80 million members, via careful optimization of several steps to increase the size of the library. Individual library members were shown to be correctly expressed and processed in yeast. High-throughput sequencing was conducted on the randomized primer used for library construction and the pure yeast SICLOPPS library isolated from Escherichia coli. A distinct guanine insertion bias was observed in the peptide-encoding, randomized sequence, which was primarily attributed to the degenerate primer used to introduce the randomized sequence. Moreover, high-throughput sequencing was performed on the library before and after the induction of cyclic peptide expression in yeast. Importantly, expression of the SICLOPPS library in S. cerevisiae caused only a marginal further sequence bias. Our work paves the way for selection studies using a large and diverse library to identify cyclic peptides of therapeutic interest that promote a specific phenotypic outcome in eukaryotic organisms, with yeast representing a beneficial model system due to its high transformation efficiency.

酵母SICLOPPS文库的优化构建,用于环肽的无偏体内筛选。
dna编码文库在发现具有药物潜力的小环化肽方面具有很大的潜力。肽和蛋白的分裂-蛋白环连接(SICLOPPS)是一种成熟的针对特定细胞内成分的环肽在体内选择的方法。然而,该方法主要用于原核细胞。相反,直接在真核细胞中进行的选择研究允许鉴定促进功能结果的环肽,而不需要定义特定的细胞靶标。在这里,我们报道了一个8000万成员的酿酒酵母特异性SICLOPPS文库的构建,通过精心优化几个步骤来增加文库的规模。个别文库成员被证明在酵母中正确表达和加工。对构建文库的随机引物和从大肠杆菌中分离到的纯酵母SICLOPPS文库进行高通量测序。在肽编码随机序列中观察到明显的鸟嘌呤插入偏倚,这主要归因于用于引入随机序列的简并引物。在酵母诱导环状肽表达前后对文库进行高通量测序。重要的是,SICLOPPS文库在酿酒酵母中的表达仅引起了边际的进一步序列偏差。我们的工作为选择研究铺平了道路,使用一个庞大而多样的文库来识别具有治疗意义的环肽,这些环肽可以促进真核生物中特定表型的结果,酵母由于其高转化效率而代表了一个有益的模型系统。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Biochemistry Biochemistry
Biochemistry Biochemistry 生物-生化与分子生物学
CiteScore
5.50
自引率
3.40%
发文量
336
审稿时长
1-2 weeks
期刊介绍: Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.
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