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Screening Combinatorial Libraries of Cyclic Peptides Using the Yeast Two-Hybrid Assay

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Yeast Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1163))

Abstract

Peptides are useful reagents for reverse analysis of protein function in a variety of organisms, as they have a dominant mode of action that can inhibit protein interactions or activities. Further, peptides are important tools for validating proteins as therapeutic targets, for determining structure/activity relationships, and for designing small molecules. Genetic selection strategies have been developed for screening combinatorial peptide libraries to rapidly isolate peptides that interact with a given target. In genetic selections and biological assays, linear peptides are not very stable and are rapidly degraded. In contrast, cyclic peptides are more stable and bind with higher affinity. Genetic selections of cyclic peptides are difficult as they are not compatible with most selection technologies. Thus, there has been limited number of applications that use cyclic peptides for the reverse analysis of protein function.

Here, we describe a protocol to isolate cyclic peptides that bind proteins in the yeast two-hybrid assay. Cyclic peptides used in the yeast two-hybrid assay are referred to as “lariat” peptides. Lariat peptides are made by blocking the intein-producing cyclic peptide reaction at an intermediate step. They consist of a lactone cyclic peptide or “noose” region connected by an amide bond to a transcription activation domain. Combinatorial libraries of >107 lariat peptides can be screened using the yeast two-hybrid assay to isolate lariat peptides for studying the function or validating the therapeutic potential of protein targets.

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  1. Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada, S7N 5E5

    Kris Barreto & C. Ronald Geyer

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  1. Kris Barreto
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  2. C. Ronald Geyer
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Correspondence to C. Ronald Geyer .

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  1. Capital Normal University College of Life Sciences, Beijing, China

    Wei Xiao

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Barreto, K., Geyer, C.R. (2014). Screening Combinatorial Libraries of Cyclic Peptides Using the Yeast Two-Hybrid Assay. In: Xiao, W. (eds) Yeast Protocols. Methods in Molecular Biology, vol 1163. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0799-1_21

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  • DOI: https://doi.org/10.1007/978-1-4939-0799-1_21

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