Mammalian Surface Display Screening of Diverse Cystine-Dense Peptide Libraries for Difficult-to-Drug Targets

  • Zachary R. Crook
  • Gregory P. Sevilla
  • Andrew J. Mhyre
  • James M. OlsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)


Many diseases are mediated by targets that are not amenable to conventional small-molecule drug approaches. While antibody-based drugs have undeniable utility, peptides of the 1–9 kDa size range (10–80 amino acids) have drawn interest as alternate drug scaffolds This is born of a desire to identify compounds with the advantages of antibody-based therapeutics (affinity, potency, specificity, and ability to disrupt protein:protein interactions) without all of their liabilities (large size, expensive manufacturing, and necessity of humanization). Of these alternate scaffolds, cystine-dense peptides (CDPs) have several specific benefits. Due to their stable intra-chain disulfide bridges, CDPs often demonstrate resistance to heat and proteolysis, along with low immunogenicity. These properties do not require chemical modifications, permitting CDP screening by conventional genetic means. The cystine topology of a typical CDP requires an oxidative environment, and we have found that the mammalian secretory pathway is most effective at allowing diverse CDPs to achieve a stable fold. As such, high-diversity screens to identify CDPs that interact with targets of interest can be efficiently conducted using mammalian surface display. In this protocol, we present the theory and tools to conduct a mammalian surface display screen for CDPs that bind with targets of interest, including the steps to validate binding and mature the affinity of preliminary candidates. With these methods, CDPs of all kinds can be brought to bear against targets that would benefit from a peptide-based intervention.

Key words

Surface display Peptides Screening Flow cytometry High throughput Drug discovery Cysteine-rich 



The author thanks Colin Correnti, Roland Strong, and Ashok Bandaranayake for helpful discussions as the manuscript was prepared. The Fred Hutch Shared Resources, particularly the Flow Cytometry and Genomics facilities, were instrumental to data generation. The author also thanks Shelli Morris and Chris Mehlin for their help in editing the manuscript. This work was funded by NIH Grants R01CA114567 (J.M.O.) and R01CA155360 (J.M.O.); A Washington Research Foundation Innovation Fellowship through the University of Washington Institute for Protein Design; NIH Fellowship T32AG00005740 (Z.R.C.); and Project Violet (


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Zachary R. Crook
    • 1
  • Gregory P. Sevilla
    • 1
  • Andrew J. Mhyre
    • 1
  • James M. Olson
    • 1
    Email author
  1. 1.Clinical Research DivisionFred Hutchinson Cancer Research CenterSeattleUSA

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