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Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Nature Protocols volume 12, pages 21892214 (2017) | Download Citation

Abstract

Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4–P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1′ position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4–P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4–8 weeks (HyCoSuL synthesis: 3–5 weeks; HyCoSuL screening per enzyme: 4–8 d; and activity-based probe synthesis: 1–2 weeks).

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Acknowledgements

This work was supported by the Polish Ministry of Science and Higher Education (grant Iuventus Plus IP2012 040172 to M.P.), the Polish National Science Centre (grant 2014/14/M/ST5/00619 to M.D.), and the US National Institutes of Health (grant R01GM099040 to G.S.S.). This project has received founding from the European Union`s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 661187. The Drag laboratory is supported by the Foundation for Polish Science.

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Affiliations

  1. Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA.

    • Marcin Poreba
    •  & Guy S Salvesen
  2. Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland.

    • Marcin Poreba
    •  & Marcin Drag

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Contributions

M.P., G.S.S. and M.D. developed the protocol, designed the research, interpreted the data and wrote the protocol. M.P. carried out the experiments in the protocol.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Marcin Poreba or Marcin Drag.

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https://doi.org/10.1038/nprot.2017.091

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