Brief Communication | Published:

Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

Nature Biotechnology volume 31, pages 331334 (2013) | Download Citation

Abstract

Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. Biotechniques 36, 1018–1022, 1024, 1026–1029 (2004).

  2. 2.

    , & Annu. Rev. Biochem. 72, 783–812 (2003).

  3. 3.

    et al. Mol. Cell Proteomics 11, O111.016253 (2012).

  4. 4.

    et al. Genomics 89, 307–315 (2007).

  5. 5.

    , , & Ribosome display: in vitro selection of protein-protein interactions in Cell Biology—A Laboratory Handbook (ed. Celis, J.) Vol. 1, 3rd Ed., 497–509 (Elsevier Academic Press, 2006).

  6. 6.

    & Oncogene 23, 7918–7927 (2004).

  7. 7.

    et al. Mol. Cell Biol. 14, 4509–4521 (1994).

  8. 8.

    , & Blood 113, 3845–3856 (2009).

  9. 9.

    et al. Nat. Biotechnol. 29, 535–541 (2011).

  10. 10.

    et al. PLoS ONE 4, e4894 (2009).

  11. 11.

    et al. Cancer Res. 65, 379–382 (2005).

Download references

Acknowledgements

We would like to thank K. Waraska, M. Cicero, S. Alian and A. Gagne for assistance with Illumina sequencing, and J. Laserson for statistical advice. Thanks to D. Zhu for help with synthesis of biotin-dasatinib, which was partially supported by US National Institutes of Health U54 CA156734 to the University of Massachusetts Boston–Dana-Farber Harvard Cancer Center U54 Comprehensive Partnership (Project 3, Co-PIs: N.S. Gray, W. Zhang, and P.L. Yang). We also thank N. Gray at Harvard Medical School for valuable advice, and H. Varmus at the National Cancer Institute for providing gefitinib reagents and advice. This work was supported in part by NIH grant 3P30CA023100-25S8 to S.K. S.J.E. is an investigator with the Howard Hughes Medical Institute.

Author information

Author notes

    • Jian Zhu
    •  & H Benjamin Larman

    These authors contributed equally to this work.

Affiliations

  1. Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • Jian Zhu
    • , H Benjamin Larman
    • , Geng Gao
    • , Alberto Ciccia
    • , Natalya Pavlova
    •  & Stephen J Elledge
  2. Department of Genetics, Harvard University Medical School, Boston, Massachusetts, USA.

    • Jian Zhu
    • , H Benjamin Larman
    • , Uri Laserson
    • , Alberto Ciccia
    • , Natalya Pavlova
    • , George Church
    •  & Stephen J Elledge
  3. Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • Jian Zhu
    • , H Benjamin Larman
    • , Geng Gao
    • , Alberto Ciccia
    • , Natalya Pavlova
    •  & Stephen J Elledge
  4. Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA.

    • H Benjamin Larman
    • , Uri Laserson
    •  & George Church
  5. Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • H Benjamin Larman
  6. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Romel Somwar
  7. Department of Chemistry, University of Massachusetts Boston, Boston, Massachusetts, USA.

    • Zijuan Zhang
    •  & Wei Zhang
  8. Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Uri Laserson
  9. Translational Neuro-Oncology Laboratories, Department of Neurosciences, Division of Neuro-Oncology, University of California, San Diego, Moores Cancer Center, La Jolla, California, USA.

    • Santosh Kesari

Authors

  1. Search for Jian Zhu in:

  2. Search for H Benjamin Larman in:

  3. Search for Geng Gao in:

  4. Search for Romel Somwar in:

  5. Search for Zijuan Zhang in:

  6. Search for Uri Laserson in:

  7. Search for Alberto Ciccia in:

  8. Search for Natalya Pavlova in:

  9. Search for George Church in:

  10. Search for Wei Zhang in:

  11. Search for Santosh Kesari in:

  12. Search for Stephen J Elledge in:

Contributions

S.J.E. and H.B.L. conceived and supervised the project. pRD human ORFeome library was constructed by J.Z., and characterized by J.Z. and H.B.L. The PLATO protocol was developed by H.B.L. and J.Z. Clinical evaluations and patient sample acquisitions were performed by S.K. Statistical analysis was performed by U.L. under the supervision of G.C. R.S. provided gefitinib-conjugated beads. PLATO candidates were confirmed by J.Z. and G.G. A.C. provided support for the validation of LYN binding candidates. N.P. provided support for the validation of PND autoantigen candidates. Z.Z. and W.Z. provided biotin-dasatinib. The manuscript was prepared by H.B.L. and J.Z., and edited by S.J.E.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stephen J Elledge.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figs. 1–6 and Supplementary Methods

Excel files

  1. 1.

    Supplementary Tables 1-4

    ORFeome enrichment by GST-LYN, ORFeome enrichment by PND patient autoantibodies, ORFeome enrichment by dasatinib, Sequences of DNA primers used in this study

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.2539

Further reading