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Monitoring regulated protein-protein interactions using split TEV

Nature Methods volume 3, pages 985993 (2006) | Download Citation



Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs). Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with 'proteolysis-only' reporters, which can be previously silent fluorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield 'transcription-coupled' reporter systems. Thus, split TEV combines the advantages of split enzyme– and reporter gene–mediated assays, and provides full flexibility with regard to the final readout. In a first biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.

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We acknowledge the excellent technical assistance from F. Herzog with cell cultures, and H. Böhli and J. Ohsam for cloning expression constructs. The GST-Nrg-1β fusion constructs were a kind gift of C. Lai (The Scripps Institute). Her2 and mouse ErbB4 templates were kindly provided by A. Ullrich (Max Planck Institute for Biochemistry) and C. Lai.

Author information

Author notes

    • Michael C Wehr
    •  & Rico Laage

    These authors contributed equally to this work.


  1. Max Planck Institute of Experimental Medicine, Hermann Rein Str. 3, D-37075 Göttingen, Germany.

    • Michael C Wehr
    • , Tobias M Fischer
    • , Klaus-Armin Nave
    •  & Moritz J Rossner
  2. Axaron Bioscience AG, INF515, D-69120 Heidelberg, Germany.

    • Rico Laage
    • , Ulrike Bolz
    • , Sylvia Grünewald
    • , Sigrid Scheek
    • , Alfred Bach
    •  & Moritz J Rossner


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M.C.W. cloned and western blot–verified TEV fragment constructs and reporters and conducted the final experiments except the TEV fragment screen; R.L. cloned TEV fragment constructs and the TM-GV reporter, and perfomed the TEV fragment screen and proof-of-principle experiments monitoring interactions at the membrane including rapamycin regulation; U.B. was involved in cloning of reporters and interaction constructs; T.M.F. cloned the Nrg1 constructs and performed the Nrg1 western blot; S.G. and S.S. contributed to initial data monitoring GPCR interactions; A.B. and K.-A.N. supported the project and contributed conceptually; M.J.R. developed the concept, cloned initial reporter constructs and supervised the project. M.C.W. and M.J.R. wrote the manuscript.

Competing interests

R.L., U.B. and A.B. are employees of Axaron Bioscience AG, Germany, which holds the patent for the described technology. S.G., S.S. and M.J.R. were temporarily employees of Axaron Bioscience AG during the initial phase of the project.

Corresponding authors

Correspondence to Rico Laage or Moritz J Rossner.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Summary of principles and properties of all membrane reporters.

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    Supplementary Fig. 2

    Comparsion of GV-ER and GV-2ER activation by full-length TEV.

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    Supplementary Fig. 3

    Western blotting of model membrane Split-TEV fusion constructs.

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    Supplementary Fig. 4

    Analysis of TM-Luc reporter activation by transmembrane and cytosolic GCN4/GBR1a/GBR2 cc domain fragment pairs.

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    Supplementary Fig. 5

    Time-dependent analysis of the rapamycin-induced FKBP-FRB interactions monitored with LucER.

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    Supplementary Fig. 6

    Specificity of PPIs in the cytosol measured with LucER in CHO cells.

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    Supplementary Fig. 7

    Comparsion of TM-GV with GV-2ER and GV-ER activated by transmembrane and cytosolic GCN4cc-TEV fragment pairs.

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    Supplementary Fig. 8

    Western blot of Split-TEV interaction pair dependent cleavage of GV-2ER.

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    Supplementary Fig. 9

    Comparing the kinetics of the rapamycin-induced FKBP-FRB interactions monitored with the GV-2ER and LucER.

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    Supplementary Note 1

    TEV protease fragments suited for transcomplementation.

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    Supplementary Note 2

    A recombinase reporter system for permanent reporter activation.

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    Supplementary Note 3

    Fluorescent 'Proteolysis-only' TEV-Reporters.

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    Supplementary Methods

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