Abstract

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure–function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.

  • Subscribe to Nature Methods for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    , , & Trends Biochem. Sci. 37, 49–57 (2012).

  2. 2.

    et al. Curr. Opin. Struct. Biol. 32, 139–146 (2015).

  3. 3.

    et al. Nat. Methods 3, 1021–1032 (2006).

  4. 4.

    ChemBioChem 15, 1691–1694 (2014).

  5. 5.

    & Annu. Rev. Biochem. 79, 413–444 (2010).

  6. 6.

    Annu. Rev. Biochem. 83, 379–408 (2014).

  7. 7.

    , , , & Proc. Natl. Acad. Sci. USA 110, 11803–11808 (2013).

  8. 8.

    et al. Angew. Chem. Int. Ed. Engl. 48, 4052–4055 (2009).

  9. 9.

    et al. Biochem. Biophys. Res. Commun. 371, 818–822 (2008).

  10. 10.

    , , , & Nat. Chem. Biol. 8, 748–750 (2012).

  11. 11.

    , , & Protein Sci. 19, 440–448 (2010).

  12. 12.

    , & Nucleic Acids Res. 35, 21–34 (2007).

  13. 13.

    , , & J. Am. Chem. Soc. 132, 14819–14824 (2010).

  14. 14.

    et al. Angew. Chem. Int. Ed. Engl. 53, 2245–2249 (2014).

  15. 15.

    et al. Angew. Chem. Int. Ed. Engl. 51, 4166–4170 (2012).

  16. 16.

    , , , & Angew. Chem. Int. Ed. Engl. 50, 3878–3881 (2011).

  17. 17.

    et al. Proc. Natl. Acad. Sci. USA 109, 16101–16106 (2012).

  18. 18.

    , , & Mol. Cell. Biol. 21, 1737–1746 (2001).

  19. 19.

    et al. Mol. Cell. Biol. 25, 945–957 (2005).

  20. 20.

    , , , & Methods Enzymol. 475, 455–514 (2010).

  21. 21.

    et al. Nat. Chem. Biol. 7, 671–677 (2011).

  22. 22.

    & Genome Res. 22, 549–556 (2012).

  23. 23.

    et al. Gigascience 1, 18 (2012).

  24. 24.

    & Bioinformatics 27, 2957–2963 (2011).

  25. 25.

    , & Nat. Biotechnol. 22, 1583–1587 (2004).

  26. 26.

    , & ACEMBL Expression System User Manual version 09.11 (EMBL, 2009).

  27. 27.

    & Biophys. J. 101, 1710–1719 (2011).

  28. 28.

    et al. J. Am. Chem. Soc. 134, 5187–5195 (2012).

  29. 29.

    et al. Mol. Cell. Proteomics 6, 2200–2211 (2007).

  30. 30.

    , & Anal. Chem. 75, 663–670 (2003).

  31. 31.

    & Nat. Biotechnol. 26, 1367–1372 (2008).

  32. 32.

    et al. Cell 163, 432–444 (2015).

  33. 33.

    , , & Mol. Cell. Proteomics 15, 2769–2778 (2016).

  34. 34.

    et al. Mol. Cell. Proteomics 10, M110.003699 (2011).

  35. 35.

    & Methods Cell Biol. 113, 169–187 (2013).

  36. 36.

    et al. Mod. Pathol. 28, 1185–1201 (2015).

  37. 37.

    et al. J. Clin. Oncol. 31, 3997–4013 (2013).

  38. 38.

    et al. J. Natl. Cancer Inst. 105, 1897–1906 (2013).

  39. 39.

    et al. Ann. Oncol. 24, 2206–2223 (2013).

Download references

Acknowledgements

We thank all members of our laboratories for helpful discussions. E.A.L., C.K., P.F.S., M.W., and S.B. acknowledge funding from the BW Stiftung. E.A.L. acknowledges additional support from the Emmy Noether program. E.A.L. and C.S. are grateful for funding by SPP1623 of the Deutsche Forschungsgemeinschaft. I.B. is funded by the European Commission Framework Programme 7 (FP7) ComplexINC project (contract no. 279039). P.S.-B. acknowledges funding from the Laura Bassi Centres of Expertise initiative for the Centre of Optimized Structural Studies, project 253275. M.W. thanks the KSOP for financial support. P.S.T. is supported by the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND. The Wellcome Trust generously funded this work through a Senior Research Fellowship to J.R. (103139), a Centre core grant (092076), and an instrument grant (108504). We also thank the members of the EMBL Genomics Core Facility for sample processing and sequencing, as well as the EMBL FACS facility for technical support.

Author information

Affiliations

  1. European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

    • Christine Koehler
    • , Gemma Estrada Girona
    • , Jonathan J M Landry
    • , Markus Hsi-Yang Fritz
    • , Ksenija Radic
    • , Jan-Erik Hoffmann
    • , Piau Siong Tan
    • , Carsten Schultz
    • , Hueseyin Besir
    • , Vladimir Benes
    • , Martin Jechlinger
    • , Jan O Korbel
    •  & Edward A Lemke
  2. Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Karlsruhe, Germany.

    • Paul F Sauter
    • , Mirella Wawryszyn
    • , Moritz Bosse Biskup
    •  & Stefan Braese
  3. European Molecular Biology Laboratory (EMBL), Grenoble, Grenoble, France.

    • Kapil Gupta
    •  & Imre Berger
  4. Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

    • Zhuo A Chen
    • , Juan Zou
    •  & Juri Rappsilber
  5. Vienna Biocenter Core Facilities (VBCF GmbH), Vienna, Austria.

    • Bence Galik
    • , Sini Junttila
    • , Peggy Stolt-Bergner
    •  & Attila Gyenesei
  6. Division of Pathology and Laboratory Medicine, European Institute of Oncology, Milan, Italy.

    • Giancarlo Pruneri
  7. Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.

    • Juri Rappsilber
  8. The School of Biochemistry, University of Bristol, Bristol, United Kingdom.

    • Imre Berger
  9. Karlsruhe Institute of Technology (KIT), Institut für Toxikologie und Genetik, Eggenstein-Leopoldshafen, Germany.

    • Stefan Braese

Authors

  1. Search for Christine Koehler in:

  2. Search for Paul F Sauter in:

  3. Search for Mirella Wawryszyn in:

  4. Search for Gemma Estrada Girona in:

  5. Search for Kapil Gupta in:

  6. Search for Jonathan J M Landry in:

  7. Search for Markus Hsi-Yang Fritz in:

  8. Search for Ksenija Radic in:

  9. Search for Jan-Erik Hoffmann in:

  10. Search for Zhuo A Chen in:

  11. Search for Juan Zou in:

  12. Search for Piau Siong Tan in:

  13. Search for Bence Galik in:

  14. Search for Sini Junttila in:

  15. Search for Peggy Stolt-Bergner in:

  16. Search for Giancarlo Pruneri in:

  17. Search for Attila Gyenesei in:

  18. Search for Carsten Schultz in:

  19. Search for Moritz Bosse Biskup in:

  20. Search for Hueseyin Besir in:

  21. Search for Vladimir Benes in:

  22. Search for Juri Rappsilber in:

  23. Search for Martin Jechlinger in:

  24. Search for Jan O Korbel in:

  25. Search for Imre Berger in:

  26. Search for Stefan Braese in:

  27. Search for Edward A Lemke in:

Contributions

C.K. planned and performed experiments. P.F.S., M.W., M.B.B., S.B., G.E.G., J.J.M.L., M.H.-Y.F., B.G., S.J., P.S.-B., G.P., A.G., H.B., V.B., J.O.K., K.G., I.B., K.R., M.J., J.-E.H., C.S., Z.A.C., J.Z., J.R., and P.S.T. provided critical instrumental and analytical expertise or reagents. C.K., I.B., and E.A.L. cowrote the manuscript with input from all authors. E.A.L. planned experiments and conceived the project.

Competing interests

A patent application comprising parts of the MultiBacTAG technology here described has been filed.

Corresponding author

Correspondence to Edward A Lemke.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1–3 and Supplementary Notes 1–4.

Excel files

  1. 1.

    Supplementary Table 4

    List of quantified cross-linked peptides between TAF11 and TAF1334→DiAzKs.