Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Genetic code expansion for multiprotein complex engineering

Nature Methods volume 13, pages 997–1000 (2016)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: An overview of the MultiBacTAG system for the expression of multidomain protein complexes in insect cells with different ncAAs for diverse applications.
Figure 2: Characterization of MultiBacTAG, click labeling of Herceptin, and detection of human cancer.
Figure 3: Crosslinking of TAF11–TAF13–TBP complex.

Similar content being viewed by others

References

  1. Bieniossek, C., Imasaki, T., Takagi, Y. & Berger, I. Trends Biochem. Sci. 37, 49–57 (2012).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Lemke, E.A. ChemBioChem 15, 1691–1694 (2014).

    Article  CAS  Google Scholar 

  5. Liu, C.C. & Schultz, P.G. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  Google Scholar 

  6. Chin, J.W. Annu. Rev. Biochem. 83, 379–408 (2014).

    Article  CAS  Google Scholar 

  7. Chatterjee, A., Xiao, H., Bollong, M., Ai, H.W. & Schultz, P.G. Proc. Natl. Acad. Sci. USA 110, 11803–11808 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Bianco, A., Townsley, F.M., Greiss, S., Lang, K. & Chin, J.W. Nat. Chem. Biol. 8, 748–750 (2012).

    Article  CAS  Google Scholar 

  11. Mukai, T., Wakiyama, M., Sakamoto, K. & Yokoyama, S. Protein Sci. 19, 440–448 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hernandez, G. Jr., Valafar, F. & Stumph, W.E. Nucleic Acids Res. 35, 21–34 (2007).

    Article  CAS  Google Scholar 

  13. Hancock, S.M., Uprety, R., Deiters, A. & Chin, J.W. J. Am. Chem. Soc. 132, 14819–14824 (2010).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E.A. Angew. Chem. Int. Ed. Engl. 50, 3878–3881 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Kraemer, S.M., Ranallo, R.T., Ogg, R.C. & Stargell, L.A. Mol. Cell. Biol. 21, 1737–1746 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P.J. & Seidel, C.A.M. Methods Enzymol. 475, 455–514 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Simpson, J.T. & Durbin, R. Genome Res. 22, 549–556 (2012).

    Article  CAS  Google Scholar 

  23. Luo, R. et al. Gigascience 1, 18 (2012).

    Article  Google Scholar 

  24. Magoč, T. & Salzberg, S.L. Bioinformatics 27, 2957–2963 (2011).

    Article  Google Scholar 

  25. Berger, I., Fitzgerald, D.J. & Richmond, T.J. Nat. Biotechnol. 22, 1583–1587 (2004).

    Article  CAS  Google Scholar 

  26. Nie, Y., Bieniossek, C. & Berger, I. ACEMBL Expression System User Manual version 09.11 (EMBL, 2009).

  27. Milles, S. & Lemke, E.A. Biophys. J. 101, 1710–1719 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Rappsilber, J., Ishihama, Y. & Mann, M. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  Google Scholar 

  31. Cox, J. & Mann, M. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  32. Tomko, R.J. Jr. et al. Cell 163, 432–444 (2015).

    Article  CAS  Google Scholar 

  33. Chen, Z.A., Fischer, L., Cox, J. & Rappsilber, J. Mol. Cell. Proteomics 15, 2769–2778 (2016).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  35. Tyagi, S. & Lemke, E.A. Methods Cell Biol. 113, 169–187 (2013).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  37. Wolff, A.C. et al. J. Clin. Oncol. 31, 3997–4013 (2013).

    Article  Google Scholar 

  38. Polley, M.Y. et al. J. Natl. Cancer Inst. 105, 1897–1906 (2013).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

Authors and 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. Christine Koehler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul F Sauter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mirella Wawryszyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gemma Estrada Girona
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kapil Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jonathan J M Landry
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Markus Hsi-Yang Fritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ksenija Radic
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jan-Erik Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zhuo A Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Juan Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Piau Siong Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Bence Galik
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Sini Junttila
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Peggy Stolt-Bergner
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Giancarlo Pruneri
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Attila Gyenesei
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Carsten Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Moritz Bosse Biskup
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Hueseyin Besir
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Vladimir Benes
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Juri Rappsilber
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Martin Jechlinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Jan O Korbel
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Imre Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Stefan Braese
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Edward A Lemke
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Edward A Lemke.

Ethics declarations

Competing interests

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

Integrated supplementary information

Supplementary Figure 1 Overview of different experimental approaches

This overview shows the three different experimental approaches to test for and achieve Amber suppression in Sf21 cells. The left panel describes the conventional MultiBac system, the middle panel the newly established GCE MultiBacTAG system and the right panel the transient transfection approach. The conventional as well as the GCE MultiBacTAG system follow the same procedures from the point of Bacmid preparation onwards, which is followed by Virus production and Sf21 cell transfection. Protein containing Amber TAG can be expressed if ncAA is added to the medium. The significant difference of both systems is the way of incorporation of the synthetase/tRNA pair. Both systems use donor plasmids (pIDC, pIDK, pIDS) and a acceptor plasmid (pACEBac1,2), which can be combined via Cre/loxP recombination (indicated with violet spheres) into one plasmid. For the conventional method, synthetase, tRNA and the protein of interest (POI) have to be cloned into the donor and acceptor plasmids. After Cre/loxP recombination the resulting plasmid can be integrated into the Tn7 site of the Bacmid. For the GCE MultiBacTAG system, the synthetase/tRNA pair is cloned into one plasmid and incorporated into the backbone of the Bacmid, while several POIs can be combined and integrated into the Tn7 site of the Bacmid. Importantly, and in contrast to the conventional method, the Tn7 attachment site remains fully accessible for integrating multigene expression constructs. In the lower middle panel this procedure is shown in more detail. Several POIs can be cloned into the donor plasmids and in the acceptor plasmids. The donor plasmids carry a different origin of replication (ori, grey boxes) than the acceptor plasmid (ori, black box) to simplify the recombination procedure. The pACEBac1,2 plasmid carries in addition the Tn7 integration sites (Tn7L, Tn7 integration left site and Tn7R, Tn7 integration right site) After recombination via the Cre/loxP sites (magenta spheres), the plasmid can be integrated via the Tn7 site. The synthetase and tRNA are cloned in a separate plasmid (pUCDM), which has its own Cre/loxP site (green sphere). This Cre/loxP recombines with the Cre/lox-P site in the Bacmid backbone. The insertion of the tRNA production module in the Cre/loxP site of the virus ensures that the number of heterologous tRNA genes produced in our system remains constant, irrespective of the nature of the target protein assembly co-expressed, which can be decisive for reproducibility and Amber suppression 1,2. The right panel shows the workflow for transient transfection of Sf21 cells. Plasmids which contain the synthetase and a POI (pIZT-PylRS-GFP(TAG)) and the tRNA expression cassette (pU6-tRNA) can be co-transfected. After adding ncAA to the cells, expression can be followed up by e.g. flow cytometry analysis.

1. Bianco, A., Townsley, F.M., Greiss, S., Lang, K. & Chin, J.W. Expanding the genetic code of Drosophila melanogaster. Nature chemical biology 8, 748-750 (2012).

2. Young, T.S., Ahmad, I., Yin, J.A. & Schultz, P.G. An enhanced system for unnatural amino acid mutagenesis in E. coli. Journal of molecular biology 395, 361-374 (2010).

Supplementary Figure 2 Comparison of different snRNAU6 genes

To illustrate the high degree of gene conservation across species (for a review on PolIII transcription, see ref. 3), the snRNA U6 genes from various organisms (Homo sapiens, Drosophila melanogaster, Spodoptera frugiperda and Bombyx mori) are shown. The snRNA genes from Homo sapiens and Drosophila were extracted from NCBI Reference Sequence Database (RefSeq), as well as snRNA U6 isoform E from Bombyx mori. The Spodoptera frugiperda snRNAU6 gene was taken out of our assembly and the first Bombyx mori snRNA sequence out of the the silkworm DB V2.0. All the genes are highly similar to each other, only few nucleotides appear different (highlighted in red). Also the sequences are equal in length, just the snRNA U6 Bombyx mori isoform E misses 15 nucleotides at the 5’ end and 13 nucleotides plus the polyT-tail at the 3’ end.

3. Schramm, L. & Hernandez, N. Recruitment of RNA polymerase III to its target promoters. Genes & development 16, 2593-2620 (2002).

Supplementary Figure 3 Different tRNA constructs tested by transient transfection

(a) In orange the U6 promoter sequences of different organism (human, Drosophila and Bombyx) are illustrated, followed by the tRNAPyl gene from Methanosarcina mazei. The corresponding 3’termination signal of each snRNA U6 gene is displayed in blue. (b) Flow cytometry results of Sf21 cells transfected with different U6-tRNA constructs and the reporter construct pIZT-PylRSWT-mCherry-GFP(Y39TAG). Each analysis is represented by three plots. The upper right plot, FSC-A (forward-scattered light area) versus SSC-A (side-scattered light area) shows scattering data and the selected gate, which contains live cells, is marked with a black eclipse. In the lower right plot, FCS-A versus SSC-W (side-scattered light width) single cells are selected with a black eclipse. The final data is shown in the left (large) diagram, which is divided into four gates. The upper left gate shows cells only expressing mCherry, the lower left one shows cells which are neither expressing mCherry nor GFP. The lower right gate contains the cell counts for GFP expressing cells only. The upper right gate contains cells which are expressing mCherry as well as GFP. The left panel shows the expression results with PrK and the right one without ncAA. Top panel: Human U6-tRNAPyl-3term. Middle panel: U6 promoter from Drosophila melanogaster (Dm). Lower panel, U6 promoter from Bombyx mori (Bm) was used. Shown data is representative of at least three independent flow cytometry experiments.

Supplementary Figure 4 Alignment of snRNAU6 regions

Shown is the sequence alignment of U6 promoter regions (orange color) with corresponding snRNA U6 genes (highlighted in green) and 3’termination signals (in blue) of Spodoptera frugiperda, aligned to U6 promoter, snRNA U6 and 3’termination of Bombyx mori. The intensity of the color represents the degree of sequence similarity (more transparency meaning lower similarity). The alignment, which was done using ClustalW 4,5, shows that the snRNA U6 gene is well conserved. The promoter regions look similar, especially at the region close to the snRNA gene, same is true for the 3’ termination signal. Note that U6-1 and U6-3 sequences show lower similarity.

4. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007).

5. Goujon, M. et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic acids research 38, W695-699 (2010).

Supplementary Figure 5 Dicistronic tRNA cassette

The dicistronic tRNA cassette was transiently transfected into Sf21 cells and analyzed with flow cytometry. (a) Schematic overview of the yeast dicistronic tRNA (tRNAArg-tRNAAsp). The 5’leader sequence is followed by tRNAArg, which contains eukaryotic PolII recognition signals (Box A and B), a 10 bp spacer sequence, tRNAAsp and a 3’termination signal. (b) ClustalW sequence alignment4,5 of yeast dicistronic tRNA6 and Sf21 dicistronic tRNA construct, found in the Sf21 genome. The tRNAArg is shown in red. In yellow, Box A and Box B are highlighted. The spacer sequence is represented in turquoise, tRNAAsp in green. (c) Flow cytometry analysis of Sf21 cells transiently transfected with pIZT-PylRSWT-mCherry-GFP(Y39TAG) and pMT-dicistronic tRNA-cassette. Shown are the flow cytometry plots with and without the tRNA expression cassette. Each analysis is represented by three plots. The upper right plot, FSC-A (forward-scattered light area) versus SSC-A (side-scattered light area) shows scattering data and the selected gate, which contains live cells, is marked with a black eclipse. In the lower right plot, FCS-A versus SSC-W (side-scattered light width) single cells are selected with a black eclipse. The left (large) plot is divided into four gates. The upper left gate shows the data point for cells, which only express mCherry, the lower left one shows cells that are not expressing mCherry nor GFP. The lower right gate contains the cell counts for GFP expressing cells only. In the upper right gate, cells are presented that are expressing mCherry, as well as GFP. There is no difference in the mCherry-GFP signal, independent if the tRNA expression cassette is used or not. Shown experiments are representative of at least three independent flow cytometry measurements.

4. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007).

5. Goujon, M. et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic acids research 38, W695-699 (2010).

6. Schmidt, O. et al. Dimeric tRNA precursors in yeast. Nature 287, 750-752 (1980).

Supplementary Figure 6 Transient transfections of Sf21 using eight different Sf21 U6 promoters for tRNA expression

Flow cytometry analysis of Sf21 cells transiently transfected with the reporter construct pIZT-PylRSWT-mCherry-GFP(Y39TAG) and one of the eight U6(Sf21)-tRNA constructs. These flow cytometry analysis show that the Amber Stop codon can be efficiently suppressed for six out of the eight found U6 Sf21 promoters. Each analysis is represented by three plots. The upper right plot, FSC-A (forward-scattered light area) versus SSC-A (side-scattered light area) shows scattering data and the selected gate, which contains live cells, is marked with a black eclipse. In the lower right plot, FCS-A versus SSC-W (side-scattered light width) single cells are selected with a black eclipse. In the lower right plot singe cells are selected with a black eclipse. The final data is shown in the left (large) diagram, which is divided into four gates. The upper left gate shows the data point for cells, which only express mCherry (mCherry only), the lower left one shows cells, which are not expressing mCherry nor GFP (double negatives). The lower right gate contains the cell counts for GFP expressing cells only (GFP only). In the upper right gate, cells are presented, which are expressing mCherry, as well as GFP, the so called double positives. There are always two panels for each promoter on top of each other. The upper panel shows the expression results with ncAA and the lower one without. Shown experiments are representative of at least three independent flow cytometry measurements.

Supplementary Figure 7 Mass spectrometry analysis of GFP39→PrK and GFP39→SCO

GFP39→TAG was expressed in Sf21 cells once in presence of PrK (a-c) and once with SCO (d-f). The cells were transfected with Bacmid-DNA prepared out of DH10MultiBacTAG cells, harboring the M. mazei PylRS WT for the expression with PrK or MM PylRS AF for SCO and the tRNAPyl expression cassette. (a) and (b) present the result of the peptide digest using trypsin and the corresponding peptide sequence, showing the incorporation of PrK at position 39 of GFP. In panel a matched peptides are highlighted in red. Panel b shows the scores of the peptide, which contains PrK instead of Tyrosine. (c) native mass result measure: 28801 g/mol, expected: 28801 g/mol. (d) and (e) present the result of the peptide digest using trypsin and the corresponding peptide sequence, showing the incorporation of SCO at position Y39 of GFP. In panel d matched peptides are highlighted in red. Panel e shows the score of the peptide, which contains SCO instead of tyrosine. (f) native mass result: measured: 28870 g/mol, expected: 28870 g/mol.

Supplementary Figure 8 Analysis of GFP39→TAG and Herceptin expressions including labeling reactions

(a) Shown are the full-size SDS-PAGE corresponding to the cropped gels of Figure 2. On the left gel GFP39→TAG expressed with and without PrK was loaded on the gel (not shown in main text figure). On the right one, GFP39→TAG was expressed with and without SCO. All GFP expressions were done in the MultiBacTAG system using the PylRS WT or PylRS AF, respectively. (b) Flow cytometry plot of mCherry-GFP39→PrK expression in the MultiBacTAGWT system with and without ncAA, here PrK was used. Both flow cytometry analyses contain three plots. The top right one, shows the scattering data for the forward scattering-area (FSC-A) versus side-scattering-area (SSC-A) gating for live cells (black eclipse). In the lower plot (FSC-A versus side-scattering-width (SSC-W)) the gate (black eclipse) selects only the single cells for further analysis. The left plot shows the signal for cells expressing GFP versus mCherry. This plot is divided into four gates, the top left one being mCherry only, meaning this gate represents cells, which only express mCherry. The lower left one counts the amount of cells, which do not express mCherry or GFP. In the lower right ones are cells, which only express GFP and in the top right one, the population is shown, which can incorporate PrK into GFP and therefore can express mCherry-GFP as a full length protein. The top plot shows the expression with PrK, resulting in 90% of single cells expressing mCherry-GFP, the lower one without PrK, where only 0.52% of the single cells can express mCherry-GFP. Shown experiments are representative of at least three independent measurements. In Panel c-f full-size SDS-PAGEs are shown corresponding to the cropped gels of Figure 2. The SDSPAGEs are stained with Coomassie Blue after an UV scan at the respective wave length. Panel c corresponds to the cropped gels of Figure 2, which show the expression of Herceptin with and without PrK using the MultiBacTAGWT system and with and without BOC with the MultiBac-TAGAF system (not shown in main text figure).The complete gel after the labeling reaction of Herceptin132→PrK and fluorescein-azide is shown in (d). Copper based click reaction can occur between Herceptin132→PrK and an azide, but not between Herceptin WT and an azide dye derivative. The first two lanes show a higher amount of both Herceptin132→PrK and WT compared to the following two lanes. The click reaction of Herceptin121→SCO with TAMRA-tetrazine is shown in panel e. The UV scan indicates that only Herceptin121→SCO can be labeled and not Herceptin WT. (f) SPDAC reaction between Herceptin121→TCO* and TAMRA-tetrazine. Shown experiments are representative of at least three independent expression experiments.

Supplementary Figure 9 Mass spectrometry analysis of Herceptin121→PrK and Herceptin132→PrK

Different mutants of Herceptin have been expressed in Sf21 cells, using the MultiBacTAGWT system and analyzed by mass spectrometry after purification. Panel (a) gives an overview of the results for all native mass analysis of Herceptin. Panel (b-e) present the mass spectrometry analysis of Herceptin121→PrK (panel b and c), as well as for Herceptin132→PrK (panel d and e). Both mutants were digested with trypsin, resulting in the peptide coverage map (upper half of panel b and panel d) for both, the heavy and light chain of the Herceptin mutants (matched peptides in bold red), as well as the peptide scores, which show the incorporation of PrK in the corresponding peptide of the heavy chain (lower part of panel b and d). (c) native mass result for Herceptin121→PrK: heavy chain (expected mass: 25834 g/mol, measure mass: 25836 g/mol)), light chain (expected mass: 23615 g/mol, measured mass: 23617 g/mol). (e) native mass result of Herceptin132→PrK: heavy chain (expected mass: 25834 g/mol, measured mass: 25836 g/mol), light chain (expected mass: 23615 g/mol, measured mass: 23617 g/mol).

Supplementary Figure 10 Mass spectrometry analysis of different Herceptin121→TAG mutants

Panel (a-d): native mass analysis of different Herceptin mutants, which have been expressed in Sf21 cells, transfected with Bacmid-DNA prepared out of DH10MultiBacTAG cells, harboring the MM PylRS AF and the tRNAPyl expression cassette. (a) shows the native mass of the Herceptin121→SCO heavy chain on the left side (expected: 25902 g/mol, measured: 25905 g/mol) and on the right side the native mass result of the light chain (expected mass: 23615 g/mol, measured mass: 23618 g/mol). Panel (b) shows the result of the native mass analysis of Herceptin121→SCO labeled with TAMRA-tetrazine. The analysis of the native mass for the heavy chain labeled with TAMRA-tetrazine is presented on the left side (expected mass: 26473 g/mol, measured mass: 26476 g/mol) and the light chain results are on the right side (expected mass: 23615 g/mol, measured mass: 23619 g/mol). Panel (c) represents the result of the native mass analysis for Herceptin121→TCO* labeled with TAMRA-tetrazine. On the left side is the result for the heavy chain (expected mass: 26476 g/mol, measured mass: 26480 g/mol) and on the right side the one for the light chain (expected mass: 23615 g/mol, measured mass: 23619 g/mol). Panel (d) is the result of native mass analysis of Herceptin121→BCN after labeling with Glycan-azide (PSZ170). The left side of panel (d) shows the results for the heavy chain (expected mass: 26539, measured mass: 26543), and the left side for the light chain (expected mass: 23615, measured mass: 23619).

Supplementary Figure 11 TAMRA labeled Herceptin121→TCO* performance to detect cancer in human tumor sections

Human tumour sections included Her2+ (a and c) and Her2- (b and d) samples. Images shown are maximum projections of 26 planes spanning 4 micrometers in total. Blue channel: DAPI, red channel: Herceptin121→TCO* labeled with TAMRA-tetrazine. An additional experiment is shown in main text Figure 2, so that a total of 3 Her2+ and 3 Her2- samples were analysed in this work.

Supplementary Figure 12 Analysis of TAF11/TAF1320→SCO labeled with Alexa488-tetrazine and Alexa594 maleimide

The WT TAF13/TAF11 complex has two native cysteins, one at position C37 TAF13 and one at position C197 TAF11. We first tested, if one cysteine can be labeled selectively. To this end, 2.5 nmol TAF11/TAF13 were incubated with a nearly stoichiometric amount of Alexa594 maleimide overnight at 4 °C. Gratifyingly, as shown by gel analysis in panel (a), a major fluorescent product is only visible at the MW of TAF13, indicating that C37 of TAF13 gets labeled selectively under those conditions. Lane 1-3 shows different loading amount of the labeling reaction. Next, MultiBacTAGAF was used to generate TAF11/TAF1320→SCO. The sample was labeled site specifically with Alexa488-tetrazine and Alexa594 maleimide. After labeling of TAF11/ TAF1320→SCO with the two dyes, the sample was loaded on a SEC column and the elution fractions were analyzed by SDS-PAGE. In panel (b) two fluorescent scans of the same SDS-PAGE are illustrated. The upper gel scan was done at an excitation of 488 nm and the lower one of 555 nm, illustrating, that TAF1320→SCO is labeled with both dye molecules, Alexa488-tetrazine and Alexa594 maleimide, respectively. Fractions 15-17 were pooled, concentrated and used for the smFRET measurement as described in the online methods.

Supplementary Figure 13 Cross-linking experiments of TAF11/TAF13/TBP complex

Shown are full size (compare to Fig. 3c) Coomassie-stained SDS-PAGE gel (top) and the corresponding anti-TAF13 and anti-TBP WBs of the cross-linking experiment of TAF11/TAF13 complex (1:1 ratio) with increasing concentration of TBP (1:1:0.625 and 1:1:1.25). In lane 3 and 4 the lower amount of TBP is indicated by a plus inside a circle. With TBP a specific double-band appears as a cross-link product which does contain TAF13 but not TBP as evident from the WBs. In (b) also an experiment using a FLAG-TBP experiment was shown (only UV exposed samples shown), and detected in addition with an anti-FLAG Ab. Consistent with the anti-TBP (a) experiment, also this WB does not detect an TBP in the cross-linked product. All shown experiments are representative of at least three independent experiments.

Supplementary Figure 14 Quantified cross-links between TAF11 and TAF1334→DiAzKs

(a) Relative abundance of cross-links from TAF1334→DiAzKs to five TAF11 regions, in sample A and B (TAF11+ TAF1334→DiAzKs +TBP) versus their respective reference (TBP absent). Sample A and B are biological replica, A0 (TAF11+ TAF1334→DiAzKs) is a biological replica of the reference. Center values are the median, error bars base on standard deviations of multiple cross-linked peptides and “n” indicates the number of quantified cross-linked peptides. (b) The relative abundance of quantified cross-linked peptide pairs between TAF1334→DiAzKs and TAF11 residues in sample A and B versus their respective reference (TBP absent). Sample A0 is a biological replica of the reference. As can be seen, the region 146-149 of TAF11 cross-links much less efficiently when TBP is incubated with the TAF11-TAF1334→DiAzKs complex prior to UV exposure, indicative of a conformational change in the TAFs depending on TBP interaction.

Supplementary Figure 15 Size exclusion binding analysis of TAF11/13 complex and TBP

Size exclusion chromatograms (SEC) on Superdex S200 10/300 column and SDS-PAGE analyses indicating the formation of TAF11/TAF13/TBP complex. Separate SEC runs are shown in different colors. TAF11/TAF13 complex is illustrated in red, TBP alone in dark blue, and the trimeric complex (TAF11/TAF13/TBP) in black. The reloading of the trimeric complex is represented by a dashed green line. All runs were followed by SDS-PAGE analyses. The colored arrows indicate the affiliation to the SEC run. Shown experiments are representative of at least three independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Tables 1–3 and Supplementary Notes 1–4. (PDF 28447 kb)

Supplementary Table 4

List of quantified cross-linked peptides between TAF11 and TAF1334→DiAzKs. (XLSX 13 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Koehler, C., Sauter, P., Wawryszyn, M. et al. Genetic code expansion for multiprotein complex engineering. Nat Methods 13, 997–1000 (2016). https://doi.org/10.1038/nmeth.4032

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.4032

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing