Advertisement

Molecular Biotechnology

, Volume 60, Issue 1, pp 31–40 | Cite as

Vectors for Expression of Signal Peptide-Dependent Proteins in Baculovirus/Insect Cell Systems and Their Application to Expression and Purification of the High-Affinity Immunoglobulin Gamma Fc Receptor I in Complex with Its Gamma Chain

  • Le T. M. Le
  • Jens R. Nyengaard
  • Monika M. Golas
  • Bjoern Sander
Original Paper

Abstract

Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER). The vectors code for N-terminal and C-terminal affinity tags including 3 × FLAG and Twin-Strep, which represent tags compatible with efficient translocation to the ER as well as with purification under mild conditions that preserve protein structure and function. As a model, we used our system to express and purify the engineered high-affinity immunoglobulin gamma Fc receptor I (CD64) in complex with its gamma subunit (γ-chain). We demonstrate that CD64 expressed in complex with the γ-chain is functional in immunoglobulin G (IgG) binding. The sedimentation of CD64 in complex with IgG suggests individual CD64/IgG complexes in addition to formation of high-molecular weight complexes. In summary, our vectors can be used as a tool for expression of membrane proteins, other secretory pathway proteins and their protein complexes.

Keywords

Recombinant protein expression Signal peptide CD64 FLAG tag Strep tag Baculovirus 

Abbreviations

CD64

Cluster of differentiation 64; high-affinity immunoglobulin gamma Fc receptor I

C-terminal

Carboxy-terminal

E. coli

Escherichia coli

ER

Endoplasmic reticulum

FBS

Fetal bovine serum

γ-chain

High-affinity immunoglobulin E receptor γ subunit

IgG

Immunoglobulin G

IPTG

Isopropyl β-d-1-thiogalactopyranoside

kDa

Kilo Dalton

MCS

Multiple cloning site

N-terminal

Amino-terminal

P/S

Penicillin/streptomycin

RNCs

Ribosomal nascent chain complexes

SDS–PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Sf21

Spodoptera frugiperda 21

Sf9

Spodoptera frugiperda 9

SRP

Signal recognition particle

X-gal

5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside

Notes

Acknowledgements

We thank Golshah Ayoubi and Susanne N. Stubbe for expert technical assistance, and Srdja Drakulic for initial help with experiments. The work has been supported by a grant from the Danish Council for Independent Research for Natural Sciences, a grant from the A.P. Møller Foundation for the Advancement of Medical Science and a grant from the Fabrikant Einar Willumsen Mindelegat to B.S. The Centre for Stochastic Geometry and Advanced Bioimaging is supported by the Villum Foundation. M.M.G. acknowledges support from the Lundbeck Foundation and the Carlsberg Foundation.

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interests related to this work.

Ethical approval

The study does not use human material, patient data or animals.

Supplementary material

12033_2017_41_MOESM1_ESM.docx (102 kb)
Supplementary material 1 (DOCX 103 kb)

References

  1. 1.
    Keenan, R. J., Freymann, D. M., Walter, P., & Stroud, R. M. (1998). Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell, 94(2), 181–191.CrossRefGoogle Scholar
  2. 2.
    Walter, P., & Blobel, G. (1982). Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature, 299(5885), 691–698.CrossRefGoogle Scholar
  3. 3.
    Blobel, G., & Dobberstein, B. (1975). Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. Journal of Cell Biology, 67(3), 835–851.CrossRefGoogle Scholar
  4. 4.
    Bohni, P. C., Deshaies, R. J., & Schekman, R. W. (1988). SEC11 is required for signal peptide processing and yeast cell growth. Journal of Cell Biology, 106(4), 1035–1042.CrossRefGoogle Scholar
  5. 5.
    Evans, E. A., Gilmore, R., & Blobel, G. U. (1986). Purification of microsomal signal peptidase as a complex. Proceedings of the National Academy of Sciences U S A, 83(3), 581–585.CrossRefGoogle Scholar
  6. 6.
    von Heijne, G. (1990). The signal peptide. Journal of Membrane Biology, 115(3), 195–201.CrossRefGoogle Scholar
  7. 7.
    von Heijne, G., & Abrahmsen, L. (1989). Species-specific variation in signal peptide design. Implications for protein secretion in foreign hosts. FEBS Letters, 244(2), 439–446.CrossRefGoogle Scholar
  8. 8.
    Wiedmann, M., Huth, A., & Rapoport, T. A. (1984). Xenopus oocytes can secrete bacterial beta-lactamase. Nature, 309(5969), 637–639.CrossRefGoogle Scholar
  9. 9.
    Martoglio, B. (2003). Intramembrane proteolysis and post-targeting functions of signal peptides. Biochemical Society Transactions, 31(Pt 6), 1243–1247.CrossRefGoogle Scholar
  10. 10.
    Froeschke, M., Basler, M., Groettrup, M., & Dobberstein, B. (2003). Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C. Journal of Biological Chemistry, 278(43), 41914–41920.CrossRefGoogle Scholar
  11. 11.
    Kurys, G., Tagaya, Y., Bamford, R., Hanover, J. A., & Waldmann, T. A. (2000). The long signal peptide isoform and its alternative processing direct the intracellular trafficking of interleukin-15. Journal of Biological Chemistry, 275(39), 30653–30659.CrossRefGoogle Scholar
  12. 12.
    Dultz, E., Hildenbeutel, M., Martoglio, B., Hochman, J., Dobberstein, B., & Kapp, K. (2008). The signal peptide of the mouse mammary tumor virus Rem protein is released from the endoplasmic reticulum membrane and accumulates in nucleoli. Journal of Biological Chemistry, 283(15), 9966–9976.CrossRefGoogle Scholar
  13. 13.
    Hiss, J. A., Resch, E., Schreiner, A., Meissner, M., Starzinski-Powitz, A., & Schneider, G. (2008). Domain organization of long signal peptides of single-pass integral membrane proteins reveals multiple functional capacity. PLoS ONE, 3(7), e2767.CrossRefGoogle Scholar
  14. 14.
    Pennock, G. D., Shoemaker, C., & Miller, L. K. (1984). Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Molecular and Cellular Biology, 4(3), 399–406.CrossRefGoogle Scholar
  15. 15.
    Smith, G. E., Summers, M. D., & Fraser, M. (1983). Production of human beta interferon in insect cells infected with a baculovirus expression vector. Molecular and Cellular Biology, 3(12), 2156–2165.CrossRefGoogle Scholar
  16. 16.
    Smith, G. E., Ju, G., Ericson, B. L., Moschera, J., Lahm, H. W., Chizzonite, R., et al. (1985). Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proceedings of the National Academy of Sciences U S A, 82(24), 8404–8408.CrossRefGoogle Scholar
  17. 17.
    Berger, I., Fitzgerald, D. J., & Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nature Biotechnology, 22(12), 1583–1587.CrossRefGoogle Scholar
  18. 18.
    Bieniossek, C., Imasaki, T., Takagi, Y., & Berger, I. (2012). MultiBac: Expanding the research toolbox for multiprotein complexes. Trends in Biochemical Sciences, 37(2), 49–57.CrossRefGoogle Scholar
  19. 19.
    Bieniossek, C., Richmond, T. J., & Berger, I. (2008). MultiBac: Multigene baculovirus-based eukaryotic protein complex production. Current Protocols in Protein Science (Chapter 5, Unit 5.20).  https://doi.org/10.1002/0471140864.ps0520s51.
  20. 20.
    Trowitzsch, S., Bieniossek, C., Nie, Y., Garzoni, F., & Berger, I. (2010). New baculovirus expression tools for recombinant protein complex production. Journal of Structural Biology, 172(1), 45–54.CrossRefGoogle Scholar
  21. 21.
    Trowitzsch, S., Palmberger, D., Fitzgerald, D., Takagi, Y., & Berger, I. (2012). MultiBac complexomics. Expert Review of Proteomics, 9(4), 363–373.CrossRefGoogle Scholar
  22. 22.
    Barford, D., Takagi, Y., Schultz, P., & Berger, I. (2013). Baculovirus expression: Tackling the complexity challenge. Current Opinion in Structural Biology, 23(3), 357–364.CrossRefGoogle Scholar
  23. 23.
    Inui, K., Zhao, Z., Yuan, J., Jayaprakash, S., Le, L. T. M., Drakulic, S., et al. (2017). Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target. Protein Science, 26(5), 997–1011.CrossRefGoogle Scholar
  24. 24.
    Altmann, F., Staudacher, E., Wilson, I. B., & März, L. (1999). Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconjugate Journal, 16(2), 109–123.CrossRefGoogle Scholar
  25. 25.
    Palmberger, D., Wilson, I. B., Berger, I., Grabherr, R., & Rendic, D. (2012). SweetBac: A new approach for the production of mammalianised glycoproteins in insect cells. PLoS ONE, 7(4), e34226.CrossRefGoogle Scholar
  26. 26.
    Nie, Y., Bellon-Echeverria, I., Trowitzsch, S., Bieniossek, C., & Berger, I. (2014). Multiprotein complex production in insect cells by using polyproteins. Methods in Molecular Biology, 1091, 131–141.CrossRefGoogle Scholar
  27. 27.
    Clem, R. J., & Passarelli, A. L. (2013). Baculoviruses: Sophisticated pathogens of insects. PLoS Pathogens, 9(11), e1003729.CrossRefGoogle Scholar
  28. 28.
    Jawhari, A., Uhring, M., Crucifix, C., Fribourg, S., Schultz, P., Poterszman, A., et al. (2002). Expression of FLAG fusion proteins in insect cells: Application to the multi-subunit transcription/DNA repair factor TFIIH. Protein Expression and Purification, 24(3), 513–523.CrossRefGoogle Scholar
  29. 29.
    Jensen, I. S., Inui, K., Drakulic, S., Jayaprakash, S., Sander, B., & Golas, M. M. (2017). Expression of Flp protein in a baculovirus/insect cell system for biotechnological applications. Protein Journal, 36(4), 332–342.CrossRefGoogle Scholar
  30. 30.
    Masuda, M., & Roos, D. (1993). Association of all three types of FC gamma R (CD64, CD32, and CD16) with a gamma-chain homodimer in cultured human monocytes. J Immunol, 151(12), 7188–7195.Google Scholar
  31. 31.
    Ernst, L. K., Duchemin, A. M., & Anderson, C. L. (1993). Association of the high-affinity receptor for IgG (Fc gamma RI) with the gamma subunit of the IgE receptor. Proceedings of the National Academy of Sciences U S A, 90(13), 6023–6027.CrossRefGoogle Scholar
  32. 32.
    Scholl, P. R., & Geha, R. S. (1993). Physical association between the high-affinity IgG receptor (Fc gamma RI) and the gamma subunit of the high-affinity IgE receptor (Fc epsilon RI gamma). Proceedings of the National Academy of Sciences U S A, 90(19), 8847–8850.CrossRefGoogle Scholar
  33. 33.
    Asaoka, Y., Hatayama, K., Tsumoto, K., Tomita, M., & Ide, T. (2012). Engineering of recombinant human Fcgamma receptor I by directed evolution. Protein Engineering, Design & Selection, 25(12), 835–842.CrossRefGoogle Scholar
  34. 34.
    Kiyoshi, M., Caaveiro, J. M., Kawai, T., Tashiro, S., Ide, T., Asaoka, Y., et al. (2015). Structural basis for binding of human IgG1 to its high-affinity human receptor FcgammaRI. Nature Communications, 6, 6866.CrossRefGoogle Scholar
  35. 35.
    Mancardi, D. A., Albanesi, M., Jönsson, F., Iannascoli, B., Van Rooijen, N., Kang, X., et al. (2013). The high-affinity human IgG receptor FcgammaRI (CD64) promotes IgG-mediated inflammation, anaphylaxis, and antitumor immunotherapy. Blood, 121(9), 1563–1573.CrossRefGoogle Scholar
  36. 36.
    Petersen, T. N., Brunak, S., von Heijne, G., & Nielsen, H. (2011). SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nature Methods, 8(10), 785–786.CrossRefGoogle Scholar
  37. 37.
    Rai, J., Pemmasani, J. K., Voronovsky, A., Jensen, I. S., Manavalan, A., Nyengaard, J. R., et al. (2014). Strep-tag II and Twin-Strep based cassettes for protein tagging by homologous recombination and characterization of endogenous macromolecular assemblies in Saccharomyces cerevisiae. Molecular Biotechnology, 56(11), 992–1003.CrossRefGoogle Scholar
  38. 38.
    Lin, T. Y., Voronovsky, A., Raabe, M., Urlaub, H., Sander, B., & Golas, M. M. (2015). Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 1854(3), 198–208.CrossRefGoogle Scholar
  39. 39.
    Brizzard, B. L., Chubet, R. G., & Vizard, D. L. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. BioTechniques, 16(4), 730–735.Google Scholar
  40. 40.
    Schmidt, T. G., Batz, L., Bonet, L., Carl, U., Holzapfel, G., Kiem, K., et al. (2013). Development of the Twin-Strep-tag(R) and its application for purification of recombinant proteins from cell culture supernatants. Protein Expression and Purification, 92(1), 54–61.CrossRefGoogle Scholar
  41. 41.
    Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Cerretti, D. P., et al. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio-Technology, 6(10), 1204–1210.CrossRefGoogle Scholar
  42. 42.
    Schmidt, T. G., & Skerra, A. (1993). The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Protein Engineering, 6(1), 109–122.CrossRefGoogle Scholar
  43. 43.
    Karakas, E., & Furukawa, H. (2014). Crystal structure of a heterotetrameric NMDA receptor ion channel. Science, 344(6187), 992–997.CrossRefGoogle Scholar
  44. 44.
    Bai, M., Trivedi, S., & Brown, E. M. (1998). Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. Journal of Biological Chemistry, 273(36), 23605–23610.CrossRefGoogle Scholar
  45. 45.
    Xu, B., Chakraborty, R., Eilers, M., Dakshinamurti, S., O’Neil, J. D., Smith, S. O., et al. (2013). High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant. PLoS ONE, 8(9), e76481.CrossRefGoogle Scholar
  46. 46.
    Hernan, R., Heuermann, K., Brizzard, B., et al. (2000). Multiple epitope tagging of expressed proteins for enhanced detection. BioTechniques, 28(4), 789–793.Google Scholar
  47. 47.
    van de Winkel, J. G., Ernst, L. K., Anderson, C. L., & Chiu, I. M. (1991). Gene organization of the human high affinity receptor for IgG, Fc gamma RI (CD64). Characterization and evidence for a second gene. Journal of Biological Chemistry, 266(20), 13449–13455.Google Scholar
  48. 48.
    Wirthmueller, U., Kurosaki, T., Murakami, M. S., & Ravetch, J. V. (1992). Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain. Journal of Experimental Medicine, 175(5), 1381–1390.CrossRefGoogle Scholar
  49. 49.
    van Vugt, M. J., Heijnen, A. F., Capel, P. J., Park, S. Y., Ra, C., Saito, T., et al. (1996). FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo. Blood, 87(9), 3593–3599.Google Scholar
  50. 50.
    Erickson, H. P. (2009). Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biological Procedures Online, 11, 32–51.CrossRefGoogle Scholar
  51. 51.
    Beekman, J. M., van der Linden, J. A., van de Winkel, J. G., & Leusen, J. H. (2008). FcgammaRI (CD64) resides constitutively in lipid rafts. Immunology Letters, 116(2), 149–155.CrossRefGoogle Scholar
  52. 52.
    Harrison, P. T., & Allen, J. M. (1998). High affinity IgG binding by FcgammaRI (CD64) is modulated by two distinct IgSF domains and the transmembrane domain of the receptor. Protein Engineering, 11(3), 225–232.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Le T. M. Le
    • 1
  • Jens R. Nyengaard
    • 1
    • 2
    • 3
  • Monika M. Golas
    • 4
    • 5
  • Bjoern Sander
    • 1
    • 3
    • 6
  1. 1.Stereology and EM Laboratory, Department of Clinical MedicineAarhus UniversityAarhus CDenmark
  2. 2.Core Center for Molecular Morphology, Department of Clinical MedicineAarhus University HospitalAarhus CDenmark
  3. 3.Centre for Stochastic Geometry and Advanced BioimagingAarhus UniversityAarhus CDenmark
  4. 4.Department of BiomedicineAarhus UniversityAarhus CDenmark
  5. 5.Department of Human GeneticsHannover Medical SchoolHannoverGermany
  6. 6.Institute of PathologyHannover Medical SchoolHannoverGermany

Personalised recommendations