Advertisement

Molecular Biotechnology

, Volume 60, Issue 11, pp 820–832 | Cite as

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5

  • Monika M. Golas
  • Sakthidasan Jayaprakash
  • Le T. M. Le
  • Zongpei Zhao
  • Violeta Heras Huertas
  • Ida S. Jensen
  • Juan Yuan
  • Bjoern Sander
Original Paper

Abstract

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Keywords

Chromatin remodeler Plasmid Recombinant protein Protein–protein interaction Nucleus Protein production 

Abbreviations

AcNPV

Autographa californica nuclear polyhedrosis virus

ATP

Adenosine triphosphate

BAF

BRM/BRG1-associated factor

BAF47

BRM/BRG1-associated factor 47

BRG1

Brahma-related gene 1

BRM

Brahma Abbreviations

CBP

Calmodulin-binding peptide

CC

Coiled coil

CE

Cytoplasmic extract

DBD

DNA-binding domain

DTT

Dithiothreitol

E

Elution fraction

EDTA

Ethylenediaminetetraacetic acid

FBS

Fetal bovine serum

FT

Flow through

GST

Glutathione S-transferase

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HSE

Heat shock element

Hsp

Heat shock protein

INI1

Integrase interactor 1

IPTG

Isopropyl β-d-1-thiogalactopyranoside

kDa

Kilo dalton

LB

Lysogeny broth

M

Marker

MBP

Maltose-binding protein

MCS

Multiple cloning site

MDa

Mega dalton

NE

Nuclear extract

NMDA

N-methyl-d-aspartate

OD600

Optical density at 600 nm

ORF

Open reading frame

PAGE

Polyacrylamide gel electrophoresis

PCR

Polymerase chain reaction

Polh

Polyhedrin

P/S

Penicillin/streptomycin

SD

Synthetic defined

SDS

Sodium dodecyl sulfate

SMARCB1

SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1

SNF5

Sucrose Non Fermentable 5

SWI/SNF

Switch/sucrose non-fermentable

TATA

TATA box

TCA

Trichloroacetic acid

TEV

Tobacco etch virus

W

Wash fraction

X-gal

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

YFP

Yellow fluorescent protein

Notes

Acknowledgements

We wish to thank Susanne Stubbe and Golshah Ayoubi for expert technical assistance, and Srdja Drakulic for support with some of the experiments. We are grateful for the access to experimental facilities at the Danish Neuroscience Centre House, Aarhus University, Denmark. This study was supported by the Sapere Aude Program of the Danish Council for Independent Research, the Lundbeck Foundation’s Fellowship Program, the A.P. Møller Foundation for the Advancement of Medical Sciences, and the Carlsbergfondet to MMG. ISJ was supported by a fellowship of the Graduate School of Health, Aarhus University.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest related to this study.

Supplementary material

12033_2018_107_MOESM1_ESM.pdf (79 kb)
Supplementary material 1 (PDF 80 KB)

References

  1. 1.
    Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., & Green, M. R. (1994). Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex. Nature, 370(6489), 477–481.CrossRefPubMedGoogle Scholar
  2. 2.
    Imbalzano, A. N., Kwon, H., Green, M. R., & Kingston, R. E. (1994). Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature, 370(6489), 481–485.CrossRefPubMedGoogle Scholar
  3. 3.
    Liu, R., Liu, H., Chen, X., Kirby, M., Brown, P. O., & Zhao, K. (2001). Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell, 106(3), 309–318.CrossRefPubMedGoogle Scholar
  4. 4.
    Peterson, C. L., Dingwall, A., & Scott, M. P. (1994). Five SWI/SNF gene products are components of a large multisubunit complex required for transcriptional enhancement. Proceedings of National Academy of Science USA, 91(8), 2905–2908.CrossRefGoogle Scholar
  5. 5.
    Phelan, M. L., Sif, S., Narlikar, G. J., & Kingston, R. E. (1999). Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits. Molecular Cell, 3(2), 247–253.CrossRefPubMedGoogle Scholar
  6. 6.
    Roberts, C. W., Galusha, S. A., McMenamin, M. E., Fletcher, C. D., & Orkin, S. H. (2000). Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid tumors in mice. Proceedings of the National Academy Science USA, 97(25), 13796–13800.CrossRefGoogle Scholar
  7. 7.
    Klochendler-Yeivin, A., Fiette, L., Barra, J., Muchardt, C., Babinet, C., & Yaniv, M. (2000). The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression. EMBO Report, 1(6), 500–506.CrossRefGoogle Scholar
  8. 8.
    Roberts, C. W., Leroux, M. M., Fleming, M. D., & Orkin, S. H. (2002). Highly penetrant, rapid tumorigenesis through conditional inversion of the tumor suppressor gene Snf5. Cancer Cell, 2(5), 415–425.CrossRefPubMedGoogle Scholar
  9. 9.
    Miller, M. D., & Bushman, F. D. (1995). HIV integration. Ini1 for integration? Current Biology, 5(4), 368–370.CrossRefPubMedGoogle Scholar
  10. 10.
    Versteege, I., Sévenet, N., Lange, J., Rousseau-Merck, M. F., Ambros, P., Handgretinger, R., … Delattre, O. (1998). Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature, 394(6689), 203–206.CrossRefPubMedGoogle Scholar
  11. 11.
    Hulsebos, T. J., Plomp, A. S., Wolterman, R. A., Robanus-Maandag, E. C., Baas, F., & Wesseling, P. (2007). Germline mutation of INI1/SMARCB1 in familial schwannomatosis. American Journal of Human Genetics, 80(4), 805–810.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Tsurusaki, Y., Okamoto, N., Ohashi, H., Kosho, T., Imai, Y., Hibi-Ko, Y., … Fukushima, Y. (2012). Mutations affecting components of the SWI/SNF complex cause Coffin-Siris syndrome. Nature Genetics, 44(4), 376–378.CrossRefPubMedGoogle Scholar
  13. 13.
    Wieczorek, D., Bögershausen, N., Beleggia, F., Steiner-Haldenstätt, S., Pohl, E., Li, Y., … Alanay, Y. (2013). A comprehensive molecular study on Coffin-Siris and Nicolaides-Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling. Human Molecular Genetics, 22(25), 5121–5135.CrossRefPubMedGoogle Scholar
  14. 14.
    Taylor, M. D., Gokgoz, N., Andrulis, I. L., Mainprize, T. G., Drake, J. M., & Rutka, J. T. (2000). Familial posterior fossa brain tumors of infancy secondary to germline mutation of the hSNF5 gene. American Journal of Human Genetics, 66(4), 1403–1406.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lee, D., Sohn, H., Kalpana, G. V., & Choe, J. (1999). Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA. Nature, 399(6735), 487–491.CrossRefPubMedGoogle Scholar
  16. 16.
    Wu, D. Y., Kalpana, G. V., Goff, S. P., & Schubach, W. H. (1996). Epstein-Barr virus nuclear protein 2 (EBNA2) binds to a component of the human SNF-SWI complex, hSNF5/Ini1. Journal of Virology, 70(9), 6020–6028.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Hwang, S., Lee, D., Gwack, Y., Min, H., & Choe, J. (2003). Kaposi’s sarcoma-associated herpesvirus K8 protein interacts with hSNF5. Journal of General Virology, 84(Pt 3), 665–676.CrossRefPubMedGoogle Scholar
  18. 18.
    Morozov, A., Yung, E., & Kalpana, G. V. (1998). Structure-function analysis of integrase interactor 1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly conserved region. Proceedings of National Academy Science USA, 95(3), 1120–1125.CrossRefGoogle Scholar
  19. 19.
    Das, S., Banerjee, B., Hossain, M., Thangamuniyandi, M., Dasgupta, S., Chongdar, N., … Basu, G. (2013). Characterization of DNA binding property of the HIV-1 host factor and tumor suppressor protein integrase interactor 1 (INI1/hSNF5). PLoS ONE 8(7), e66581.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Das, S., Cano, J., & Kalpana, G. V. (2009). Multimerization and DNA binding properties of INI1/hSNF5 and its functional significance. Journal of Biological Chemistry, 284(30), 19903–19914.CrossRefPubMedGoogle Scholar
  21. 21.
    Yan, L., Xie, S., Du, Y., & Qian, C. (2017). Structural insights into BAF47 and BAF155 complex formation. Journal of Molecular Biology, 429(11), 1650–1660.CrossRefPubMedGoogle Scholar
  22. 22.
    Mesa, P., Deniaud, A., Montoya, G., & Schaffitzel, C. (2013). Directly from the source: Endogenous preparations of molecular machines. Current Opinion in Structure Biology, 23(3), 319–325.CrossRefGoogle Scholar
  23. 23.
    Vijayachandran, L. S., Viola, C., Garzoni, F., Trowitzsch, S., Bieniossek, C., Chaillet, M., … Richmond, T. J. (2011). Robots, pipelines, polyproteins: Enabling multiprotein expression in prokaryotic and eukaryotic cells. Journal of Structural Biology, 175(2), 198–208.CrossRefPubMedGoogle Scholar
  24. 24.
    Rai, J., Pemmasani, J. K., Voronovsky, A., Jensen, I. S., Manavalan, A., Nyengaard, J. R., … Sander, B. (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.CrossRefPubMedGoogle Scholar
  25. 25.
    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.CrossRefPubMedGoogle Scholar
  26. 26.
    Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., & Séraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnology, 17(10), 1030–1032.CrossRefPubMedGoogle Scholar
  27. 27.
    Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S., & Huang, B. (2016). A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proceedings of the National Academy Sciences USA, 113(25), E3501–E3508.CrossRefGoogle Scholar
  28. 28.
    Murray, V., Chen, J., Huang, Y., Li, Q., & Wang, J. (2010). Preparation of very-high-yield recombinant proteins using novel high-cell-density bacterial expression methods. Cold Spring Harbor Protocols, 2010(8), pdb-prot5475.CrossRefPubMedGoogle Scholar
  29. 29.
    Dalton, A. C., & Barton, W. A. (2014). Over-expression of secreted proteins from mammalian cell lines. Protein Science, 23(5), 517–525.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    He, Y., Wang, K., & Yan, N. (2014). The recombinant expression systems for structure determination of eukaryotic membrane proteins. Protein and Cell, 5(9), 658–672.CrossRefPubMedGoogle Scholar
  31. 31.
    Peti, W., & Page, R. (2007). Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expression and Purification, 51(1), 1–10.CrossRefPubMedGoogle Scholar
  32. 32.
    Correa, A., & Oppezzo, P. (2011). Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: Advantages of high-throughput screening. Biotechnology Journal, 6(6), 715–730.CrossRefPubMedGoogle Scholar
  33. 33.
    Brondyk, W. H. (2009). Selecting an appropriate method for expressing a recombinant protein. Methods Enzymology, 463, 131–147.CrossRefGoogle Scholar
  34. 34.
    Weidner, M., Taupp, M., & Hallam, S. J. (2010). Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Journal of Visualized Experiments, 36, 1862.Google Scholar
  35. 35.
    Ahmad, M., Hirz, M., Pichler, H., & Schwab, H. (2014). Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology, 98(12), 5301–5317.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Bill, R. M. (2014). Playing catch-up with Escherichia coli: Using yeast to increase success rates in recombinant protein production experiments. Frontiers in Microbiology, 5, 85.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Hacker, D. L., & Balasubramanian, S. (2016). Recombinant protein production from stable mammalian cell lines and pools. Current Opinion Structure Biology, 38, 129–136.CrossRefGoogle Scholar
  38. 38.
    Baser, B., & van den Heuvel, J. (2016). Assembling multi-subunit complexes using mammalian expression. Advances Experimental Medicine Biology, 896, 225–238.CrossRefGoogle Scholar
  39. 39.
    Dyson, M. R. (2016). Fundamentals of expression in mammalian cells. Advances Experimental Medicine Biology, 896, 217–224.CrossRefGoogle Scholar
  40. 40.
    van Oers, M. M., Pijlman, G. P., & Vlak, J. M. (2015). Thirty years of baculovirus-insect cell protein expression: From dark horse to mainstream technology. Journal of General Virology, 96(Pt 1), 6–23.CrossRefPubMedGoogle Scholar
  41. 41.
    Miller, L. K. (1988). Baculoviruses for foreign gene expression in insect cells. Biotechnology, 10, 457–465.PubMedGoogle Scholar
  42. 42.
    Li, J., Happ, B., Schetter, C., Oellig, C., Hauser, C., Kuroda, K., … Doerfler, W. (1990). The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells. Veterinary Microbiology, 23(1–4), 73–78.CrossRefPubMedGoogle Scholar
  43. 43.
    Berger, I., Fitzgerald, D. J., & Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nature Biotechnology, 22(12), 1583–1587.CrossRefPubMedGoogle Scholar
  44. 44.
    Fitzgerald, D. J., Berger, P., Schaffitzel, C., Yamada, K., Richmond, T. J., & Berger, I. (2006). Protein complex expression by using multigene baculoviral vectors. Nature Methods, 3(12), 1021–1032.CrossRefPubMedGoogle Scholar
  45. 45.
    Friesen, P. D., & Miller, L. K. (1985). Temporal regulation of baculovirus RNA: Overlapping early and late transcripts. Journal of Virology, 54(2), 392–400.PubMedPubMedCentralGoogle Scholar
  46. 46.
    Knebel, D., Lübbert, H., & Doerfler, W. (1985). The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: Activation by viral gene products and sensitivity to DNA methylation. EMBO Journal, 4(5), 1301–1306.CrossRefPubMedGoogle Scholar
  47. 47.
    Smith, G. E., Summers, M. D., & Fraser, M. J. (1983). Production of human beta interferon in insect cells infected with a baculovirus expression vector. Molecular and Cellular Biology, 3(12), 2156–2165.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Inui, K., Zhao, Z., Yuan, J., Jayaprakash, S., Le, L. T., Drakulic, S., … Golas, M. M. (2017). Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target. Protein Science, 26(5), 997–1011.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Le, L. T., Nyengaard, J. R., Golas, M. M., & Sander, B. (2018). 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. Molecular Biotechnology, 60(1), 31–40.CrossRefPubMedGoogle Scholar
  51. 51.
    Karakas, E., & Furukawa, H. (2014). Crystal structure of a heterotetrameric NMDA receptor ion channel. Science, 344(6187), 992–997.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Johnsson, N., & Varshavsky, A. (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of National Academy Science USA, 91(22), 10340–10344.CrossRefGoogle Scholar
  53. 53.
    Wellhausen, A., & Lehming, N. (1999). Analysis of the in vivo interaction between a basic repressor and an acidic activator. FEBS Letter, 453(3), 299–304.CrossRefGoogle Scholar
  54. 54.
    Zhao, X., Li, G., & Liang, S. (2013). Several affinity tags commonly used in chromatographic purification. Journal of Analytical Methods Chemistry, 2013, 8.CrossRefGoogle Scholar
  55. 55.
    Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., & Tan, S. (2005). Comparison of affinity tags for protein purification. Protein Expression Purification, 41(1), 98–105.CrossRefPubMedGoogle Scholar
  56. 56.
    Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R., & Stüber, D. (1988). Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nature Biotechnology, 6(11), 1321–1325.CrossRefGoogle Scholar
  57. 57.
    Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Cerretti, D. P., … Conlon, P. J. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Nature Biotechnology, 6, 1204–1210.CrossRefGoogle Scholar
  58. 58.
    Einhauer, A., & Jungbauer, A. (2001). The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. Journal of Biochemical Biophysical Methods, 49(1–3), 455–465.CrossRefPubMedGoogle Scholar
  59. 59.
    Maroux, S., Baratti, J., & Desnuelle, P. (1971). Purification and specificity of porcine enterokinase. Journal of Biological and Chemistry, 246(16), 5031–5039.Google Scholar
  60. 60.
    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.PubMedGoogle Scholar
  61. 61.
    Junttila, M. R., Saarinen, S., Schmidt, T., Kast, J., & Westermarck, J. (2005). Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells. Proteomics, 5(5), 1199–1203.CrossRefPubMedGoogle Scholar
  62. 62.
    Voss, S., & Skerra, A. (1997). Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification. Protein Engineering, 10(8), 975–982.CrossRefPubMedGoogle Scholar
  63. 63.
    Khavari, P. A., Peterson, C. L., Tamkun, J. W., Mendel, D. B., & Crabtree, G. R. (1993). BRG1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription. Nature, 366(6451), 170–174.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Monika M. Golas
    • 1
    • 4
  • Sakthidasan Jayaprakash
    • 1
  • Le T. M. Le
    • 2
  • Zongpei Zhao
    • 1
  • Violeta Heras Huertas
    • 1
  • Ida S. Jensen
    • 1
  • Juan Yuan
    • 1
  • Bjoern Sander
    • 2
    • 3
    • 5
  1. 1.Department of BiomedicineAarhus UniversityAarhus CDenmark
  2. 2.Stereology and Electron Microscopy LaboratoryAarhus UniversityAarhus CDenmark
  3. 3.Center for Stochastic Geometry and Advanced BioimagingAarhus UniversityAarhus CDenmark
  4. 4.Department of Human GeneticsHannover Medical SchoolHannoverGermany
  5. 5.Institute of PathologyHannover Medical SchoolHannoverGermany

Personalised recommendations