Skip to main content
SpringerLink
Log in

Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Marston, F. A. O. (1986) The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem. J. 240, 1–12.

    PubMed  CAS  Google Scholar 

  2. Edwards, A. M., Arrowsmith, H. C., Christendat, D., Dharamsi, A., Friesen, J. D., Greenblatt, J. F., and Vedadi, M. (2000) Protein production: feeding the crystallographers and NMR spectroscopists. Nat. Struct. Biol. 7, 970–972.

    Article  PubMed  CAS  Google Scholar 

  3. Thomas, J. G., Ayling, A., and Baneyx, F. (1997) Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. Appl. Biochem. Biotech. 66, 197–238.

    CAS  Google Scholar 

  4. Nishihara, K., Kanemori, M., Kitagawa, M., Yanagi, H., and Yura, T. (1998) Chaperone coexpression plasmids: Differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl. Environ. Microbiol. 64, 1694–1699.

    PubMed  CAS  Google Scholar 

  5. Nishihara, K., Kanemori, M., Yanagi, H., and Yura, T. (2000). Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66, 884–889.

    Article  PubMed  CAS  Google Scholar 

  6. Li, Z. Y., Liu, C. P., Zhu, L. Q., Jing, G. Z., and Zhou, J. M. (2001) The chaperone activity of trigger factor is distinct from its isomerase activity during co-expression with adenylate kinase in Escherichia coli. FEBS Letters 506, 108–112.

    Article  PubMed  CAS  Google Scholar 

  7. Ikura, K., Kokubo, T., Natsuka, S., et al. (2002) Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli. Prep. Biochem. Biotechnol. 32, 189–205.

    Article  PubMed  CAS  Google Scholar 

  8. Haslbeck, M., Schuster, I., and Grallert, H. (2003) GroE-dependent expression and purification of pig heart mitochondrial citrate synthase in Escherichia coli. J Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 786, 127–136.

    Article  CAS  Google Scholar 

  9. Xu, H. M., Zhang, G. Y., Ji, X. D., Cao, L., Shu, L., and Hua, Z. C. (2005) Expression of soluble, biologically active recombinant human endostatin in Escherichia coli. Protein Expr. Purif. 41, 252–258.

    Article  PubMed  CAS  Google Scholar 

  10. Hartl, F. U. (1996) Molecular chaperones in cellular protein folding. Nature 381, 571–580.

    Article  PubMed  CAS  Google Scholar 

  11. Hartl, F. U. and Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852–1858.

    Article  PubMed  CAS  Google Scholar 

  12. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K., and Hartl, F. U. (1992) Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 356, 683–689.

    Article  PubMed  CAS  Google Scholar 

  13. Blum, P., Ory, J., Bauernfeid, J., and Krska, J. (1992) Physiological consequences of DnaK and DnaJ over-production in Escherichia coli. J. Bacteriol. 174, 7436–7444.

    PubMed  CAS  Google Scholar 

  14. Thomas, J. G. and Baneyx, F. (1996) Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines. Mol. Microbiol. 21, 1185–1196.

    Article  PubMed  CAS  Google Scholar 

  15. Netzer, W. J. and Hartl, F. U. (1997) Recombination of protein domains facilitated by co-translational folding in eukaryotes. Nature 388, 343–349.

    Article  PubMed  CAS  Google Scholar 

  16. Kolb, V. A., Makeyev, E. V., and Spirin, A. S. (1994) Folding of firefly luciferase during translation in a cell-free system. The EMBO J. 13, 3631–3637.

    CAS  Google Scholar 

  17. Kolb, V. A., Makeyev, E. V., and Spririn, A. S. (2000) Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system. J. Biol. Chem. 275, 16,597–16,601.

    Article  CAS  Google Scholar 

  18. Agashe, V. R., Guha, S., Chang, HC., et al. (2004) Function of trigger factor and DnaK in multidomain protein folding: Increase in yield at the expense of folding speed. Cell 117, 199–209.

    Article  PubMed  CAS  Google Scholar 

  19. Hesterkamp, T., Hauser, S., Lutcke, H., and Bukan, B. (1996) Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains. Proc. Natl. Acad. Sci. USA 93, 4437–4441.

    Article  PubMed  CAS  Google Scholar 

  20. Schirmer, E. C., Glover, J. R., Singer, M. A., and Lindquist, S. (1996) HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21, 289–296.

    Article  PubMed  CAS  Google Scholar 

  21. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

    Article  PubMed  CAS  Google Scholar 

  22. Martin, G. A., Kawaguchi, R., Lam, Y., DeGiovanni, A., Fukushima, M., and Mutter, W. (2001) High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/translation system. Biotechniques 31, 948–953.

    PubMed  CAS  Google Scholar 

  23. Shelton, D. L., Sutherland, J., Gripp, J., et al. (1995) Human trks; Molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins. J. Neurosci. 15, 477–491.

    PubMed  CAS  Google Scholar 

  24. Spiess, M., Schwarz, A. L., and Lodish, H. F. (1985) Sequence of human asialoglycoprotein receptor cDNA. J. Biol. Chem. 260, 1979–1982.

    PubMed  CAS  Google Scholar 

  25. Baneyx, F. (1999) Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421.

    Article  PubMed  CAS  Google Scholar 

  26. Beissinger, M. and Buchner, J. (1998) How chaperones fold proteins. Biol. Chem. 379, 245–259.

    PubMed  CAS  Google Scholar 

  27. Jakob, U., Muse, W., Eser, M., and Bardwell, J. C. A. (1999) Chaperone activity with a redox switch. Cell 96, 341–352.

    Article  PubMed  CAS  Google Scholar 

  28. Hoffmann, J., Linke, K., Graf, P. C. F., Lilie, H., and Jakob, U. (2004) Identification of a redox-regulated chaperone network. The EMBO J. 23, 160–168.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biological Science Laboratories, Toray Research Center Inc., 1111 Tebiro, Kamakura, 248-8555, Kanagawa, Japan

    Sumiyo Endo, Yusuke Tomimoto, Hiroyuki Shimizu, Yoshitaka Taniguchi & Takuo Onizuka

Authors
  1. Sumiyo Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yusuke Tomimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroyuki Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yoshitaka Taniguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takuo Onizuka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takuo Onizuka.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Endo, S., Tomimoto, Y., Shimizu, H. et al. Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system. Mol Biotechnol 33, 199–209 (2006). https://doi.org/10.1385/MB:33:3:199

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/MB:33:3:199

Index Entries

Search

Navigation