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.

  • Research Article
  • Published:

Purification of recombinant proteins by fusion with thermally-responsive polypeptides

Nature Biotechnology volume 17, pages 1112–1115 (1999)Cite this article

Abstract

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.

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: Characterization of the inverse transition of ELP fusion proteins.
Figure 2: Purification by inverse transition cycling.
Figure 3: Gene sequences.

Similar content being viewed by others

References

  1. Nilsson, B., Forsberg, G., Moks, T., Hartmanis, M. & Uhlén, M. Fusion proteins in biotechnology and structural biology. Curr. Opin. Struct. Biol. 2, 569–575 (1992).

    Article  CAS  Google Scholar 

  2. Uhlén, M. & Moks, T. Gene fusions for purpose of expression: an introduction. Methods Enzymol. 195, 129–143 (1990).

    Article  Google Scholar 

  3. Maina, C.V. et al. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose binding protein. Gene 74, 365–373 (1988).

    Article  CAS  Google Scholar 

  4. Smith, D.B. & Johnson, K.S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40 (1988).

    Article  CAS  Google Scholar 

  5. Tsao, K.W., deBarbieri, B., Hanspeter, M. & Waugh, D.W. A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli. Gene 169, 59–64 (1996).

    Article  CAS  Google Scholar 

  6. Smith, P.A. et al. A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli. Nucleic Acids Res. 26, 1414–1420 (1998).

    Article  CAS  Google Scholar 

  7. LaVallie, E.R. et al. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11, 187–193 (1993).

    CAS  PubMed  Google Scholar 

  8. Ong, E. et al. The cellulose-binding domains of cellulases: tools for biotechnology. Trends Biotechnol. 7, 239–243 (1989).

    Article  CAS  Google Scholar 

  9. Smith, M.C., Furman, T.C., Ingolia, T.D. & Pidgeon, C. Chelating peptide-immobilized metal ion affinity chromatography. J. Biol. Chem. 263, 7211–7215 (1988).

    CAS  PubMed  Google Scholar 

  10. Kim, J-S. & Raines, R.T. Ribonuclease S-peptide as a carrier in fusion proteins. Protein. Sci. 2, 348–356 (1993).

    Article  CAS  Google Scholar 

  11. Su, X., Prestwood, A.K. & McGraw, R.A. Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system. Biotechniques 13, 756–762 (1992).

    CAS  PubMed  Google Scholar 

  12. Nilsson, J., Ståhl, S., Lundeberg, J., Uhlén, M. & Nygren, P.Å. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1–16 (1997).

    Article  CAS  Google Scholar 

  13. Urry, D.W. Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition due to internal chain dynamics. J. Protein. Chem. 7, 1–34 (1988).

    Article  CAS  Google Scholar 

  14. Urry, D.W. Free energy transduction in polypeptides and proteins based on inverse temperature transitions. Prog. Biophys. Mol. Biol. 57, 23–57 (1992).

    Article  CAS  Google Scholar 

  15. Urry, D.W. Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J. Phys. Chem. B 101, 11007–11028 (1997).

    Article  CAS  Google Scholar 

  16. McPherson, D.T., Xu, J. & Urry, D.W. Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP. Protein Expr. Purif. 7, 51–57 (1996).

    Article  CAS  Google Scholar 

  17. Hoffman. A.S. Applications of thermally-reversible polymers and hydrogels in therapeutics and diagnostics. J. Controlled Release 6, 297–305 (1987).

    Article  CAS  Google Scholar 

  18. Chen, J.P. & Hoffman A.S. Protein-polymer conjugates II. Affinity precipitation separation of immunogammaglobulin by a poly(N-isopropylacrylamide)-protein A conjugate. Biomaterials 11, 631–634 (1990).

    Article  CAS  Google Scholar 

  19. Chilkoti, A., Chen, G-H., Stayton, P.S. & Hoffman, A.S. Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein. Bioconjugate Chem. 5, 504–507 (1994).

    Article  CAS  Google Scholar 

  20. Urry, D.W. et al. Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity. J. Am. Chem. Soc. 113, 4346–4348 (1991).

    Article  CAS  Google Scholar 

  21. Urry, D.W., Trapane, T.L. & Prasad, K.U. Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies. Biopolymers 24, 2345–2356 (1985).

    Article  CAS  Google Scholar 

  22. Vertesy, L., Oeding, V., Bender, R., Zepf, K. & Nesemann, G. Tendamistat (HOE 467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158. Eur. J. Biochem. 141, 505–512 (1984).

    Article  CAS  Google Scholar 

  23. Porath, J. Immobilized metal ion affinity chromatography. Prot. Expr. Purif. 3, 262–282 (1992).

    Article  Google Scholar 

  24. Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W. & Wingfield, P.T. Current protocols in protein science. (John Wiley & Sons, New York; 1995).

    Google Scholar 

  25. Hartmeier, W. Immobilized biocatalysts. (Springer-Verlag, Berlin; 1988).

    Book  Google Scholar 

  26. Diamandis, E.P. & Christopoulos, T.K. Immunoassay. (Academic Press, San Diego, CA; 1996).

    Google Scholar 

  27. Dewhirst, M.W. & Samulski, T.V. Current Concepts: Hyperthermia in the treatment of cancer. (The UpJohn Co., Kalamazoo, MI; 1998).

    Google Scholar 

  28. Hauck, M.L., Dewhirst, M.W., Bigner, D.D. & Zalutsky, M.R. Local hyperthermia improves uptake of a chimeric monoclonal antibody in a subcutaneous xenograft model. Clin. Cancer Res. 3, 63–70 (1997).

    CAS  PubMed  Google Scholar 

  29. Cope, D.A., Dewhirst, M.W., Friedman, H.S., Bigner, D.D. & Zalutsky, M.R. Enhanced delivery of a monoclonal antibody F(ab′)2 fragment to subcutaneous human glioma xenografts using local hyperthermia. Cancer Res. 50, 1803–1809 (1990).

    CAS  PubMed  Google Scholar 

  30. Ausubel, F.M. et al. Current protocols in molecular biology (John Wiley & Sons, New York; 1995).

    Google Scholar 

  31. Holmgren, A. & Bjornstedt, M. Enzymatic reduction-oxidation of protein disulfides by thioredoxin. Methods Enzymol. 107, 295–300 (1984).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R.T. Piervincenzi for the thioredoxin–tendamistat gene. This work was supported by Duke University through its provision of start-up funds to A.C., by the Whitaker Foundation, the North Carolina Biotechnology Center (ARIG no. 9605-ARG-0050), and the National Institutes of Health (1R21-GM-057373-01). We also thank the Whitaker Foundation for support of D.E.M. as a graduate fellow.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Box 90281, Duke University, Durham, 27708-0281, NC

    Dan E. Meyer & Ashutosh Chilkoti

Authors
  1. Dan E. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashutosh Chilkoti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ashutosh Chilkoti.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meyer, D., Chilkoti, A. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17, 1112–1115 (1999). https://doi.org/10.1038/15100

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/15100

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