Research Article | Published:

Purification of recombinant proteins by fusion with thermally-responsive polypeptides

Nature Biotechnology volume 17, pages 11121115 (1999) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , , & Fusion proteins in biotechnology and structural biology. Curr. Opin. Struct. Biol. 2, 569–575 (1992).

  2. 2.

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

  3. 3.

    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).

  4. 4.

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

  5. 5.

    , , & A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli. Gene 169, 59–64 (1996).

  6. 6.

    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).

  7. 7.

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

  8. 8.

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

  9. 9.

    , , & Chelating peptide-immobilized metal ion affinity chromatography. J. Biol. Chem. 263, 7211–7215 (1988).

  10. 10.

    & Ribonuclease S-peptide as a carrier in fusion proteins. Protein. Sci. 2, 348–356 (1993).

  11. 11.

    , & Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system. Biotechniques 13, 756–762 (1992).

  12. 12.

    , , , & Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr. Purif. 11, 1–16 (1997).

  13. 13.

    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).

  14. 14.

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

  15. 15.

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

  16. 16.

    , & 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).

  17. 17.

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

  18. 18.

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

  19. 19.

    , , & Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein. Bioconjugate Chem. 5, 504–507 (1994).

  20. 20.

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

  21. 21.

    , & Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies. Biopolymers 24, 2345–2356 (1985).

  22. 22.

    , , , & Tendamistat (HOE 467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158. Eur. J. Biochem. 141, 505–512 (1984).

  23. 23.

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

  24. 24.

    , , , & Current protocols in protein science. (John Wiley & Sons, New York; 1995).

  25. 25.

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

  26. 26.

    & Immunoassay. (Academic Press, San Diego, CA; 1996).

  27. 27.

    & Current Concepts: Hyperthermia in the treatment of cancer. (The UpJohn Co., Kalamazoo, MI; 1998).

  28. 28.

    , , & Local hyperthermia improves uptake of a chimeric monoclonal antibody in a subcutaneous xenograft model. Clin. Cancer Res. 3, 63–70 (1997).

  29. 29.

    , , , & Enhanced delivery of a monoclonal antibody F(ab′)2 fragment to subcutaneous human glioma xenografts using local hyperthermia. Cancer Res. 50, 1803–1809 (1990).

  30. 30.

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

  31. 31.

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

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

Affiliations

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

    • Dan E. Meyer
    •  & Ashutosh Chilkoti

Authors

  1. Search for Dan E. Meyer in:

  2. Search for Ashutosh Chilkoti in:

Corresponding author

Correspondence to Ashutosh Chilkoti.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/15100

Further reading