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.

  • Protocol
  • Published:

Recombinant protein purification by self-cleaving aggregation tag

Abstract

A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 °C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50–100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8–24 h depending on specific process parameters.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Details of the fusion junction between the intein C-terminus and the N-terminus of the target protein (the GFP sequence is given as an example target protein).
Figure 2: SDS–PAGE analysis of samples taken during GFP purification.
Figure 3: In-process samples for GFP purification.

Similar content being viewed by others

References

  1. Banki, M.R., Feng, L. & Wood, D.W. Simple bioseparations using self-cleaving elastin-like polypeptide tags. Nat. Methods 2, 659–661 (2005).

    Article  CAS  Google Scholar 

  2. Urry, D.W., Long, M.M. & Sugano, H. Cyclic analog of elastin polyhexapeptide exhibits an inverse temperature transition leading to crystallization. J. Biol. Chem. 253, 6301–6302 (1978).

    CAS  PubMed  Google Scholar 

  3. Meyer, D.E. & Chilkoti, A. Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides. Biomacromolecules 5, 846–851 (2004).

    Article  CAS  Google Scholar 

  4. Luan, C.H., Harris, R.D., Prasad, K.U. & Urry, D.W. Differential scanning calorimetry studies of the inverse temperature transition of the polypentapeptide of elastin and its analogues. Biopolymers 29, 1699–1706 (1990).

    Article  CAS  Google Scholar 

  5. Luan, C.H., Parker, T.M., Prasad, K.U. & Urry, D.W. Differential scanning calorimetry studies of NaCl effect on the inverse temperature transition of some elastin-based polytetra-, polypenta-, and polynonapeptides. Biopolymers 31, 465–475 (1991).

    Article  CAS  Google Scholar 

  6. Meyer, D.E. & Chilkoti, A. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat. Biotechnol. 17, 1112–1115 (1999).

    Article  CAS  Google Scholar 

  7. Hyun, J., Lee, W.K., Nath, N., Chilkoti, A. & Zauscher, S. Capture and release of proteins on the nanoscale by stimuli-responsive elastin-like polypeptide “switches”. J. Am. Chem. Soc. 126, 7330–7335 (2004).

    Article  CAS  Google Scholar 

  8. Lin, M., Rose-John, S., Grotzinger, J., Conrad, U. & Scheller, J. Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: purification via inverse transition cycling. Biochem. J. 398, 577–583 (2006).

    Article  CAS  Google Scholar 

  9. Nath, N. & Chilkoti, A. Fabrication of a reversible protein array directly from cell lysate using a stimuli-responsive polypeptide. Anal. Chem. 75, 709–715 (2003).

    Article  CAS  Google Scholar 

  10. Shimazu, M., Mulchandani, A. & Chen, W. Thermally triggered purification and immobilization of elastin-OPH fusions. Biotechnol. Bioeng. 81, 74–79 (2003).

    Article  CAS  Google Scholar 

  11. Sun, X.L., Haller, C.A., Wu, X., Conticello, V.P. & Chaikof, E.L. One-pot glyco-affinity precipitation purification for enhanced proteomics: the flexible alignment of solution-phase capture/release and solid-phase separation. J. Proteome Res. 4, 2355–2359 (2005).

    Article  CAS  Google Scholar 

  12. Trabbic-Carlson, K., Liu, L., Kim, B. & Chilkoti, A. Expression and purification of recombinant proteins from Escherichia coli: comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Sci. 13, 3274–3284 (2004).

    Article  CAS  Google Scholar 

  13. Ford, C.F., Suominen, I. & Glatz, C.E. Fusion tails for the recovery and purification of recombinant proteins. Protein Expr. Purif. 2, 95–107 (1991).

    Article  CAS  Google Scholar 

  14. Ge, X. et al. Self-cleavable stimulus responsive tags for protein purification without chromatography. J. Am. Chem. Soc. 127, 11228–11229 (2005).

    Article  CAS  Google Scholar 

  15. Wood, D.W., Wu, W., Belfort, G., Derbyshire, V. & Belfort, M. A genetic system yields self-cleaving inteins for bioseparations. Nat. Biotechnol. 17, 889–892 (1999).

    Article  CAS  Google Scholar 

  16. Wood, D.W. et al. Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor. Biotechnol. Prog. 16, 1055–1063 (2000).

    Article  CAS  Google Scholar 

  17. Chong, S. et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271–281 (1997).

    Article  CAS  Google Scholar 

  18. Paulus, H. Protein splicing and related forms of protein autoprocessing. Annu. Rev. Biochem. 69, 447–496 (2000).

    Article  CAS  Google Scholar 

  19. Banki, M.R. & Wood, D.W. Inteins and affinity resin substitutes for protein purification and scale up. Microb. Cell Fact. 4, 32 (2005).

    Article  Google Scholar 

  20. Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.Q. & Perler, F.B. Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques 27 110–114, 116, 118–120 (1999).

    Article  CAS  Google Scholar 

  21. Sharma, S.S., Chong, S.R. & Harcum, S.W. Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system. Appl. Biochem. Biotechnol. 126, 93–117 (2005).

    Article  CAS  Google Scholar 

  22. Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523–533 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Michael Perl for help in troubleshooting advice. This work was supported by US Army Research Office grant W911NF-04-1-0056, and was partially supported by an NSF graduate research fellowship.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to David W Wood.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, WY., Mee, C., Califano, F. et al. Recombinant protein purification by self-cleaving aggregation tag. Nat Protoc 1, 2257–2262 (2006). https://doi.org/10.1038/nprot.2006.314

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.314

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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