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Recombinant protein purification by self-cleaving aggregation tag

Nature Protocols volume 1, pages 2257–2262 (2006)Cite this article

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.

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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.

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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.

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Authors and Affiliations

  1. Department of Chemical Engineering, A-217 Engineering Quadrangle, Princeton University, New Jersey, 08544-5263, USA

    Wan-Yi Wu, Courtney Mee, Reza Banki & David W Wood

  2. Department of Chemistry and Physics, St Francis College, 180 Remsen Street, Brooklyn Heights, 11201, New York, USA

    Filomena Califano

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  1. Wan-Yi Wu
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  4. Reza Banki
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Correspondence to David W Wood.

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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

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