Recombinant protein purification by self-cleaving aggregation tag

Nature Protocols
Wan-Yi WuD W Wood

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 degrees 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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Citations

May 8, 2010·Applied Microbiology and Biotechnology·Skander Elleuche, Stefanie Pöggeler
Jan 27, 2011·Biotechnology Letters·Yifeng Li
Jul 4, 2012·The Journal of Biological Chemistry·Patricia Carvajal-VallejosStefan R Schmidt
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Aug 19, 2011·Journal of Controlled Release : Official Journal of the Controlled Release Society·Yizhe ChenDarin Y Furgeson
Jun 28, 2011·Journal of Controlled Release : Official Journal of the Controlled Release Society·Guoyong SunJ Andrew MacKay
Apr 3, 2010·Trends in Biotechnology·Baley A FongDavid W Wood
Aug 20, 2015·Applied Biochemistry and Biotechnology·Daniel DemonteSheldon Park
Apr 10, 2013·Biotechnology and Bioengineering·Dongli GuanZhilei Chen
Sep 17, 2013·Protein Science : a Publication of the Protein Society·Mark R BellJames E Strickler
Jun 1, 2010·Protein Science : a Publication of the Protein Society·Baley A FongDavid W Wood
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Aug 8, 2013·Protein Expression and Purification·Tianyang MuXuexun Fang
Jan 15, 2014·Protein Expression and Purification·Jie ZhangShuang Quan Zhang
May 30, 2013·Journal of Controlled Release : Official Journal of the Controlled Release Society·Pu ShiJ Andrew MacKay
Dec 21, 2010·Protein Expression and Purification·Wan-Yi WuDavid W Wood
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May 26, 2018·Journal of Agricultural and Food Chemistry·Herui GaoYingfeng An
Sep 9, 2019·ACS Biomaterials Science & Engineering·Sergio AcostaJosé Carlos Rodríguez-Cabello

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