Recombinant protein purification by self-cleaving aggregation tag.
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.
Wu
Wan-Yi
WY
Department of Chemical Engineering, A-217 Engineering Quadrangle, Princeton University, New Jersey 08544-5263, USA.
Mee
Courtney
C
Califano
Filomena
F
Banki
Reza
R
Wood
David W
DW
D004926:Escherichia coli / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000302:isolation & purification; D047668:Inteins; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification*
D010455:Peptides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins