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Protein expression and purification in Escherichia coli

NF Natalia Fili
YH Yukti Hari-Gupta
ÁS Ália dos Santos
AC Alexander Cook
SP Simon Poland
SA Simon M. Ameer-Beg
MP Maddy Parsons
CT Christopher P. Toseland
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Recombinant constructs were expressed in E.coli BL21 DE3 cells (Invitrogen) in Luria Bertani media. Proteins were purified by affinity chromatography (HisTrap FF, GE Healthcare). The purest fractions were desalted through a PD10 column (GE Healthcare) to remove imidazole before treatment with TEV protease for 4 h at 25 °C. The samples were then passed through a second HisTrap column. The cleaved protein was further purified through a Superdex 200 16/600 column (GE Healthcare). Note: TEV cleavage was not performed when the His-tag was needed for pull-down experiments.

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