Purification and on-column refolding of EGFP overexpressed as inclusion bodies in Escherichia coli with expanded bed anion exchange chromatography.
The enhanced green fluorescent protein (EGFP) was over-expressed in Escherichia coli as inclusion bodies to increase its quantity and to facilitate its purification. Insoluble EGFP has been purified on Q Hyper Z matrix by expanded bed adsorption after solubilization in 8 M urea. The adsorption was made in expanded bed mode to avoid centrifugation. EBA-column refolding was done by elimination of urea and elution with NaCl. The EGFP was obtained as a highly purified soluble form with similar behavior in fluorescence and electrophoresis as native EGFP.
Cabanne
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C
Ecole Supérieure de Technologie des Biomolécules de Bordeaux (ESTBB), Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. charlotte.cabanne@estbb.u-bordeaux.fr
Noubhani
A M
AM
Hocquellet
A
A
Dole
F
F
Dieryck
W
W
Santarelli
X
X
D000327:Adsorption; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014508:Urea
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014508:Urea