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High-yield production and purification of recombinant T7-tag mature streptavidin in glucose-stressed E. coli

Auteur(s)
Humbert, N.
Schurmann, P.
Zocchi, A.
Ward, T. R.
Date de parution
2008
In
Methods Mol Biol
No
418
De la page
101
A la page
10
Mots-clés
Résumé
The overexpression of toxic recombinant proteins is often problematic, leading to either low production levels or inclusion bodies. Streptavidin is no exception and thus the highest production level reported to date for streptavidin is 70 mg/L of functional protein. Herein, we report on the production in Escherichia coli and the purification of a recombinant mature streptavidin bearing a T7-tag. Optimization of critical parameters, including the glucose concentration, the pH and the time of induction as well as the use of BL21(DE3)pLysS cell strain, affords up to 120 mg/L functional streptavidin in soluble form. The yield can be further increased by an osmotic stress during the preculture by adding highly concentrated glucose before the inoculation of the culture medium, thus affording reproducibly 230 mg/L of soluble streptavidin. A single denaturing-renaturing step and affinity chromatography afford highly active tetrameric protein with >3.8/4.0 active sites.
Identifiants
https://libra.unine.ch/handle/123456789/22639
Type de publication
journal article