Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Gene Année : 1995

Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12.

Résumé

A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase. The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin. After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis. Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase. The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin. After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis. Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.
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Dates et versions

hal-00313224 , version 1 (27-08-2008)

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  • HAL Id : hal-00313224 , version 1

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M. Scarabel, F. Penin, Christelle Bonod-Bidaud, D. Negre, Aj Cozzone, et al.. Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12.. Gene, 1995, 153, pp.9-15. ⟨hal-00313224⟩
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