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Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12
Ist Teil von
Gene, 1995-02, Vol.153 (1), p.9-15
Ort / Verlag
Netherlands: Elsevier B.V
Erscheinungsjahr
1995
Link zum Volltext
Quelle
Access via ScienceDirect (Elsevier)
Beschreibungen/Notizen
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 chomatography 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.