Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12.
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.
Scarabel
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Institut de Biologie et Chimie des Protéines, C.N.R.S., Lyon, France.
Penin
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Bonod-Bidaud
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Nègre
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D
Cozzone
A J
AJ
Cortay
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JC
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D056968:Lac Repressors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005978:Glutathione; D005982:Glutathione Transferase; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014357:Trypsin; C489416:FruR protein, E coli; C068837:FruR protein, Bacteria; D056968:Lac Repressors