Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12.
The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.
Wee
S
S
Department of Biochemistry, University of California, Berkeley 94720.
Neilands
J B
JB
Bittner
M L
ML
Hemming
B C
BC
Haymore
B L
BL
Seetharam
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007501:Iron