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Biology of Metals

, Volume 1, Issue 1, pp 62–68 | Cite as

Expression, isolation and properties of Fur (ferric uptake regulation) protein ofEscherichia coli K 12

  • Sechan Wee
  • J. B. Neilands
  • Michael L. Bittner
  • Bruce C. Hemming
  • Barry L. Haymore
  • Ramnath Seetharam
Original Articles

Summary

The clonedfur (ferric uptake regulation) gene ofEscherichia 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 × 10−12 M. A much smaller value, 2.5 × 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.

Key words

Fur repressor Iron regulation 

Abbreviations

Bistris

2-[bis(2-hydroxyethyl)aminol-2-(hydroxymethyl)-propane-1,3-diol

DMSO

dimethylsulfoxide

EDTA

ethylenediaminetetraacetate

Hepes

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

kb

kilobase

kDa

kilodalton

LB

Luria broth

PMSF

phenylmethylsulfonyl fluoride

SDS-PAGE

sodium dodecyl sulfate/polyacrylamide gel electrophoresis

Tris

2-amino-2-hydroxymethylpropane1,3-diol

TEMED

tetramethylethylenediamine

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Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Sechan Wee
    • 1
  • J. B. Neilands
    • 1
  • Michael L. Bittner
    • 2
  • Bruce C. Hemming
    • 2
  • Barry L. Haymore
    • 2
  • Ramnath Seetharam
    • 2
  1. 1.Department of BiochemistryUniversity of CaliforniaBerkeleyUSA
  2. 2.Monsanto CompanySt. LouisUSA

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