Purification and characterization of the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K12.

T. J. Larson, S. Z. Ye, D. L. Weissenborn, H. J. Hoffmann, H. Schweizer

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42 Scopus citations

Abstract

The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda. This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene. The repressor was purified 40-fold to homogeneity from an induced strain. The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography. Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor. The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions. Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis. Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM. Interaction of the repressor with DNA was studied using band-shift electrophoresis. The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes. Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate.

Original languageEnglish (US)
Pages (from-to)15869-15874
Number of pages6
JournalJournal of Biological Chemistry
Volume262
Issue number33
StatePublished - Nov 25 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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