PMID:3316209
| Citation |
Larson, TJ, Ye, SZ, Weissenborn, DL, Hoffmann, HJ and Schweizer, H (1987) Purification and characterization of the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K12. J. Biol. Chem. 262:15869-74 |
|---|---|
| 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. |
| Links | |
| Keywords |
Escherichia coli/genetics; Escherichia coli/metabolism; Genes; Genes, Bacterial; Genes, Regulator; Glycerophosphates/metabolism; Plasmids; Protein Binding; Repressor Proteins/genetics; Repressor Proteins/isolation & purification; Repressor Proteins/metabolism; Transcription Factors/genetics |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<protect><annotationlinks/></protect>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
See also
References
See Help:References for how to manage references in EcoliWiki.
