Abstract
The TraJ protein encoded by the conjugative plasmid F has been designated an Escherichia coli K-12 envelope protein that participates in a mechanism of gene regulation. We have purified the TraJ protein, using an Flac::lambda traJ lysogen that overproduces the protein after heat induction of the prophage. Sufficient TraJ protein was synthesized within 40 min after induction to follow the purification by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Our purification exploited the observation that the TraJ protein remains insoluble after repeated Triton X-100/EDTA extractions of crude envelope fractions. The protein was then solubilized in sodium dodecyl sulfate at 60 degrees C and fractionated further by gel filtration and hydroxylapatite chromatography, both in the presence of sodium dodecyl sulfate. After hydroxylapatite chromatography, the protein was greater than 95% pure. The identity of the purified protein was confirmed by analysis of its NH2-terminal amino acid sequence, which was the same as that predicted from the partial nucleotide sequence of the traJ gene (Thompson, R., and Taylor, L. (1982) Mol. Gen. Genet. 188, 513-518). This analysis also indicated that the TraJ protein is localized in the cell without proteolytic modification of its NH2-terminus. We discuss the possible significance of these observations with respect to the cellular functions of the TraJ protein.
Highlights
From the Department of Molecular Biology, Division of Biological Sciences, AlbertEinstein College of Medicine, Bronx, New York 10461
The TraJ protein encoded by the conjugative plasmid F has been designated an Escherichia coli K-12 envelope protein that participates in a mechanism of gene regulation
Sufficient TraJ protein was synthesized within40 min after induction to follow the purification by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis
Summary
JYALEKB phage EMXl was maintained as alysogen by homologous recombination with the tra region of JCFLO in EM1500. Phage were prepared from EM1501 (EmX1) and EM1590 (EMX1b)y induction ofthe prophage at 42 "C Singleplaques from these lysates were tested for their ability to complement traAl[Am] and traJ90[Am] by infection complementation of EM1501 and EM1590, as described above. The pellet fraction from the second Triton/EDTA extraction was suspended in 5 ml of 10 mM Tris-HCI buffer, pH 7.2, containing 2% SDS, 5% 2-mercaptoethanol, 10% (v/ v) glycerol; heated at 60"Cfor30min [25];and fractionated by sedimentation for 1 h at 100,000 X g and 25 "C. Isolation of Radiolabeled TraJ Protein for NH2-terminal Sequencing-A 5-ml culture of EMX3 lysogen waslabeled between 10 and min after a temperature shift to "C. Radioactivity was determined by liquid scintillation spectrometry after addition of 5 ml of Aquasol to each sample.Otherwise, theanilinothiazolinonederivatives were
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