Overproduction in Escherichia coli K-12 and purification of the TraJ protein encoded by the conjugative plasmid F.
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.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D003090:Coliphages / Q000235:genetics; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005144:F Factor; D008242:Lysogeny; D009154:Mutation; D014176:Protein Biosynthesis; D013045:Species Specificity
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C488999:TraJ protein, E coli; C041427:TraJ protein, bacteria; D004262:DNA Restriction Enzymes