Purification and characterization of pro-TraTp, the signal sequence-containing precursor of a secreted protein encoded by the F sex factor.
The structural gene for the F sex factor outer membrane surface exclusion protein ( traT ) was cloned onto a high-copy-number plasmid where it is expressed from the phage lambda promoter pL. Conditional control over expression was provided by a temperature-sensitive lambda cI repressor. Induction of pL produced large quantities of the traT gene product ( TraTp ) and, in rich growth media, even larger amounts of a higher-molecular-weight form of TraTp . This polypeptide was purified and characterized as a pro- TraTp precursor, which contains at its amino terminus a typical signal-like sequence, which is not present in the mature form of TraTp as isolated from the outer membrane of F-containing cells. Accumulation of pro- TraTp seemed not to result from the jamming of export sites, as in another system for obtaining precursors of secreted proteins, but rather from overwhelming kinetically the ability of the cell to process exported proteins. Although pro- TraTp appeared to be successfully translocated to the outer membrane, it was defective in forming the oligomeric structure required for surface exclusion function. The procedure used is not a general method but can be applied to certain other secreted proteins.
D001425:Bacterial Outer Membrane Proteins; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005144:F Factor; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000032:analysis*; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; C036297:TraT protein, E coli