Purification and characterization of the wild-type and mutant carboxy-terminal domains of the Escherichia coli Tar chemoreceptor.
The carboxy-terminal half of the Escherichia coli Tar chemoreceptor protein was cloned into an overproducing plasmid with the transcription of the insert under the control of the strong hybrid tac promoter. Two dominant mutations in the tar gene, which result in "tumble-only" (tar-526) or "swim-only" (tar-529) phenotypes and which are postulated to produce proteins locked in specific signalling modes, were introduced separately onto the overproducing plasmid. After induction with isopropyl-beta-D-thiogalactopyranoside, cells containing the plasmids produced about 10% of their soluble cellular protein as the carboxy-terminal fragments. A scheme to purify the overproduced fragments was developed. Typical yields of pure fragment were 5, 30, and 20 mg per liter of induced culture for the wild type, 526 mutant, and 529 mutant, respectively. Fast-protein liquid chromatography-gel filtration analysis of the pure fragments showed that they all existed as oligomers (ca. 103,000 daltons), possibly trimers or tetramers (monomer size is 31,000 daltons). However, the 529 mutant fragment showed an additional oligomeric form (240,000 daltons) corresponding approximately to an octamer. When chromatographed in the presence of 1% octylglucoside, all three fragments showed an identical single oligomeric size of about 135,000 daltons. Further differences between the fragments such as ion-exchange behavior and susceptibility to degradation were found. Taken together, these results suggest that conformational differences between the 529 mutant fragment and the other fragments exist and that these differences may correlate with the phenotypic effects of the tar-529 mutation.
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D002628:Chemoreceptor Cells; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli