title: Preparation and properties of pure, full-length IclR protein of Escherichia coli. Use of time-of-flight mass spectrometry to investigate the problems encountered.
abstract: IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time-of-flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned iclR gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N-terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize Omp T proteolysis, or by eliminating it through mutation of the host strain, we can isolate full-length IclR protein (lacking only the N-terminal methionine residue). Full-length IclR is a much better DNA-binding protein than the truncated versions: it binds the aceBAK operator sequence 44-fold more tightly, presumably because of additional contacts that the N-terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Canada.
authors.forename: L J
authors.initials: LJ
authors.lastname: Donald
authors.forename: I V
authors.initials: IV
authors.lastname: Chernushevich
authors.forename: J
authors.initials: J
authors.lastname: Zhou
authors.forename: A
authors.initials: A
authors.lastname: Verentchikov
authors.forename: N
authors.initials: N
authors.lastname: Poppe-Schriemer
authors.forename: D J
authors.initials: DJ
authors.lastname: Hosfield
authors.forename: J B
authors.initials: JB
authors.lastname: Westmore
authors.forename: W
authors.initials: W
authors.lastname: Ens
authors.forename: H W
authors.initials: HW
authors.lastname: Duckworth
authors.forename: K G
authors.initials: KG
authors.lastname: Standing
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009876:Operon; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C067479:IclR protein, E coli; D014357:Trypsin