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Preparation and properties of pure, full‐length Ic1R protein of escherichia coli. Use of time‐of‐flight mass spectrometry to investigate the problems encountered
Author(s) -
Donald Lynda J.,
Chernushevich Igor V.,
Zhou Jie,
Verentchikov Anatoli,
PoppeSchriemer Nancy,
Hosfield David J.,
Westmore John B.,
Ens Werner,
Duckworth Harry W.,
Standing Kenneth G.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560050815
Subject(s) - chemistry , cyanogen bromide , escherichia coli , peptide sequence , mass spectrometry , trypsin , biochemistry , amino acid , peptide , chromatography , peptide mass fingerprinting , protein mass spectrometry , electrospray ionization , gene , enzyme , proteomics
IclR protein, the repressor of the ace BAK 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 icl R 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 OmpT 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 ace BAK 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.