PREPARATION AND PROPERTIES OF PURE, FULL-LENGTH ICLR PROTEIN OF ESCHERICHIA-COLI - USE OF TIME-OF-FLIGHT MASS-SPECTROMETRY TO INVESTIGATE THE PROBLEMS ENCOUNTERED

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 ma de on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is tha t predicted from the gene sequence, except that the protein has suffer ed truncation by removal of the N-terminal eight or, in some cases, ni ne amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coil protease OmpT We f ind that, by taking precautions to minimize OmpT proteolysis, or by el iminating 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 truncat ed versions: it binds the aceBAK operator sequence 44-fold more tightl y, presumably because of additional contacts that the N-terminal resid ues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins pr oduced from cloned genes, especially where proteolysis or other covale nt modification is a concern. This technique gives mass spectra from c omplex peptide mixtures that can be analyzed completely, without any f ractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene.