Characterization of Serratia marcescens nuclease isoforms by plasma desorption mass spectrometry.
Isoforms of Serratia marcescens nuclease found in the natural nuclease produced by S. marcescens and in recombinant nuclease produced by Escherichia coli were structurally characterized by peptide mapping using plasma desorption mass spectrometry. The nuclease isoforms produced and secreted from S. marcescens B10M1, which are present in much greater amounts than in S. marcescens W225 nuclease produced by E. coli, were characterized completely and the information used to facilitate characterization of the recombinant nuclease isoforms. After purification of the nuclease the isoforms were separated on a DEAE-cellulose anion-exchange column and then digested with endoproteinase Lys-C. The peptides generated were isolated by reverse-phase HPLC and their molecular masses determined by plasma desorption mass spectrometry. Comparison of the peptides from the native nuclease, Sm2, and the two isoforms, Sm1 and Sm3, revealed that they differed only in the N-terminus, the latter being found to lack three amino acids in Sm1 and one amino acid in Sm3. No interior post-translational changes were found in either of the three isoforms. Using this information we were able to confirm that Sm1, the isoform lacking three amino acids, was also present in very small amounts in recombinant S. marcescens W225 nuclease produced and excreted by E. coli.
Department of Biotechnology, Technical University of Denmark, Lyngby.
J
J
Pedersen
M
M
Filimonova
P
P
Roepstorff
K
K
Biedermann
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000302:isolation & purification; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012706:Serratia marcescens / Q000201:enzymology*
D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase