ABSTRACT
Isoforms of the natural and recombinant nucleases of Serratia marcescens were characterized by their molecular mass, which was determined by electrospray mass spectrometry. The natural nuclease was isolated from the S. marcescens B10M1 culture, whereas the recombinant nuclease was obtained from Escherichia coli MT102 cells carrying plasmid p403-SD2 with the nuclease gene nuc. The primary structure for each of the isoforms isolated from the nuclease preparations was determined by comparing its molecular mass with that of known amino acid sequence, which was determined from the nucleotide sequence of the nuc gene. Both preparations included identical nuclease variants with N-terminal amino acid residues removed. The number of isoforms in the natural nuclease was, however, significantly greater than in the recombinant nuclease. The structures of some of the isoforms were confirmed by N-terminal analysis.
Subject(s)
Endodeoxyribonucleases/chemistry , Endoribonucleases/chemistry , Mass Spectrometry/methods , Chromatography, DEAE-Cellulose , Electrophoresis, Polyacrylamide Gel , Endodeoxyribonucleases/genetics , Endoribonucleases/genetics , Escherichia coli/genetics , Isoelectric Focusing , Molecular Weight , Plasmids , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
The primary structures of nucleases Sm1, Sm2, and Sm3 produced by Serratia marcescens were completely characterized using plasma desorption mass spectrometry (PDMS) of proteolytic peptides isolated by reverse-phase HPLC. The isoforms were separated by anion-exchange chromatography on DEAE cellulose and subjected to hydrolysis by the lysine-specific endoproteinase Lys-C. Comparative analysis of the peptides identified by PDMS showed that all three nucleases are N-terminal variants of the same protein: Sm2 represents a "mature" protein form, whereas Sm1 and Sm3 lack three and one N-terminal amino acid residues, respectively.
