title: [Expression, purification and characterization of non-specific Serratia nuclease in Escherichia coli].
abstract: To efficiently produce non-specific nuclease (NU) of Serratia marcescens through recombinant overexpression approach and to characterize the purified NU. The nuclease gene was amplified from the genomic DNA of Serratia marcescens by PCR and fused into vector pMAL-c4X with maltose binding protein (MBP) tag. The recombinant vector verified by DNA sequencing was transformed into Escherichia coli BL21. The expressed MBP-NU was purified through the amylose resin and its catalytic characters were analyzed. The results showed the NU gene had 97% identities with the reported S. marcescens nuclease gene and intracellularly expressed in E. coli BL21. The optimal expression conditions were 37 degrees C, 0.75 mmol/L IPTG with 1.5 h induction. The purified MBP-NU exhibited non-specific nuclease activity, able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Its optimal temperature was 37 degrees C and optimal pH 8.0. From 1 L culture broth 10.8 mg NU could be purified with a specific activity of 1.11x10(6) U/mg. The catalytic activity of NU was not inhibited by reagents such as EDTA (0.5 mmol/L), PMSF (1 mmol/L) and KCl (150 mmol/L) commonly used in protein purification.
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling 712100, China. pengchen@nwsuaf.edu.cn
authors.forename: Peng
authors.initials: P
authors.lastname: Chen
authors.forename: Haiyan
authors.initials: H
authors.lastname: Yang
authors.forename: Huijing
authors.initials: H
authors.lastname: Li
authors.forename: Longyu
authors.initials: L
authors.lastname: Yang
authors.forename: Xuejun
authors.initials: X
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D004706:Endodeoxyribonucleases / Q000096:biosynthesis* / Q000235:genetics; D004722:Endoribonucleases / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012706:Serratia marcescens / Q000201:enzymology*
chemical_list: D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease