Expression, Purification And Activity Analysis Of Non-Specific Serratia Nuclease Overexpressed In Escherichia Coli

The non-specific serratia nuclease(NU) is one of the most important extracellular proteins in Serratia marcescens. The non-specific serratia nuclease(NU) can degrade all forms of DNA and RNA (single-stranded, double stranded, linear or circular). With its high intrinsic activity and broad substrate tolerance, the NU has a strong toxic effects to its host cell, resulting in the recombinant expression difficult. In this communication, we constructed both the secretion and intracellular prokaryotic expression vectors of the NU, and obtained the optimum expression condition and analyzed the characteristics of the catalytic activity of the NU.We obtained the following results:We tried to construct the secretion vectors pllp-OmpA-NU and pET22b-NU, but failed because of the trace amounts of its intracellular basal level expression.In the process of building the intracellular expression vector pMAL-c4X-NU, the NU coding sequence was successfully amplified from the genomic DNA of Serratia marcescens by PCR and sequenced. The NU sequence showed 97% identities compared with S.marcescens nuclease gene reported. The molecular weight of MBP-NU expressed in E.coli BL21 was about 78 kDa. The MBP-NU was purified through the amylose resin and its optimum expression condition were 37℃, 0.75 mmol/L IPTG with 1.5h induction. The purity of the purified MBP-NU was 93.8% by Bandscan.The characterization of the purified MBP-NU exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear, and showed that its optimal temperature and pH and the concentration of Mg2+ were 37℃and pH 8.0 and 5.0mmol/L Mg2+ respectively. The specific activity was 1.11×106 U/mg, and 10.875mg target protein can be purified from per liter. The MBP-NU was not the protease activity. The results indicated that the enzymatic activity was not inhibited by enzyme inhibitors such as EDTA (0.5mmol/L), PMSF (1mmol/L),β-mercaptoethanol (50 mmol/L) and KCl (150 mmol/L).The experiments showed that the MBP-NU have the same hydrolysis efficiency as Benzonase. In the recombinant expression and purification experiments of PPA, the internal standard DNA template can not be detected by PCR in 5 minutes, when the enzyme concentration was 20 U/mL.Through this study, we successfully built the efficient intracellular recombinant expression system of serratia nuclease, and its expression is much higher than the existing system of secreted. These results laid the foundation for using enzymatic degradation nucleic acid at low cost in the protein purification process.