Establishment Of Removal System For Non-specific Serratia Nuclease

Nucleic acid contamination has become a big problem during the process of expression and preparation of recombinant protein. Traditional methods for removal of nucleic acid are high cost and complicated while nuclease shows high efficiency and convenience. S. marcescens nucleases are widely used due to its high activity and strong stability. However, the arising problem is how to efficiently remove the trace amount nuclease left in the reaction system. In the research, based on the high affinity between Strep tag II and streptavidin, the systems for expressing S. marcescens nucleases fused with Strep tag II and streptavidin were established and the highly efficient removal system was established based on the immobili zed streptavidin mutant. The results are as follow:The primers were designed based on the sequence of S. marcescens nuclease published in GenBank and the sequence of Strep tag II was fused into the forward primer. The nuclease gene was amplified by PCR and ligated into expression vector pLLP-OmpA. The verified recombinant vector pLLP-OmpA+snu was transformed into Escherichia coli BL21 and induced by IPTG, and the recombinant nuclease was overexpressed in the form of inclusion body. The purified inclusion body was dissolved in 8 M urea and renatured by gradient dialysis. The ratio of purity of renatured protein from inclusion body was approximate 60%. The renatured nuclease was further purified through DEAE- Sepharose and the final yield of the fusion protein was 18.695 mg/L.Catalytic characteristics analysis results of S. marcescens fused with Strep tag II showed optimal temperature of recombinant nuclease was 37 ℃ and optimal pH was 8.0 and its specific activity was 1.53 ×105 U/mg. The catalytic activity was not inhibited by urea of 500 mmol/L while was inhibited by EDTA. Mg2+ was necessary for the catalytic activity of the nuclease while inhibited by over 50 mmol/L MgCl2. Evaluation on the effects of differe nt bivalent metal ic cations on catalytic activities showed that the activity was strongly enhanced by Mn2+, Mg2+ and lightly enhanced by Co2+, Ni2+ and Fe2+ while not by Zn2+, Cu2+ and Ca2+.The streptavidin gene was synthetized and ligated into expression vector pET-28 a. By Quikchange site-directed mutagenesis three amino acids were mutated and the three mutants separately were E44V、S45T、V47R. The mutational recombinant vector was named pET28 aStM which was transformed into Escherichia coli BL21(DE3). The streptavidin mutant was overexpressed in the form of inclusion body. The purified streptavidin mutant was obtained by the purification, dissolution, renaturation. The streptavidin mutant was then coupled to Sepharose CL 6B and the effect of removal system was verified with the streptavidin mutant chromatographic column and S. marcescens nuclease fused with Strep tag II. The result showed that 10 μg recombinant nuclease were completely removed by 3 mL streptavidin mutantSepharose CL 6B. Finally the removal system for S. marcescens nuclease has been completed and provides the foundation for the practice application of S. marcescens nuclease.