Expression, Purification And Activity Analysis Of Nuclease Pi In Escherichia Coli And Pichia Pastoris

Nuclease P1(NP1, EC3.1.30.1) has nonspecifically cleaved single-stranded RNA andDNA into5'-mononucleotides(5'-AMP,5'-GMP,5'-CMP and5'-UMP), and5'-nucleotidesand derivates have been widely used in food and biomedical industry. Currently, low-purityNP1has been used in industry, and high-purity NP1has been purified by thermal deactivation,ultrafiltration, ion-exchange chromatography. The prokaryotic expression vectors andeukaryotic expression vectors had been constructed and recombinant NP1was expressed andpurified from Escherichia coli strains T7Express and Origami B(DE3) and Pichia pastorisstrain GS115. To further clarify the biological function of recombinant NP1, this papercarried out the following studies:1. We spliced22oligonucleotides to get a synthetic NP1(Nuclease P1) gene throughoverlapping PCR. Then five prokaryotic expression vectors had been constructed (pET-21a-MBP-NP1-His,pET-21a-NP1-His,pET-21a-NP1,pMAL-p4X-NP1and pMAL-p4X-pre-NP1).2. The recombinant vectors had been transformed into Escherichia coli host strains T7Express and Origami B(DE3). Then the recombinant proteins had been purified by Amyloseor Ni-NTA affinity chromatography. The results showed five recombinant NP1wereexpressed in soluble form from Origami B(DE3) strain. The specific activity of recombinantNP1from Origami B(DE3) strain was higher than T7Express strain (75.5U/mg:51.5U/mg).When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased upto258.1U/mg and139.2U/mg.3. The thermal inactivation experiment demonstrated that the recombinant NP1was quitestable, and it retained more than90%of original activity after incubated for30min at80℃.Zn2+, Co2+, Fe2+(2.0mmol/L) had different levels of activation while2.0mmol/L Mn2+, Ca2+,Ni2+, Mg2+, Cu2+had different levels of inhibition for recombinant NP1. Zn2+(2.0mmol/L)could increase enzyme activity (to119.1%), on the contrary, the enzyme activity was reducedby2.0mmol/L Cu2+(to63.1%).4. The encoding sequence of NP1was cloned into pPIC9k to obtain pPIC9k-NP1-His and pPIC9k-MF-pre-NP1. The recombinant vectors were linearized and electroporated intoPichia pastoris strain GS115competent cells. After histidine-dificient medium and G-418selection, PCR analysis and optimization of methanol inducing time,the high level expressionstrain of GS115/pPIC9k-MF-pre-NP1was obtained.5. The recombinant NP1was expressed by methanol inducing. Through activity analysisthe results revealed the expression level of recombinant protein had reached to top at72hour,inoculation amount OD600=2.0after inducing by0.5%methanol. The recombinant NP1wasobtained after thermal inactivation,40%(NH4)2SO4precipitation, dialysis and vacuumfreeze-drying. The activity of recombinant NP1was up to201.9U/mL and2.9-foldpurification with35.7%enzyme recovery.Our research lays the foundation for the application of NP1in food, biomedical industryand molecular biology research.