Prokaryotic Expression And Activity Study Of NS2B-NS3 Protease Complex Of ZIKA Virus

ObjectiveTo obtain protease complex containing the NS2B cofactor region(NS2BH)and the NS3 protease domain(NS3pro)of ZIKA virus by prokaryotic genetic engineering and to investigate the protease activity of the recombinant protein for further research on screening ZIKA virus inhibitors and establish the process of expression and purification.Methods1.Using overlapping PCR to amplify NS2BH and NS3pro.The PCR products were inserted into the pET-28b vectors to construct prokaryotic expression system that express linked active NS2BH-NS3pro complex.In the linked protease termed'NS28H-G4TG4-NS3pro' in this study NS2B and NS3pro are covalently linked via a Gly4ThrGly4 linker.2.The plasmid insert was confirmed by DNA sequencing.Recombinant pET-28b-NS2BH-G4TG4-NS3pro was individually transformed into E.coli BL21 to induce protein expression with IPTG.The recombinant protein was purified by Ni-NTA affinity chromatography.3.A Matrix-assisted laser desorption ionization/time of flight mass spectrometry(MALDI-TOF MS)assay was used to identify molecular structure of NS2BH-G4TG4-NS3pro,and their peptide mass fingerprintings were compared to Swissprot database.4.The protease activitiy of NS2BH-G4TG4-NS3pro was measured using a fluorogeneic substrate Bz-Nle-Lys-Arg-Arg-AMC,based on fluorescence resonance transfer technique.Results1.Sequence result indicated that the plasmids encoding genes of NS2BH and NS3 protease domain were successfully inserted into pET-28b and the sequence were consistent with that in GeneBank.High yields of soluble protein was obtained through expression.After the purification of Ni-NTA resin,SDS-PAGE analysis showed that the relative molecular masses of NS2BH-G4TG4-NS3pro and NS2BH/NS3pro was closed to the expected protein achieved;2.MALDI-TOF MS assay indicated that the NS2BH-G4TG4-NS3pro match to genome polyprotein of ZIKA virus and the PMF confirmed the recombinant protein was the desired protease complex indeed.3.Enzymatic activity study demonstrated that the NS2BH-G4TG4-NS3pro have the ability to catalyze the substrate emitting a strong fluorescent signal while the fluorescence readings of the three control were low.Conclusion1.The NS2BH-G4TG4-NS3pro target sequence was amplified successfully by overlapping PCR.2.The prokaryotic expression system that express linked active NS2BH-NS3 protease complex were constructed successfully by genetic engineering technology.High yields of soluble protein could be obtained through expression.3.The NS2BH-G4TG4-NS3pro protease complex exhibited high protease activity and laying the foundation for further research on screening ZIKA virus inhibitors.