Cloning, Expression And Activity Etection Of Papaya Methionine Sulfoxide Reductase B1(PaMsrB1)

Papaya ringspot virus (PRSV), a member of the aphid-transmitted genus Potyvirus, is the most widespread and destructive virus disease affecting papaya cultivation worldwide. It was demonstrated that PRSV infection leads to the accumulation of ROS papaya in vivo, and the virus-encoded PRSV nuclear inclusion protein a protease (NIa-Pro) contains4Met residues, one of which is predicted to be particularly prone to oxidation into Met sulfoxide (MetSO). Plant methionine sulfoxide reductase B1(MsrB1) protects the photo synthetic apparatus from oxidative damage by scavenging ROS to repair Met-oxidized proteins. However the chloroplast-localized papaya PaMsrB1interacts with interacts with NIa-Pro, disturbing import of PaMsrB1into chloroplasts. These findings led us to hypothesize that PaMsrBl catalyzes the reduction of oxidized PRSV NIa-Pro by ROS, representing a novel mechanism of PRSV towards the host defense.In order to obtain biologically active recombinant proteins PaMsrB1, Pichia pastoris expression system and Escherichia coli expression system were used to express the recombinant protein, and detect its activity. In Pichia pastoris expression system, PaMsrBl gene was cloned using cDNA as the template that was synthesized by reverse transcription of total RNA of papaya leaves. It was transplanted to expression vector pGAPZa B, eukaryotic expression vector of pGAPZa-B1was constructed successfully. And recombinant protein PaMsrB1was highly expressed in Pichia pastoris GS115with a secreted form.500mL expression supernatant was purified by Ni2+-NTA agarose affinity chromatography, concentration of0.65mg/mL Pichia pastoris recombinant proteins PaMsrBl was obtained. SDS-PAGE and Western Blot analysis showed that specific bands appeared at22.7kDa and purified recombinant protein was expressed successfully.In Escherichia coli expression system, PaMsrBl gene was transplanted to expression vector pET28a(+), prokaryotic expression vector pET28-B1was constructed successfully. Then it was transformed into the expression strain BL21(DE3), at20℃,25℃,28℃,30℃,37℃with0.1mM of IPTG induced2h,4h,6h,8h and10h,respectively. The recombinant proteins PaMsrB1was highly expressed in inclusion body form and the optimal induced condition was0.1mM of IPTG induced2h at37℃.200mL culture was centrifuged to collect the cells, after ultrasonic fragmentation and denatured with8M urea, it was purified by Ni2+-NTA agarose affinity chromatography, and concentration of0.65mg/mL Escherichia coli recombinant proteins PaMsrB1was obtained. SDS-PAGE and Western Blot analysis showed that specific bands appeared at22.7kDa and purified recombinant protein was expressed successfully. To detect the biological activity of the recombinant protein PaMsrB1, a protection detection against H2O2-mediated stress showed that recombinant strain pGAPB1could resistant to2.5mM H2O2oxidative stress in vivo, and induced recombinant strain pETPB1could resistant to300mM H2O2oxidative stress in vivo, respectively. Dabsyl-MetSO was synthesized by chemical methods for thin layer chromatography assay and HPLC analysis, and the results indicated that recombinant proteins PaMsrB1had reductase activity that could reduce MetSO back to Met in vitro, which lay a foundation of further study on the mechanism of PaMsrB1antioxidant in papaya chloroplasts.