The Effect Of Inhibiting Hydrolysis Of Endogenous Protease On The Accumulation Of Recombinant RPA In Plants

Plant bioreactors are considered as the most economical and safety natural bioreactors.The plant virus expression system is a new platform which has great potential,using plants as a reactor to produce foreign proteins.The endogenous protease hydrolyzes the exogenous recombinant protein intracellularly,resulting in a low accumulation of the products,which is the "bottleneck"of the platform towards commercialization.The predecessor studies have shown that specific protective tags can increase the accumulation of exogenous recombinant fusion proteins in the cell,and co-expression protease inhibitors(PIs)can protect recombinant proteins in situ.Based on this,we regard rPA as the research object and start the research from the aspects of protective labeling and co-expression of PIs.By constructing viral expression vectors,which fused with different types of protective tags with rPA,the research is focus on analyzing the expression of recombinant fusion proteins in inoculated leaves and exploring the effect of protective tags on the accumulation of recombinant proteins in plants.Through constructing a protease inhibitor plant binary expression vector and co-inoculating it with a viral expression vector,the thesis can explore the effects of co-expressing protease inhibitors on the accumulation of recombinant proteins in plants.The main findings and conclusions are as follows:(1)The HFB-tag fusion expression strategy is failed to detect stable expression in the leaves of the recipient plant which named N.benthamiana.It is speculated that the role of HFB tags in the expression of foreign genes may be affected the types of foreign genes that fused with them,consider that there may have "incompatibility" between the HFB tags and rPA.(2)The ELP tag did not effectively increase and protect the intracellular accumulation of the recombinant rPA which fused with it.Considering that the length of the ELP tag has a certain degree of correlation with the protective effect of its fusion protein,it is speculated that the ELP30 tag that used in the experiment may not be the optimal tag "companion" for recombinant rPA,and the suitable ELP tag about rPA needs further experimental screening.(3)The Zera tag increased the expression of rPA protein which fused with it in a certain extent.Because it needs to add a reducing agent during the extraction process to transform from insoluble particles to soluble form,it is recommended that it should be treated cautious in practical applications.(4)In a co-expression system composed of non-replicating viral vectors with PIs,the three tested PIs(SlCYS8,NbPR4 and HsTIMP),all had significant competitive inhibitory effects on the accumulation of recombinant rPA expressed in the endoplasmic reticulum or extracellular space.The competitive inhibitory effect of co-expression of PIs is proved normally.The degree of competitive inhibition of co-expression of PIs are of high level.It is speculated that competitive inhibition masks the in situ protection of exogenous recombinant proteins by PIs,causing the observed accumulation of recombinant proteins to be lower than their normal accumulation level.(5)The competitive inhibitory effect of co-expression of PIs is directly related to the abundance of PIs.The competitive inhibitory effect weakens when the abundance of PIs decreases.By changing the inoculation ratio of PIs in the co-inoculation mixed bacterial solution in vitro,the degree of competition inhibition effect can be alleviated in a certain extent,to improve the accumulation of co-expressed exogenous recombinant protein.(6)In the co-expression system consisting of replicating virus with PIs,the accumulation of recombinant rPA was not significantly inhibited.Meanwhile,the accumulation of recombinant rPA in the endoplasmic reticulum showed a positive correlation with the concentration of PIs inoculation,indicating that virus replication Down-regulating the relative abundance of PIs in vivo,can effectively weaken the competitive inhibition effect The accumulation of exogenous recombinant proteins of the three PIs increased by 3 to 12-folds,indicating that the co-expressing replicative viral vector strategy is worth,and also have practical application potential,it provides new ideas to make people use the PIs reasonably.The relevant research results provide a reference for solving the key common problems behind the "bottleneck" faced by the commercialization process of plant virus expression platforms,it also has the theoretical and practical foundation for promoting the commercialization process of plant virus expression systems.