Co-expression With Replicating Vector Overcoming Competitive Effects Derived by a Companion Protease Inhibitor in Plants.

Jiexue Ma,Zhiying Li,Sheng Wang,Xiangzhen Ding

doi:10.3389/fpls.2021.699442

Abstract

Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the in situ protective effects of a companion PI. Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.

Highlights

In recent years, plants are gradually regarded as a viable alternative for producing recombinant proteins (RPs; Shanmugaraj et al, 2020), due to their potential for low energy requirement, reduced animal pathogen contamination risks, and post-translational modifications (Schillberg and Finnern, 2021)
We explored the competitive effects behind the protease inhibitor (PI)-RP system by co-expressing rPA with those unrelated PIs in N. benthamiana leaves
RPA expressed by E. coli, positive control, showed a major band with a position corresponding to 39.0 kDa

Summary

Introduction

Plants are gradually regarded as a viable alternative for producing recombinant proteins (RPs; Shanmugaraj et al, 2020), due to their potential for low energy requirement, reduced animal pathogen contamination risks, and post-translational modifications (Schillberg and Finnern, 2021). The RPs can be essentially produced in plants by either stable transformation or transient expression (Gils et al, 2005). The latter approach is more efficient in terms of time consumption as well as batch processing (Kopertekh and Schiemann, 2019). Various RPs have been expressed transiently in plants in recent years, including proteins for therapeutic, diagnostic, research, and industrial applications. Several plant-derived RPs have reached or are close to the market (Fox, 2012; Schillberg and Finnern, 2021), but the relatively low yields of RPs limited more of them to be commercialized (Zischewski et al, 2016; Schillberg and Finnern, 2021)

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Conclusion