The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Abstract

BackgroundXylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs).Methodology/Principal FindingsTobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase.Conclusion/SignificanceIn the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.

Molecular farming on rescue of pharma industry for next generations

“…The advantage of using such a domain is that it forms protein aggregates that can accumulate in the ER. Zera-fused approach was recently used to purify Xylanase enzyme from transgenic tobacco (Llop-Tous et al, 2011). More simple and cost-effective approaches consist in the fusion of target molecules to oleosin proteins to enhance the accumulation of the target proteins onto the surface of the oil bodies.…”

Plant Molecular Farming: Much More than Medicines

“…The secretory pathway in seeds is ideal for molecular farming because it includes specialized storage organelles that allow proteins to accumulate and become encapsulated in a protective matrix (37,42,43). These storage organelles can even be induced ectopically in tissues that are not adapted for storage functions and provide a simple strategy for producing active insoluble enzyme polymers (44). For pharmaceutical applications, the inclusion of additional peptide tags changes the nature of the product and attracts additional regulatory scrutiny, but this is not an issue for nonpharmaceutical products and protein targeting can therefore be exploited fully to maximize yields and to accumulate a recombinant protein in a suitable compartment (Figure 1 Targeting strategies for the expression of recombinant proteins.…”

Plant biotechnology for lignocellulosic biofuel production

“…Subcellular targeting signals in ER, vacuole and chloroplast also had substantial impact on the accumulation of fungal cellobiohydrolase I (CBH1), CBH2, and bacterial endoglucanase (EG) in sugarcane (Harrison et al, 2011). Targeting products into Dai et al (2000aDai et al ( ,b, 2005 RbcS protein bodies could also result in high-level accumulation: for example, up to 9% of total protein for Thermobifida fusca xylanase cel6A (Llop-Tous et al, 2011). Many bacterial and fungal CWD enzymes were highly expressed when targeted to chloroplasts (Verma et al, 2010).…”