Abstract
BackgroundThe N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.ResultsWe analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.ConclusionsThis study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.
Highlights
The N-terminal proline-rich domain (Zera) of the maize storage protein g-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest
As expected, ZeraDsRed expression induced the formation of new organelles similar to that described for other Zera-derived fusions [17]
Immunoelectron microscopy of transformed leaf epidermal sections labeled with a polyclonal antibody against Zera detected protein bodies as electron dense structures embedded in the cytoplasm and surrounded by other organelles including mitochondria and chloroplasts (Figure 1c)
Summary
The N-terminal proline-rich domain (Zera) of the maize storage protein g-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. Several approaches have been used for the improvement of yields and stability of recombinant proteins in tissue expression and biopolymers They are composed of a repeating pentapeptide ‘VPGXG’ sequence [12] that accumulate in ERderived protein bodies, when fused to a target protein and can be isolated by ‘inverse transition cycling’ [13,14]. HFBI fusions were able to increase the accumulation of GFP in plants through the formation of novel protein bodies and purified by surfactant-based aqueous two-phase system
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