Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves.
Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves.
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DOI：
10.1186/1471-2229-12-36
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发表时间：
2012-03-16
影响因子：
5.3
通讯作者：
Peltier JB
中科院分区：
文献类型：
--
作者：
Joseph M;Ludevid MD;Torrent M;Rofidal V;Tauzin M;Rossignol M;Peltier JB
玉米贮藏蛋白γ-玉米醇溶蛋白的N-末端富含脯氨酸结构域（Zera）在与目的蛋白融合时能够诱导内质网（ER）衍生的蛋白体（PB）的形成。这种包封使得重组融合蛋白能够逃避降解，并通过梯度纯化促进其从植物生物质中回收。本工作的目的是评估诱导的PB是否与重组蛋白一起包封额外的蛋白质。PB的蛋白质组成的详尽分析，预计将有助于更好地了解PB的形成和优化重组蛋白的纯化方法，从这些细胞器。我们分析了本氏烟草叶片中诱导的PBs的蛋白质组与Zera融合的荧光标记蛋白（DsRed）的瞬时转化。在基于碘克沙醇的密度梯度上分离具有其周围ER膜的完整PB，并通过共聚焦和电子显微镜验证其完整性。分离的PB的SDS-PAGE分析表明，Zera-DsRed占PB蛋白质丰度的85%左右。进行PB的差异提取以深入分析其蛋白质组和结构。除了Zera-DsRed之外，还鉴定了195种其他蛋白质，包括通过ER驻留或运输并在Zera-DsRed聚合物内招募的广泛蛋白质。本研究表明，Zera融合蛋白仍是烟草叶片新形成的细胞器的主要蛋白组分。分析还揭示了PB中存在意想不到的蛋白质多样性，这些蛋白质来源于不溶性Zera-DsRed聚合物形成，包括ER驻留蛋白和分泌蛋白，以及最可能由重组蛋白超载诱导的分泌应激反应。PBs蛋白组成的知识可能有助于优化分子农业应用中重组蛋白的下游纯化。
The 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. We 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. This 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.
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影响因子：
56.9
作者：
LI, XX;WU, YJ;OKITA, TW
通讯作者：
OKITA, TW
影响因子：
3.8
作者：
Conley, Andrew J.;Joensuu, Jussi J.;Brandle, Jim E.
通讯作者：
Brandle, Jim E.
影响因子：
3.7
作者：
Christensen A;Svensson K;Thelin L;Zhang W;Tintor N;Prins D;Funke N;Michalak M;Schulze-Lefert P;Saijo Y;Sommarin M;Widell S;Persson S
通讯作者：
Persson S
影响因子：
5.1
作者：
Alvarez, M. Lucrecia;Topal, Emel;Cardineau, Guy A.
通讯作者：
Cardineau, Guy A.
影响因子：
7.4
作者：
Joensuu, Jussi J.;Conley, Andrew J.;Menassa, Rima
通讯作者：
Menassa, Rima