proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves蛋白质组学描述烟叶的内质reticulum-derived蛋白质的身体.pdfVIP

Joseph et al. BMC Plant Biology 2012, 12:36 /1471-2229/12/36 RESEARCH ARTICLE Open Access Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves 2 2 2 1 1 1 Minu Joseph , M Dolors Ludevid , Margarita Torrent , Valérie Rofidal , Marc Tauzin , Michel Rossignol and Jean-Benoit Peltier 1* Abstract Background: 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. 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. Results: 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 tr