The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

Virgilio, Maddalena de; Marchis, Francesca De; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

doi:10.1093/jxb/ern143

Cited by 56 publications

(77 citation statements)

References 51 publications

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“…These results taken together demonstrated that the formation of PBs-like was essential to obtain a 59 higher F1-V accumulation in N. benthamiana leaves. We confirmed the feasibility of using Zera Ò in Nicotiana benthamiana leaves as was previously reported for stable transformed Arabidopsis thaliana (Geli et al 1994) and Nicotiana tabacum leaves (Virgilio et al 2008).…”

Section: Discussionsupporting

confidence: 89%

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

et al. 2009

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Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

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Section: Discussionsupporting

confidence: 89%

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

et al. 2009

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“…These results seem to suggest the existence of a translational negative regulation against zeolin(Cys-) and Dzeolin proteins. The inability to detect Dzeolin is difficult to explain because unstable proteins should be detectable at least at 0-h chase, as shown for ER-localized proteins (de Virgilio et al 2008). One possible explanation is that Dzeolin mRNA translation is very inefficient because of aberrant RNA folding.…”

Section: Production Of Tobacco Transplastomic Plants Expressing Threementioning

confidence: 99%

“…In case of positive results, the next step would be to fuse zeolin with a protein of interest and express this new fusion protein in the chloroplast. This strategy was successfully used in the ER of tobacco leaves to accumulate the human immunodeficiency virus negative factor (Nef) protein, where Nefcontaining PBs were formed by fusing the Nef sequence to zeolin (de Virgilio et al 2008). Zeolin accumulation in the chloroplast upon plastid transformation was on the contrary unsatisfactory, as no zeolin PBs were detected and higher degradation of the recombinant protein was observed with respect to the form expressed in the ER (Bellucci et al 2007).…”

Section: Introductionmentioning

confidence: 99%

A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane

et al. 2010

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Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

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“…However, the high number of HIV-1 subtypes and several other factors complicate the design of a successful vaccine (Young et al 2006;Walker and Burton 2008). For such a purpose, various HIV proteins have been expressed in plant cells using either transient or stable transformation (Zhang et al 2000(Zhang et al , 2002Pérez-Filgueira et al 2004;Karasev et al 2005;Obregon et al 2006;Shchelkunov et al 2006;Greco et al 2007;Marusic et al 2007; Barbante et al 2008;de Virgilio et al 2008;McCabe et al 2008;Zhou et al 2008).…”

Section: Introductionmentioning

confidence: 99%

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Scotti

Alagna

Ferraiolo

et al. 2009

Planta

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Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.

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The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

Cited by 56 publications

References 51 publications

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

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