Plastid Proteostasis and Heterologous Protein Accumulation in Transplastomic Plants

Marchis, Francesca De; Pompa, Andrea; Bellucci, Michele

doi:10.1104/pp.112.203778

Cited by 35 publications

(29 citation statements)

References 113 publications

(111 reference statements)

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“…This is especially relevant when protein instability constitutes the primary limiting factor [40] as occurs with the HPV E7 antigen that shows properties shared by ''natively unfolded'' or ''intrinsically disordered'' proteins [41].…”

Section: Discussionmentioning

confidence: 99%

Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts

et al. 2014

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Human papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death in women worldwide, and its E7 antigen is the major candidate for a therapeutic vaccine. The large scale production of E7 by molecular farming that would lead to the development of a safe and inexpensive vaccine is impaired by its low accumulation level in the plant cell. To enhance antigen production in the plastids, two alternative strategies were carried out: the expression of E7 as a translational fusion to β-glucuronidase enzyme and redirection of E7 into the thylakoid lumen. The use of the β-glucuronidase as a partner protein turned out to be a successful strategy, antigen expression levels were enhanced between 30 and 40 times relative to unfused E7. Moreover, best accumulation, albeit at a high metabolic cost that compromised biomass production, was obtained redirecting E7 into the thylakoid lumen by the incorporation of the N-terminal transit peptide, Str. Following this approach lumenal E7 production exceeded the stromal by two orders of magnitude. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts.

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Section: Discussionmentioning

confidence: 99%

Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts

et al. 2014

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“…Furthermore, ediii-1 and ediii-3 are controlled by the same regulatory elements, suggesting that transcript abundance is determined by factors within the coding sequence and/or by intrinsic factors depending on the leaf's developmental stage. It is well established that, in plastids, high mRNA accumulation does not necessarily result in high protein levels (Eberhard et al 2002;Kahlau and Bock 2008), because translation control is more dominant than transcriptional regulation (De Marchis et al 2012). Also, high turnover can lead to rapid degradation of proteins (Kim et al 1994).…”

Section: Discussionmentioning

confidence: 99%

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

et al. 2016

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Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia Ò ) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotypespecific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIIIexpressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway Ò plastid transformation vector for inducible transgene expression.

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“…Pulse-chase revealed that Gag was stable in the stromal environment for at least 8 h [53], but no information on the stability of the other constructs in the stroma is available. It should also be underlined that the stromal environment can adversely affect the folding of foreign proteins, thus leading to low accumulation, as it has been observed for certain secretory proteins expressed in transplastomic plants [54]. …”

Section: Discussionmentioning

confidence: 99%

Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco

Virgili-López

Langhans

Bubeck

et al. 2013

IJMS

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Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.

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Plastid Proteostasis and Heterologous Protein Accumulation in Transplastomic Plants

Cited by 35 publications

References 113 publications

Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts

Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco

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