The Chloroplasts as Platform for Recombinant Proteins Production

Scotti, Nunzia; Bellucci, Michele; Cardi, Teodoro

doi:10.1007/978-3-642-39426-3_10

Cited by 10 publications

(5 citation statements)

References 179 publications

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“…Whether or not N-terminal sequence differences between EDIII variants account for the observed differences in protein accumulation, remains to be investigated. In general, accumulation of foreign proteins in plastids depends on the rates of transcription, translation and protein stability (Scotti et al 2013;Nakakura et al 2016), and the recombinant protein accumulation level is nearly impossible to predict (Bock 2014). Unexpectedly, the immunoblot analyses revealed the presence of additional protein bands with higher molecular masses than the predicted 13 kDa for EDIII-1 and EDIII-3, respectively.…”

Section: Discussionmentioning

confidence: 95%

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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Section: Discussionmentioning

confidence: 95%

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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“…In fact, compared to its cyanobacterial ancestors, it retains only a small portion of genes (110–120) which can be classified into three major groups: protein synthesis genes (e.g., rRNAs, RNA polymerase subunits, tRNAs, ribosomal proteins), photosynthesis-related genes, and other genes. Although plastids share several features with bacteria, the regulation of gene expression is rather complex and is mainly controlled at post-transcriptional level, in particular during the initiation phase of translation [ 9 , 13 ].…”

Section: Plastid Transformation Technologymentioning

confidence: 99%

“…In the last decades, the plant-based platform for the production of recombinant proteins has had a huge boost due to low production costs, and the development of several strategies to overcome the main limitations of the system [ 6 , 7 ]. Among them, the production of recombinant enzymes by plastid transformation offers multiple attractions to biotechnologists [ 8 , 9 , 10 ], including the potential for a very high protein accumulation level [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Plastid Transformation: New Challenges in the Circular Economy Era

Tamburino

Marcolongo²,

Sannino

et al. 2022

IJMS

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In a circular economy era the transition towards renewable and sustainable materials is very urgent. The development of bio-based solutions, that can ensure technological circularity in many priority areas (e.g., agriculture, biotechnology, ecology, green industry, etc.), is very strategic. The agricultural and fishing industry wastes represent important feedstocks that require the development of sustainable and environmentally-friendly industrial processes to produce and recover biofuels, chemicals and bioactive molecules. In this context, the replacement, in industrial processes, of chemicals with enzyme-based catalysts assures great benefits to humans and the environment. In this review, we describe the potentiality of the plastid transformation technology as a sustainable and cheap platform for the production of recombinant industrial enzymes, summarize the current knowledge on the technology, and display examples of cellulolytic enzymes already produced. Further, we illustrate several types of bacterial auxiliary and chitinases/chitin deacetylases enzymes with high biotechnological value that could be manufactured by plastid transformation.

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“…Gene integration occurs precisely via homologous recombination by flanking the genetic construct of interest with sequences immediately upstream and downstream the chosen insertion site, which avoids unwanted mutation of host genes. In many plants with strict maternal plastid inheritance, transgene containment is very much improved due to rare pollen transmission, and backcrossing the transgene to different genotypes is facilitated (Scotti et al 2013).…”

Section: Introductionmentioning

confidence: 99%

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

et al. 2015

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A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

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The Chloroplasts as Platform for Recombinant Proteins Production

Cited by 10 publications

References 179 publications

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

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

Plastid Transformation: New Challenges in the Circular Economy Era

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

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