High‐level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes

Zhou, Fei; Badillo-Corona, Jesús Agustín; Karcher, Daniel; González-Rábade, Nuria; Piepenburg, Katrin; Borchers, A-M Inka; Maloney, Alan; Kavanagh, Tony A.; Gray, John C.; Bock, Ralph

doi:10.1111/j.1467-7652.2008.00356.x

Cited by 171 publications

(242 citation statements)

References 43 publications

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“…HIVp24 is expected to be a key component of an AIDS vaccine (Obregon et al 2006) and HCV core protein is a target of vaccine development for Hepatitis C disease (Elmowalid et al 2007). The HIVp24 sequence used here had the additional advantage of being optimized for plastid expression (Zhou et al 2008).…”

Section: Discussionmentioning

confidence: 99%

“…1a) were engineered based on the pZF7lox plastid vector (Zhou et al 2008). After the initial steps of construction, the coding sequences for the PGL35-HCV core protein and PGL35-HIVp24 were subcloned into the pZF7lox plastid vector by excision and replacement of HIVp24-nef.…”

Section: Chloroplast Expression Constructsmentioning

confidence: 99%

“…4). A dilution series of a total extract of tobacco line pZF5.5A (Zhou et al 2008) expressing HIVp24 to 4 % of total soluble protein (1, 2 and 3 lg) was used as reference. RbcL is used as a loading control and is weaker in the tobacco line pZF5.5A due to the lesser amount of total protein loaded.…”

Section: Expression and Quantification Of Fusion Proteinsmentioning

confidence: 99%

“…The plastid transformation vectors constructed in this study are based on the pZF7lox plastid vector (Zhou et al 2008). The pZF7lox plastid transformation vectors are derived from plasmids pZSJH1 (Birch-Machin et al 2004) and pRB95 (Ruf et al 2001).…”

Section: Cloning Proceduresmentioning

confidence: 99%

“…Coding sequences for Hepatitis C virus antigen-core protein from pSV-sport HCV CE1E2 plasmid (kindly provided by Dr. Bruno Martoglio, Novartis Pharma AG, Basel, Switzerland) was amplified using HCV-F forward primer introducing a NdeI restriction site at the 5 0 end and with 3 0 -end HCV-R reverse primer introducing a XbaI restriction site. The codon-optimized synthetic HIVp24 (Zhou et al 2008) from pCR4TOPO-p24 was amplified using the HIVp24-F forward primer introducing a NdeI restriction site at the 5 0 -end and with HIVp24-R reverse primer introducing a XbaI restriction at the 3 0 -end. Both the HCV and HIVp24 amplification products were digested with the NdeI and XbaI and ligated into the corresponding site of pLITMUS28i-PGL35.…”

Section: Cloning Proceduresmentioning

confidence: 99%

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Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

et al. 2012

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Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C-terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35-HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35-HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes.

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Section: Discussionmentioning

confidence: 99%

Section: Chloroplast Expression Constructsmentioning

confidence: 99%

Section: Expression and Quantification Of Fusion Proteinsmentioning

confidence: 99%

Section: Cloning Proceduresmentioning

confidence: 99%

Section: Cloning Proceduresmentioning

confidence: 99%

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Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

et al. 2012

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Metabolic Engineering of Photosynthetic Cells – in Collaboration with Nature

Sørensen

Møller

2021

Metabolic Engineering

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Dicistronic expression of human proinsulin–protein A fusion in tobacco chloroplast

Yarbakht

Jalali-Javaran

Nikkhah

et al. 2014

Biotech and App Biochem

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Different expression systems such as bacteria and mammalian cells have been used to produce pharmaceutical proteins. In recent years, the use of plants as bioreactors offers efficient and economical systems in recombinant protein production. Furthermore, because of the large number of plastid copies in plants, chloroplast engineering functions as an effective method to increase recombinant protein expression. Because the commercially available insulin for treatment does not contain C-peptide, which is of great importance for type 1 diabetic patients, the current study introduces the human proinsulin gene fused with protein A into the tobacco chloroplast genome using the biolistic method. To achieve homoplasmy, three rounds of selection and regeneration of transforming cells were performed on the medium that contained spectinomycin antibiotic and hormones. The PCR analysis indicated the presence of the proinsulin gene in transplastomic plants. The reverse-transcription PCR analysis confirmed the expression of the proinsulin-protein A fusion at the transcription level. Immunoblot assays of leaf-derived protein extracts confirmed that the target gene expression is up to 0.2% of the total soluble protein. Our study showed that protein A fusion is not as efficient as other reported fusions. The transplastomic plants were also confirmed for homoplasmy using Southern blot analysis.

show abstract

High‐level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes

Cited by 171 publications

References 43 publications

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

Metabolic Engineering of Photosynthetic Cells – in Collaboration with Nature

Dicistronic expression of human proinsulin–protein A fusion in tobacco chloroplast

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