Expression of single-chain antibodies in transgenic plants

Galeffi, Patrizia; Lombardi, Alessio; Donato, Monica Di; Latini, Arianna; Sperandei, Maria; Cantale, Cristina; Giacomini, Patrizio

doi:10.1016/j.vaccine.2004.11.025

Cited by 38 publications

(28 citation statements)

References 10 publications

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“…The Agrobacterium-mediated transformation of tobacco leaf discs was performed according to a protocol manual (Horsch et al 1998). …”

Section: Tobacco Transformationmentioning

confidence: 99%

“…Recently, the successful use of plants as expression systems for a number of biopharmaceuticals and therapeutic plantibodies has opened new vistas for medical protein and peptide production. These include cancer treatments (Khoudi et al 1999;Torres et al 1999;Vaquero et al 1999;Stoger et al 2000;Galeffi et al 2005), the treatment of dental caries (Ma et al 1998), B-cell lymphoma treatment (McCormick et al 1999), the production of microbial toxins (Almquist et al 2006), anticoagulant treatment, the production of blood substitutes, hepatitis C and B treatment, wound repair and the control of cell proliferation (Cramer et al 1999;Schinkel et al 2005), treatment of neutropenia/cystic fibrosis/liver disease/hemorrhage, the production of trypsin inhibitors for transplantation surgery (Ruggiero et al 2000), the production of growth hormones (Wang et al 2005;Ding et al 2006), the treatment of anemia/liver cirrhosis/burns, the production of opiates as antihyperanalgesics for surgical use (Kusnadi et al 1997), production of collagen (Ruggiero et al 2000), and antimicrobial activity (Chong and Langridge 2000).…”

Section: Introductionmentioning

confidence: 99%

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Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

et al. 2008

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Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots by fibrinolysis and in activating the transition from plasminogen to plasmin in blood vessels. The recombinant tissue-plasminogen activator (rTPA) produced in plants is a soluble protein derived from human t-PA. In order to produce rTPA protein in plants, a DNA fragment encoding the t-PA protein was cloned into a plant binary plasmid that included the cauliflower mosaic virus 35S promoter, a tobacco etch virus (TEV) leader sequence, an N-terminal signal peptide of the alfala glucose-regulated endoplasmic reticular protein, and an 35S terminator. The insertion of the rTPA gene in genomic DNA of phospinothricin-resistant tobacco plants was confirmed by polymerase chain reaction (PCR). The presence of the rTPA-specific transcript in the total RNAs of transgenic plants leaves was verified by reverse transcription (RT)-PCR. Immunoblot analysis of the transformed tobacco leaves revealed single bands of 68-kDa rTPAs. Enzyme-linked immunosorbent assay experiments demonstrated that the highest level of rTPA expression was 0.0014% of the total soluble protein in tobacco plants. Fibrinolysis of rTPA was confirmed by fibrin plate assay and zymography. These studies demonstrate that plants can be employed as bioreactor systems for the production of an enzymatically active t-PA with properties similar to the recombinant t-PA produced by mammalian cells.

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“…The Agrobacterium-mediated transformation of tobacco leaf discs was performed according to a protocol manual (Horsch et al 1998). …”

Section: Tobacco Transformationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

et al. 2008

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“…Agrobacterium-mediated transient expression is also a promising method for the simultaneous production of multiple proteins 2, [25][26][27] . For example, plant transient expression systems have been used to produce tumor-specific recombinant antibodies 28,29 , a glycosylated recombinant antibody against the epidermal growth factor receptor 30 , and a monoclonal antibody specific for anthrax protective antigen 31,32 . Co-infiltration of Nicotiana benthamiana plants with a target gene and a suppressor of gene silencing results in enhanced target protein expression 33,34 .…”

Section: Introductionmentioning

confidence: 99%

Optimization and Utilization of <em>Agrobacterium</em>-mediated Transient Protein Production in <em>Nicotiana</em>

Shamloul

Trusa

Mett

et al. 2014

JoVE

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Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

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“…Later on, the cloning of the genes for both chains in a single event was reported, producing a plant that expresses the whole functional antibody (Table 6.1 ). Functional antibody fragments are also expressed, the fragment antigen binding (Fab), containing one of the two identical combining sites of the immunoglobulin, the antibody single bivalent fragments F(ab')2 containing the two combining sites, fragments consisting of only the variable regions of the L and H chains (Fv), that retain the antigen binding capacity, single-chain variable fragments (scFv) consisting of the VL and VH domains linked by a fl exible peptide, maintaining the binding capacity (Seddas-Dozolme et al 1999 ;Eeckhout et al 2004 ;Galeffi et al 2005 ), and single-domain antibodies (VHH) from camelids (Marconi and Alvarez 2014 ). Secretory antibodies (IgA) are also expressed (Larrick et al 2001 ).…”

Section: Antibodiesmentioning

confidence: 99%

Molecular Farming in Plants

Álvarez

2014

Plant Biotechnology for Health

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Molecular farming can generally be defi ned as the production of molecules (proteins, fatty acids) for the pharmaceutical and chemical industries in transgenic organisms (plants, animals, etc.).Molecular farming in plants has advantageous aspects as biosecurity, they do not bear pathogens for humans or animals, and they do not produce toxins. Also, plant protein synthetic machinery is able to produce complex glycosylated proteins as they have a glycosylation pattern with slight difference respect of that of mammals, and can perform foldings.When displayed in in vitro conditions, molecular farming have the characteristic advantages of that type of culture, mainly the capacity of working under Good Manufactory practices as is required by the pharmaceutical industry. The critical aspects of plants for molecular farming are the different glycosylation patterns respect to mammals, the duration of the productive process, and the relatively low yields.The approval by the FDA of the fi rst medicine to be used in humans, taliglucerase alfa (ELELYSO®), has give an impulse to this technology but there are some drawbacks to be addressed, particularly related to low yields and regulatory aspects.

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Expression of single-chain antibodies in transgenic plants

Cited by 38 publications

References 10 publications

Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

Expression and Characterization of Human Tissue-Plasminogen Activator in Transgenic Tobacco Plants

Optimization and Utilization of <em>Agrobacterium</em>-mediated Transient Protein Production in <em>Nicotiana</em>

Molecular Farming in Plants

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