Expression of a Ricin Toxin B Subunit: Insulin Fusion Protein in Edible Plant Tissues

Carter, James E.; Odumosu, Oludare; Langridge, William H. R.

doi:10.1007/s12033-009-9217-1

Cited by 17 publications

(8 citation statements)

References 48 publications

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“…In our experiments, fusion protein RTB–M130 was detected as a band with a molecular mass of about 70 kDa (calculated mass of the protein without the N-terminal signal peptide–32.6 kDa), and it was therefore presumably in aggregated form. Similar expression behavior of RTB in a fusion protein was observed by Carter et al ( 2010 ), where it was fused with the C-terminal end of proinsulin (Ins) in transgenic potato tubers. Those authors showed that the fusion protein Ins–RTB apparently aggregates in large molecular complexes, being detected as bands of more than 200 kDa (estimated mass of Ins–RTB-−38.2 kDa).…”

Section: Discussionsupporting

confidence: 75%

“…The authors believed that aggregation of Ins–RTB occurs due to the formation of a large number of chaotic intermolecular disulfide bonds. Nevertheless, Ins–RTB retained the ability to bind to asialofetuin (Carter et al, 2010 ). In our experiments, RTB–M130 also retained the ability to bind to asialofetuin and elicit an immune response in mice.…”

Section: Discussionmentioning

confidence: 99%

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Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

et al. 2018

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The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

et al. 2018

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“…Similar accumulation levels were found in this study using the Arabidopsis seed system, with a maximum yield of 0.70% TSP. Human proinsulin has also been expressed previously in plants as a fusion with either CTB (Arakawa et al, 1998;Ruhlman et al, 2007) or RTB (Carter et al, 2010). The highest yields of up to 16% TSP were obtained in transplastomic tobacco plants (Ruhlman et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Non‐food/feed seeds as biofactories for the high‐yield production of recombinant pharmaceuticals

Morandini

Avesani

Bortesi

et al. 2011

Plant Biotechnology Journal

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SummaryWe describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food ⁄ feed crops. A vector designed for direct subcloning by Gateway Ò recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67 ⁄ 65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67 ⁄ 65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

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“…Th2 immunity can be induced by the lectin B-chain of type-2 RIP from Ricinus communis , which binds to D-galactose containing glycans, including many glycoproteins expressed on the surface of enterocytes. This property motivated the genetic linking of the ricin B-chain with the coding region of the proinsulin gene, expressed as a fusion protein in E. coli [34] or in Solanum tuberosum (potato plant) [35]. Oral administration of this recombinant fusion protein to prediabetic NOD mice suppressed the auto-immune insulitis, associated with the Th2 immune response, whereas administration of insulin only did not interfere in the course of the disease [34].…”

Section: Overview Of Immunomodulatory Plant Lectinsmentioning

confidence: 99%

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties

et al. 2013

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Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.

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Expression of a Ricin Toxin B Subunit: Insulin Fusion Protein in Edible Plant Tissues

Cited by 17 publications

References 48 publications

Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

Non‐food/feed seeds as biofactories for the high‐yield production of recombinant pharmaceuticals

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties

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