Recombinant human acid ?-glucosidase stored in tobacco seed is stable, active and taken up by human fibroblasts

Reggi, Serena; Marchetti, Stefano; Patti, Tamara; Amicis, Francesca De; Cariati, Roberta; Bembi, Bruno; Fogher, Corrado

doi:10.1007/s11103-004-6832-x

Cited by 49 publications

(31 citation statements)

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“…GLOB, which is the soybean basic 7S globulin promoter (DDBJ accession no. AX006477), was used for the seed-specific expression of antigenic proteins according to Reggi et al [19]. The chimeric constructs pBIpGLOB-F18 and pBIpGLOB-VT2eB (Fig.…”

Section: Methodsmentioning

confidence: 99%

Expression of verocytotoxicEscherichia coliantigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

et al. 2013

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Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

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Section: Methodsmentioning

confidence: 99%

Expression of verocytotoxicEscherichia coliantigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

et al. 2013

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“…Receptor-mediated uptake via a salvage pathway allows the enzyme to be sequestered intracellularly and subsequently targeted correctly to the human cell lysosome. Some of these enzymes have been produced as recombinant proteins in plants (Cramer et al 1999;Downing et al 2006;Reggi et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Ectopic expression of a conifer Abscisic Acid Insensitive3 transcription factor induces high-level synthesis of recombinant human α-l-iduronidase in transgenic tobacco leaves

Kermode

Zeng

et al. 2007

Plant Mol Biol

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We are examining various plant-based systems to produce enzymes for the treatment of human lysosomal storage disorders. Constitutive expression of the gene encoding the human lysosomal enzyme, alpha-L-iduronidase (IDUA; EC 3.2.1.76) in leaves of transgenic tobacco plants resulted in low-enzyme activity, and the protein appeared to be subject to proteolysis. Toward enhancing production of this recombinant enzyme in vegetative tissues, transgenic tobacco plants were generated to co-express a CaMV35S:Chamaecyparis nootkatensis Abscisic Acid Insensitive3 (CnABI3) gene construct, along with the human gene construct. The latter contained regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene (5'-flanking, signal-peptide-encoding, and 3'-flanking regions). Ectopic synthesis of the CnABI3 protein led to the transactivation of the arcelin promoter and accordingly high activity (e.g., 25,000 pmol/min/mg total soluble protein) and levels of recombinant IDUA mRNA and protein were induced in leaves of transgenic tobacco, particularly in the presence of 150-200 microM S-(+)-ABA. Synthesis of human IDUA containing a carboxy-terminal ER retention (SEKDEL) sequence was also inducible by ABA in leaves co-transformed with the CnABI3 gene. As compared to the natural S-(+)-ABA, two persistent ABA analogues, (+)-8' acetylene ABA and (+)-8'methylene ABA, led to greater levels of beta-glucuronidase (GUS) reporter activities in leaves co-expressing the CnABI3 gene and a vicilin:GUS chimeric gene. In contrast, (+)-8' acetylene ABA and natural ABA appeared to be equally effective in stimulating the CnABI3-induced expression of an arcelin:GUS gene, and of the human IDUA gene, the latter also driven by arcelin-gene-regulatory sequences. Various stress-related treatments, particularly high concentrations of NaCl, had an even greater effect than ABA in promoting accumulation of human IDUA in co-transformed tobacco leaves. This strategy provides the means of enhancing the yields of recombinant proteins in transgenic plant vegetative tissues and potentially in cultured plant cells. The human recombinant protein can be readily induced in the presence of chemicals such as NaCl that can be added to cell cultures or even whole plants without a significant increase in production costs.

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“…However, the targeting of recombinant proteins in seeds is not yet fully understood. For example, KDEL-tagged as well as secretory proteins have been detected in protein storage vacuoles, in the cell wall, in ER-derived compartments, or in a combination of the three (Herman et al, 1990;Philip et al, 1998Philip et al, , 2001Wright et al, 2001;Reggi et al, 2005;Downing et al, 2006;Petruccelli et al, 2006;Van Droogenbroeck et al, 2007;Abranches et al, 2008;Schmidt and Herman, 2008;Loos et al, 2011). As a consequence of this atypical targeting and deposition, seed-derived recombinant products may carry unusual N-glycosylation profiles (Van Droogenbroeck et al, 2007;Rademacher et al, 2008;Ramessar et al, 2008).…”

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Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

et al. 2011

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Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custommade human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.Recombinant monoclonal antibodies (mAbs) are of high therapeutic potential and have thus become a major product of the pharmaceutical industry (Aggarwal, 2009). In addition to full-length mAbs, antibodies are being engineered to alter their size, pharmacokinetics, specificity, valency, effector functions, etc. in order to better suit the intended applications (for review, see Filpula, 2007;Harmsen and De Haard, 2007). Of particular interest among these engineered fragments are single-chain variable fragments (scFvs), fusions of variable heavy and variable light domains that retain an antigen-binding function. Due to their smaller size as compared with their full-length counterparts, these molecules penetrate target tissues better and can even bind to intracellular targets. Furthermore, multimerization via disulfide bonds and/or multimerization domains allows for the production of divalent or higher order antibody-like molecules, where increased avidity

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Recombinant human acid ?-glucosidase stored in tobacco seed is stable, active and taken up by human fibroblasts

Cited by 49 publications

References 50 publications

Expression of verocytotoxicEscherichia coliantigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Expression of verocytotoxicEscherichia coliantigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Ectopic expression of a conifer Abscisic Acid Insensitive3 transcription factor induces high-level synthesis of recombinant human α-l-iduronidase in transgenic tobacco leaves

Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

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