High level accumulation of single‐chain variable fragments in the cytosol of transgenic <i>Petunia hybrida</i>

Jaeger, Geert De; Buys, Emmanuel; Eeckhout, Dominique; Wilde, Chris De; Jacobs, Anni; Kapila, Jyoti; Angenon, Geert; Montagu, Marc Van; Geräts, Tom; Depicker, Anna

doi:10.1046/j.1432-1327.1999.00060.x

Cited by 91 publications

(56 citation statements)

References 49 publications

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“…Once folded into the native structure, the disulfide bridges are inaccessible for reduced glutathione due to a tight surrounding structure (33, 38). However, proper folding does not always guarantee disulfide bridge formation, as illustrated by the absence of disulfide bridges in two other functional cytosolic scFv antibody fragments in stably transformed tobacco and petunia plants (39,40). That folding in our scFv antibody is accurate is emphasized by the 202Ј linker derivative with the cysteine residue.…”

Section: Discussionmentioning

confidence: 92%

Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol

Schouten¹,

Roosien²,

Bakker³

et al. 2002

Journal of Biological Chemistry

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Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fragment consists of the variable light and heavy chain domains from a mouse IgG antibody, which are connected by a flexible linker peptide. In each domain one disulfide bridge is present. The functionality of antibodies, which are normally secreted via the oxidizing environment of the endoplasmic reticulum, depends on the formation of intramolecular disulfide bridges. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. In contrast, transient expression in tobacco protoplasts resulted in a cytosolic scFv lacking disulfide bridges, which had a substantially reduced affinity for the antigen. This indicates that functionality rather than stability is determined by the presence of disulfide bridges in the in planta-expressed scFv antibody. The controversial observation of disulfide bond formation in the cytosol is discussed.Disulfide bond formation and disulfide rearrangements are reversible processes and are kinetically and thermodynamically affected by the redox state of the environment (1-3). In eukaryotic cells, subcellular compartmentation plays an important role in the regulation of these reversible thiol-disulfide exchange reactions. Glutathione is the most abundant nonprotein thiol in eukaryotic cells, and the preferential transport of the disulfide form (GSSG) compared with the reduced form (GSH) into the ER 1 lumen is thought to be responsible in maintaining redox compartmentation between the ER and cytosol. For murine hybridoma cells, it was shown that in the secretory pathway the ratio of reduced glutathione to the disulfide form (GSH/GSSG) ranged from 1 to 3, whereas the overall cellular GSH/GSSG ratio ranged from 30 to 100 (3). In vitro studies show that the redox environment of the ER corresponds with the optimum for refolding of disulfide-bonded proteins (3). In the ER, disulfide formation and rearrangements are further enhanced by protein disulfide isomerase (4).As soon as the N-terminal part of a nascent polypeptide chain enters the oxidizing environment of the ER, folding starts with the aid of a full array of molecular chaperones and folding catalysts. Failure of disulfide formation has an adverse effect on various processes ranging from protein folding, oligomerization, and intracellular transport to secretion (5-10). In extracellular proteins containing cysteine residues, there is rarely more than one free thiol group (11). Free thiols are considered extremely reactive in the oxidizing extracellular environment and may lead to disastrous polymerization or make folding more complex (11). Th...

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Section: Discussionmentioning

confidence: 92%

Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol

Schouten¹,

Roosien²,

Bakker³

et al. 2002

Journal of Biological Chemistry

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“…Since no signal peptide was attached to the GR sequence in addition to the CaMV 35S promoter sequence, GR has very likely not migrated out of the cytosol after its mRNA was translated [3]. Recombinant proteins often accumulate only at a very low amount in the cytosol despite that the transgene is efficiently transcribed and the protein is also stable in this cellular compartment [8,10,13,33,37]. Possible reasons are incorrect folding of proteins and the presence of the ubiquitinproteasome proteolytic pathway for recognition and degradation of incorrectly folded proteins [23,46].…”

Section: Resultsmentioning

confidence: 99%

Use of Transgenic Oryzacystatin-I-Expressing Plants Enhances Recombinant Protein Production

Pillay

Kibido

Plessis

et al. 2012

Appl Biochem Biotechnol

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Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter-GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein very likely due to reduced activity of endogenous cysteine protease activity.

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“…Plants lack an immune system, but with the development of genetic engineering it is now possible to express entire or recombinant antibodies in planta (2,7,8,18,22,25,30). This approach has been used with variable success for the control of several pathogens (5,19,26,29,34,38) and seems particularly well suited for the control of phytopathogenic mollicutes.…”

Section: Discussionmentioning

confidence: 99%

“…Expression of full-size IgGs could not be obtained in the cytoplasm, probably because of its reducing environment, preventing the formation of disulfide bonds (8,18). On the contrary, several, but not all, recombinant antibody (scFv) molecules have been functionally expressed in the cytoplasm (2,5,7,25,27,30), and because of their ease of construction, their relatively small size, which facilitates diffusion, and no requirement for assembly, they have become the best candidates for plantibody production. However, the effect of scFv molecules on morphology, growth, and metabolism of mollicutes has not been evaluated.…”

Section: Discussionmentioning

confidence: 99%

Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri

Malembic

Saillard

Bové

et al. 2002

Appl Environ Microbiol

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Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed.Plant-pathogenic mollicutes include the genus Spiroplasma, comprising organisms culturable in complex artificial media and showing a helical morphology, and the Candidatus genus phytoplasma, containing large numbers of pleiomorphic organisms which, until now, have resisted in vitro cultivation (4, 9). Hence, spiroplasmas are the most-studied phytopathogenic mollicutes, and S. citri (23) is the model organism for this important group of plant pathogens.Mollicutes are eubacteria without a cell wall and thus delimited only by a cytoplasmic membrane. This characteristic has been linked with the fact that metabolism and growth of mollicutes were strongly inhibited by antibodies directed against membrane proteins (3,13,14,19,36,37). This is why expression of antibodies in plants is an attractive strategy to control phytopathogenic mollicutes, especially since at this time, there is no other curative method. In 1998, we produced tobacco plants expressing single-chain variable fragment (scFv) recombinant antibodies against the stolbur phytoplasma, via the secretory pathway (apoplastic route), and some resistance to infection could be observed (20). Further analysis of the plants revealed that the resistance was not complete and that phytoplasmas could invade the tobacco plant. Chen and Chen (5) successfully expressed an scFv recombinant antibody directed against Spiroplasma kunkelii, the agent of corn stunt disease, in the cytoplasm (symplasmic route) of maize, but no resistance to the corn stunt spiroplasma was obtained.Difficulty in targeting functional antibodies into the phloem sieve tubes, where phytoplasmas reside, is probably one reason for failure, but it cannot be ruled out that scFv recombinant antibodies are not or are less efficient than polyclonal or monoclonal antibodies in inhibiting metabolism and growth of mollicutes. Indeed, Chen and Chen (5) showed that monoclonal antibodies were less efficient than polyclonal antibodies in inhibiting growth and metabolism of S. kunkelii.For the study presented here, we have constructed an S. citri-specific scFv recombinant antibody from a hybridoma cell line producing a monoclonal antibody against ...

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High level accumulation of single‐chain variable fragments in the cytosol of transgenic Petunia hybrida

Cited by 91 publications

References 49 publications

Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol

Formation of Disulfide Bridges by a Single-chain Fv Antibody in the Reducing Ectopic Environment of the Plant Cytosol

Use of Transgenic Oryzacystatin-I-Expressing Plants Enhances Recombinant Protein Production

Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri

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