Chris De Wilde

Plants as bioreactors for protein production: avoiding the problem of transgene silencing.

Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.

Production of antibodies and antibody fragments in plants.

Our current knowledge allows the generation of transgenic plants that efficiently produce heterologous proteins from plant, bacterial, fungal or animal origin. Among all types of recombinant proteins, antibodies are particularly attractive because of their ability to specifically recognize and bind virtually any type of antigen. Plants show several advantages as a large-scale antibody production system: they can be grown easily and inexpensively in large quantities that can be harvested, stored and processed by using existing infrastructures. Isolation and purification of plant-made antibodies, if necessary, allow fundamental, industrial, and therapeutical applications. In the past, we and others have successfully generated antibody-producing plants. The maximal accumulation levels of antibodies and antibody fragments that we observed are 1-5% of the extracted proteins. Currently, several biotechnological companies grow field crops to produce antibodies for ex planta applications on an industrial scale.

The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance

Immunomodulation is a molecular technique that allows the interference with cellular metabolism or pathogen infectivity by the ectopic expression of genes encoding antibodies or antibody fragments. In recent years, several reports have proven the value of this tool in plant research for modulation of phytohormone activity and for blocking plant-pathogen infection. Efficient application of the plantibody approach requires different levels of investigation. First of all, methods have to be available to clone efficiently the genes coding for antibodies or antibody fragments that bind the target antigen. Secondly, conditions to obtain high accumulation of antigen-binding antibodies and antibody fragments in plants are being investigated and optimized. Thirdly, different strategies are being evaluated to interfere with the function of the target molecule, thus enabling immunomodulation of metabolism or pathogen infectivity. In the near future, optimized antibody gene isolation and expression, especially in reducing subcellular environments, such as the cytosol and nucleus, should turn immunomodulation into a powerful and attractive tool for gene inactivation, complementary to the classical antisense and co-suppression approaches.

Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants.

Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells. Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5%. T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells. A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining. When selection for transformation competence is applied, the picture is completely different. Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome. Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors.

Bacterial and plant-produced scFv proteins have similar antigen-binding properties

A gene encoding a single‐chain variable (scFv) antibody fragment was expressed as a cytoplasmic and endoplasmic reticulum‐targeted protein in transgenic tobacco plants. In both cases, the scFv accumulated up to 0.01% of total soluble protein (TSP). The same scFv fragment was also produced in the periplasm of Escherichia coli. Measurement of the affinity by ELISA indicates that the affinity of the bacterially made scFv is about 80‐fold lower than that of the parental Fab fragment. The results suggest that the affinity of the plant‐produced scFv fragments is reduced to a similar extent, implying that all the plant‐produced scFv fragments are antigen binding.

Intact antigen-binding MAK33 antibody and Fab fragment accumulate in intercellular spaces of Arabidopsis thaliana

Abstract The in planta localization of a full-size immunoglobulin G (IgG) antibody and of its derived F ab fragment were determined using immunogold electron microscopy on transgenic Arabidopsis thaliana plants. Although it is widely assumed that antibodies, following assembly in the lumen of the endoplasmic reticulum, migrate via the secretory pathway to the cell surface, until now their subcellular in planta localization has not been demonstrated. Here, we show that both IgG and F ab fragment accumulate within intercellular spaces of leaf mesophyll cells of Arabidopsis . Using immunoblot analysis on leaf intercellular fluid, we demonstrate that these microscopically detected proteins are integral and functional antibodies or F ab fragments. The fact that a full-size IgG is not physically restricted from passage through the cell wall, despite having a molecular mass of 146 kD, questions the generally accepted limitations in cell wall porosity. These results are of particular significance in any plantibody approach that intends to modulate extracellular antigen function.

Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants

To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinant antibodies, constructs were engineered for accumulating full-size IgGs and Fab fragments in the plant cell apoplast or endoplasmic reticulum (ER). An in-house transformation protocol was worked out for the efficient co-transformation of potato root explants. Accumulation levels in tubers of up to 0.5% of total soluble protein were found for antibodies targeted to the ER whereas five-fold lower accumulation levels were found for antibodies targeted for secretion. Additionally, different aspects important for the commercial exploitation of potato tubers as a heterologous production system were analysed. Tubers could be stored for up to 6 months without significant loss of antibody amount or activity. Minor variations in antibody accumulation levels were observed in tubers that originated from the same transformant. Most isolated IgGs and Fab fragments bound the antigen and had the correct molecular weight when compared with the hybridoma-derived standard. Processing to greenhouse or field trials, including in vitro propagation of a selected transformant, required only approximately 9 months from the start of transformation, a time frame in which hundreds of kilograms of transgenic potato tubers could easily be obtained. Small-scale purification of IgG was possible by using standard laboratory techniques. Thus, molecular farming in potato tubers can be a viable production system for economic production of clinically or industrially interesting macromolecules, such as antibodies.

Generation of Single-Copy T-DNA Transformants in Arabidopsis by the CRE/loxP Recombination-Mediated Resolution System

We investigated whether complex T-DNA loci, often resulting in low transgene expression, can be resolved efficiently into single copies by CRE/loxP-mediated recombination. An SB-loxP T-DNA, containing two invertedly oriented loxP sequences located inside and immediately adjacent to the T-DNA border ends, was constructed. Regardless of the orientation and number of SB-loxP-derived T-DNAs integrated at one locus, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy. Seven transformants with a complex SB-loxP locus were crossed with a CRE-expressing plant. In three hybrids, the complex T-DNA locus was reduced efficiently to a single-copy locus. Upon segregation of the CRE recombinase gene, only the simplified T-DNA locus was found in the progeny, demonstrating DNA had been excised efficiently in the progenitor cells of the gametes. In the two transformants with an inverted T-DNA repeat, the T-DNA resolution was accompanied by at least a 10-fold enhanced transgene expression. Therefore, the resolution of complex loci to a single-copy T-DNA insert by the CRE/loxP recombination system can become a valuable method for the production of elite transgenic Arabidopsis thaliana plants that are less prone to gene silencing.

Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens

The recognition of the T-DNA left border (LB) repeat is affected by its surrounding sequences. Here, the LB regions were further characterized by molecular analysis of transgenic plants, obtained after Agrobacterium tumefaciens-mediated transformation with T-DNA vectors that had been modified in this LB region. At least the 24-bp LB repeat by itself was insufficient to terminate the T-strand synthesis. Addition of the natural inner and/or outer border regions to at least the LB repeat, even when present at a distance, enhanced the correct recognition of the LB repeat, reducing the number of plants containing vector backbone sequences. In tandem occurrence of both the octopine and nopaline LB regions with their repeats terminated the T-strand synthesis most efficiently at the LB, yielding a reproducibly high number of plants containing only the T-DNA. Furthermore, T-strand synthesis did not terminate efficiently at the right border (RB) repeat, which might indicate that signals in the outer RB region inhibit the termination of T-strand synthesis at the RB repeat.

Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida

© 1997 Federation of European Biochemical Societies.