Udo Conrad

Immunomodulation of ABA function affects early events in somatic embryo development

Abstract Immunomodulation of abscisic acid (ABA) function during somatic embryogenesis of Nicotiana plumbaginifolia has been used to demonstrate for the first time the effect of this phytohormone on early embryonic events. A homozygous transgenic line constitutively expressing an anti-abscisic acid (ABA) single chain fragment variable antibody in the endoplasmic reticulum was established. Development of somatic embryos from the transgenic line and the wild type was compared. The ABA biosynthesis mutants aba1 and aba2 and wild type cultures treated with the ABA biosynthesis inhibitor fluridone were also used for the comparative investigations. The development of embryonic structures was disturbed in the early stages of all cultures in which ABA function was blocked or which were ABA-deficient. After ABA complementation of the in vitro cell cultures normal somatic embryo development was restored.

Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFvs) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFvs accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFvs in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFvs expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFvs in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFvs at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.

Production of spider silk proteins in tobacco and potato

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420–3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability—a property that is used to purify the spidroins by a simple and efficient procedure.

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single‐chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a ‘wilty’ phenotype, was put under control of the seed‐specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild‐type plants apart from their seeds. Anti‐ABA scFv embryo development differed markedly from wild‐type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild‐type embryos. Anti‐ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti‐ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild‐type seeds but calculated free ABA levels were near‐zero until 21 days after pollination. We show for the first time seed‐specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

Optimization of scFv antibody production in transgenic plants.

BACKGROUND Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such as bacterial or mammalian cell culture. In order to explore transgenic plants for large-scale production and storage of recombinant antibodies we tried to optimize the accumulation and stability of functionally active single chain Fv (scFv) antibodies in transgenic tobacco plants. OBJECTIVES Two different scFv antibodies which were expressed in different plant organs and plant cell compartments have been used for the study. Accumulation levels and antibody properties such as stability and antigen-binding activity were investigated. STUDY DESIGN For ubiquitous expression in tobacco plants, transcription of the scFv genes was controlled by the strong cauliflower mosaic virus (CaMV) 35S promoter. We used seed specific legumin B4 (LeB4) and the unknown seed protein (USP) promoters from Vicia faba for storage organ specific expression. RESULTS High accumulation of the two different scFv proteins in transgenic tobacco plants was only achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 4-6.8% of total soluble proteins (TSP) in leaves and up to 3-4% in ripe tobacco seeds. Transgenic tobacco seeds as well as tobacco leaves facilitated stable storage of ER-accumulated scFvs over an extended (seeds) or a short (leaves) period of time. Functionally active scFv proteins could be extracted after harvesting of the leaf material--drying and storage for 1 week at room temperature. Both the amount and the binding activity of the scFv proteins remained unchanged. CONCLUSION A plant expression system where the scFv-proteins are targeted in the ER provides not only the highest accumulation level of active single chain Fv antibodies ever reported but also a short- or long-term storage of the foreign protein in the harvested plant material.

Seed-specific transcription factors ABI3 and FUS3: molecular interaction with DNA

In Arabidopsis thaliana (L.) Heynh. the seed-specific transcription factors ABI3 and FUS3 have key regulatory functions during the development of mature seeds. The highly conserved RY motif [DNA motif CATGCA(TG)], present in many seed-specific promoters, is an essential target of both regulators. Here we show that, in vitro, the full-length ABI3 protein, as well as FUS3 protein, is able to bind to RY-DNA and that the B3 domains of both transcription factors are necessary and sufficient for the specific interaction with the RY element. Flanking sequences of the RY motif modulate the binding, but the presence of an RY sequence alone allows the specific interaction of ABI3 and FUS3 with the target in vitro. Transcriptional activity of ABI3 and FUS3, measured by transient promoter activation, requires the B3 DNA-binding domain and an activation domain. In addition to the known N-terminal-located activation domain, a second transcription activation domain was found in the B1 region of ABI3.

Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application

Elastin-like polypeptides (ELPs) are highly biocompatible and exhibit a potentially highly useful property: that of a thermally responsive reversible phase transition. These characteristics make ELPs attractive for drug delivery, appealing as materials for tissue repair or engineering, and improve the efficiency with which recombinant proteins can be purified. ELP fusion proteins (referred to as ELPylation) inherit the reversible phase transition property. ELPylation technology recently has been extended to plant cells, and a number of plant-based expression systems have been evaluated for the production of ELPylated proteins. Here, we discuss recent developments in ELP technology and the substantial potential of ELPs for the deployment of transgenic plants as bioreactors to synthesize both biopharmaceuticals and industrial proteins.

Potato tubers as a biofactory for recombinant antibodies

Potato tubers have been successfully used for high-level production of a recombinant single-chain Fv (ScFv) antibody. Ubiquitous high-level expression was achieved under control of the CaMV 35S promoter through retention of the scFv protein in the endoplasmic reticulum. Recombinant antibodies accumulated up to 2% of total soluble tuber protein. After 1.5 years of tuber storage at 4 °C still half of the amount of scFv present in freshly harvested tubers was detectable. Its specific activity did not decrease during tuber storage. Recombinant protein could be efficiently purified from crude extracts by affinity chromatography.

Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation.

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and ‘inverse transition cycling’. Laboratory scale extraction of 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein. As a possible application, as well as to demonstrate biocompatibility, the growth of anchorage-dependent mammalian cells on spider silk-elastin coated culture plates was compared with conventional coatings such as collagen, fibronectin and poly-D-lysine. The anchorage-dependent chondrocytes showed similar growth behaviour and a rounded phenotype on collagen and on spider silk-elastin coated plates and the proliferation was remarkably superior to untreated polystyrene plates.

Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview

During the past two decades, antibodies, antibody derivatives and vaccines have been developed for therapeutic and diagnostic applications in human and veterinary medicine. Numerous species of dicot and monocot plants have been genetically modified to produce antibodies or vaccines, and a number of diverse transformation methods and strategies to enhance the accumulation of the pharmaceutical proteins are now available. Veterinary applications are the specific focus of this article, in particular for pathogenic viruses, bacteria and eukaryotic parasites. We focus on the advantages and remaining challenges of plant-based therapeutic proteins for veterinary applications with emphasis on expression platforms, technologies and economic considerations.