Research Group Phytoantibodies
Head: Prof. Dr. Udo Conrad
The research group is concerned with the production of recombinant proteins in transgenic plants and with simple and innovative methods for the purification of these proteins. In addition, the group is working on the targeted degradation of specific proteins in plants in order to block their function.
An important subject area includes work on Molecular F(Ph)arming. One focus of this work is the expression of repetitive proteins in plants in order to produce new biomaterials. Spider silk proteins are in the focus of interest here. During evolution, spiders have perfected the production and use of very different protein-based silk materials. These silk fibres have extraordinary properties that far outperform technical fibres in terms of strength and elasticity. The silk used by spiders as a holding thread and as a frame thread for their nets has very special mechanical properties: it is five times stronger than steel and three times tougher than Kevlar (p-Aramid), the best man-made synthetic fibre. The catching thread silk, e.g. the flagelliform silk, is less firm than the carrying thread silk, but can be stretched to several times its length before it tears. The silk used as a catch thread therefore has a very high elasticity and stretchability. It can therefore absorb the kinetic energy of the prey very well. The work has therefore concentrated on the expression of the main constituents of the silk, the proteins MaSp1 (Scheller et al., 2001) and MaSp2 as well as the most important constituent of the flagelliform silk, the protein FLAG. It is assumed that the size of the bearing silk proteins is a key factor for the mechanical properties of spun fibres, as all spiders investigated so far produce very high molecular weight spider silk proteins. For this reason, the production of spider silk proteins of native size (over 200 kDa) in plants is of central interest. As a method we use posttranslational linkage in vitro by transglutaminase (Weichert et al., 2014) and in vivo by intein-based protein splicing (Hauptmann et al., 2013a, Hauptmann et al., 2013b). "Protein splicing" was also used for seed-specific accumulation of stable high molecular weight spider silk protein multimers (Weichert et al., 2016). ELPylated spider silk derivatives proved to be non-cytotoxic and non-hemolytic biopolymers (Hauptmann et al., 2015). Mechanical characterization is performed by cooperation partners at Fraunhofer IWM Halle (Junghans et al., 2006; Junghans et al., 2008, Weichert et al., 2014, Hauptmann et al., 2013).
A second focus is the production of therapeutic antibodies and vaccines in plants. Transgenic plants have been used as production tools for therapeutic proteins for 20 years. In particular, the development of scalable and inexpensive purification systems is an essential condition for the further use of this technology. For both classes of therapeutic proteins, ELPylation has been established as a suitable method for expression enhancement and efficient purification (Floss et al., 2008, Floss et al. 2009a, Floss et al. 2009b, Floss et al. 2010a, Floss et al. 2010b, Conrad et al., 2011, Phan and Conrad, 2011).
Current work on avian influenza antigens focuses on the production of highly immunogenic haemagglutinin multimers in plants. This work is done in cooperation with the Institute for Biotechnology in Hanoi, Vietnam, with the company NAVETCO, Ho Chi Minh City, Vietnam and with the Friedrich-Löffler-Institute on Riems. We were able to show that haemagglutin trimers produced in plants induce potentially neutralizing immune responses in mice (Phan et al., 2013). By exploiting the S-tag-S-protein interaction, we succeeded in producing haemagglutinin oligomers in planta that induced neutralizing immune responses in mice (Phan et al., 2917). Derated immune responses could be produced with raw extracts from Nicotiana benthamiana plants. This minimization of downstream processing enables rapid vaccine production at low cost, as required for veterinary applications. Furthermore, we have generated oligomers in vitro using Streptag-StrepTactin interaction, which are also neutralizing immune responses in mice (Phan et al., 2018).
Another focus of our work is the degradation of specific proteins in plants. We have shown that GFP (green fluorescent protein) can be specifically degraded in cytosol (Baudisch et al., 2018). GFP is recognized by a specific nanobody (VHH). This nanobody is fused with an F-box protein. The F-box protein-nanobody fusion protein with the bound GFP (F-box from Drosophila melanogaster) is transported into the proteasome with the aid of plant proteins and degraded. This also functions with GFP fusion proteins, as can be seen from the comparison of 1a) and 1b) in the figure.
In a similar approach, we fused the specific nanobody with the protein BTB (from human cells) and in this way we also induced the degradation of a GFP fusion protein, as shown by the comparison of 2a) and 2b). By such experiments from the toolbox of synthetic biology, we aim to specifically inhibit selected protein functions or to influence the interaction with pathogens. Here, we are cooperating with the Chromosome Structure and Function group at IPK and with AIPlanta, Neustadt.
Phage Display Technology
In the phytoantibody group recombinant antibodies are generated by phage display screening. Various semisynthetic libraries (scFv and VH, VHH) are available for this purpose. In this way, various recombinant antibodies have been generated and characterized within the framework of national and international cooperations. This includes recombinant antibodies against plant pathogen antigens and antigens of human pathogens as well as recombinant antibodies against phytohormones and regulatory proteins.
Inverse Transition Cycling
ELPylated proteins can be purified by utilizing special properties of ELP. This is achieved by exploiting the fact that ELPs are reversible depending on salt concentration and temperature. At lower temperatures and without salt they dissolve again. The precipitates are separated by centrifugation (cITC) or filtration (mITC). Scalable protocols have been developed for various applications (spider silk proteins, avian flu antigens) (Phan and Conrad, 2011, Heppner et al., 2016).