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Project 6: Development and production of VHH-Fc antibodies as functional tools for plant biotechnological research
Promotor: Ann Depicker, VIB Dept. of Plant Systems Biology, Ghent University
Co-promotor: Serge Muyldermans, VIB Dept. of Molecular and Cellular Interactions, Vrije Universiteit Brussel
Keywords: Camel antibodies (nanobodies, VHH), bivalent antibodies, transgenic seeds, molecular farming, proteomics
Host University: Ghent University
Antibodies are very important tools in scientific research, especially for protein detection, quantification, localization and purification. However, until now, very few primary antibodies are available for plant biotechnical research. The aim of this project is therefore to develop nanobodies (VHH) or derived antibodies against several plant proteins to use as functional tools in plant biotechnological projects. In this PhD research, proteins important in the "Systems Biology of Yield" and "Functional Proteomics" groups in the VIB-PSB department, will be produced in a recombinant protein production system, and the purified proteins will be used to immunize a dromedary (VIB-MCI). Based on the isolated lymphocytes, a VHH display library will be constructed and screened for specific VHHs. The positive clones will be analyzed in detail (VIB-MCI). First, the use of the VHHs in ELISA, the affinity for the plant target and the specificity versus cross reactivity will be carefully monitored (VIB-MCI). Then the functionality for localization and immunoprecipitation will be evaluated (VIB-PSB). For some applications, such as ELISA's or Westerns, the nanobodies with a his tag can be used as such. Also, the use of the VHHs as immunomodulating intrabodies will be considered (intrabodies, intracellularly expressed antibodies). For other applications, the VHH antibodies will be fused to an Fc-fragment. These VHH-Fc fusions will make the antigen-antibody interaction stronger because of avidity, resulting in a higher sensitivity. Moreover, the Fc-fragment will allow amplification of the signal via the multiple available Fc specific secondary antibodies. Production of the VHH-Fc fusions will be done in the Pichia Pastoris and/or Arabidopsis thaliana seed production platforms (VIB-PSB). The produced VHH-Fcs will be evaluated for their functionality as primary antibodies in immunolocalisation experiments and for identification of interacting proteins in the plant proteome research projects (VIB-PSB). This project brings together the expertise of the VIB research groups of Serge Muyldermans (camelid antibodies) in the MCI-VIB department at the VUB , and of Ann Depicker (heterologous protein production in plant seeds, plant experimental tools), Dirk Inze (Systems Biology of Yield) and Geert De Jaeger (Functional Proteomics) at the PSB-VIB department at the UGent.
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